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Sample records for real-time quantitative measurement

  1. EVALUATION OF QUANTITATIVE REAL TIME PCR FOR THE MEASUREMENT OF HELICOBATER PYLORI AT LOW CONCENTRATIONS IN DRINKING WATER

    Science.gov (United States)

    Aims: To determine the performance of a rapid, real time polymerase chain reaction (PCR) method for the detection and quantitative analysis Helicobacter pylori at low concentrations in drinking water.Methods and Results: A rapid DNA extraction and quantitative PCR (QPCR)...

  2. Quantitative real-time imaging of glutathione

    Science.gov (United States)

    Glutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown. Here, we report a reversible reaction-based fluorescent probe—designated as RealThiol (RT)—that can quantitatively monitor the real-time ...

  3. Measurement of bacterial gene expression in vivo by laser capture microdissection and quantitative real-time RT-PCR

    DEFF Research Database (Denmark)

    Schou, Kirstine Klitgaard; Jensen, Tim Kåre; Angen, Øystein

    2007-01-01

    Due to the relative small number of bacterial pathogens present in an infected host, exploration of pathogen gene expression in vivo is challenging. This study reports the development of a protocol for quantifying bacterial gene expression in vivo in Actinobacillus pleuropneumoniae using laser ca...... capture microdissection and real-time quantitative RT-PCR....

  4. Testing vaccines in human experimental malaria: statistical analysis of parasitemia measured by a quantitative real-time polymerase chain reaction.

    NARCIS (Netherlands)

    Hermsen, C.C.; Vlas, S.J. de; Gemert, G.J.A. van; Telgt, D.S.C.; Verhage, D.F.; Sauerwein, R.W.

    2004-01-01

    Clinical trials are an essential step in evaluation of safety and efficacy of malaria vaccines, and human experimental malaria infections have been used for evaluation of protective immunity of Plasmodium falciparum malaria. In this study, a quantitative real-time polymerase chain reaction was used

  5. Real-time quantitative measurement of the mode beating of an injection-seeded optical parametric oscillator

    OpenAIRE

    Mahnke, Peter; Wirth, Martin

    2010-01-01

    In this paper we present the dependency of a quantitative measurement of the first-order longitudinal mode beating of an injection-seeded optical parametric oscillator (OPO) on its injection-seeding state. We show the correlation of the intensity of the first-order longitudinal mode beating and the side-mode suppression of an injection-seeded OPO. We further demonstrate that the mode-beating intensity can be used as an indicator for the spectral purity of an injection-seeded OPO.

  6. Optimization and validation of DNA extraction and real-time PCR assay for the quantitative measurement of residual host cell DNA in biopharmaceutical products.

    Science.gov (United States)

    Hu, B; Sellers, J; Kupec, J; Ngo, W; Fenton, S; Yang, T-Y; Grebanier, A

    2014-01-01

    Host cell DNA contamination occurs during the production of biopharmaceuticals and must be controlled and monitored for the purity and safety of the drug products. A sodium iodide-based DNA extraction and a subsequent real time PCR assay were developed and validated for the quantitative measurement of residual host cell DNA impurity in monoclonal antibody therapeutic products. A sodium iodide-based commercial kit was optimized for the removal of interfering matrices. Several incubation steps from the kit protocol were combined and a neutralization buffer was introduced to protein digestion step to eliminate any precipitation from the detergent. The elimination of the two washing steps significantly reduced assay variability from loss of DNA pellets. The optimized DNA extraction procedure can recover DNA close to 100% for DNA concentrations from 10 to 100,000pg/mL. Of the published sequences of repetitive interspersed nuclear elements, we identified a nucleotide mismatch from the published CHO probe. Correction of this nucleotide increased DNA amplification by a thousand fold. The optimized assay was further validated for the quantitation of residual CHO DNA according to ICH guidelines with preset assay acceptance criteria. The method met all assay acceptance criteria and was found linear, accurate and precise for the quantitation of residual CHO in the linear range of 10-100,000pg DNA/mL. LOQ was measured at 10pg DNA/mL and LOD at 1pg DNA/mL. No matrix interference to our validated assay was detected from bioreactor harvest, Protein A eluate or eluate from ion exchange columns. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Prognostic impact of tumour burden measured by quantitative real-time PCR from sentinel lymph nodes of melanoma patients: data from 10-year follow-up.

    Science.gov (United States)

    Eigentler, Thomas Kurt; Hinderer, Joachim; Noor, Seema; Garbe, Claus; Leiter, Ulrike

    2017-04-01

    Sentinel node (SN) biopsy is regarded as standard of care for patients (pts) with cutaneous melanoma ≥1.0 mm of thickness. In the recent AJCC classification, findings in the SN are simply classified as positive or negative. In our analyses, we were interested whether quantitative real-time PCR (qRT-PCR) is able to predict disease-free survival (DFS) and overall survival (OS) depending on tumour burden in the SN. One hundred and forty-five pts were analysed using qRT-PCR for tyrosinase. Results were analysed using accelerated failure time survival model and cox proportional hazards models using the R statistics framework. Forty-one pts (28%) were positive according to qRT-PCR. In total, 12 of 41 pts showed tumour deposits in the SN using S100 and/or HMB-45-labelled immunohistochemistry as well. One patient had micrometastases detected by immunohistochemistry staining but failed in the qRT-PCR. After 10 years of follow-up, 34 patients recurred and 27 patients died. Significant differences for DFS and OS were detected for sex, increasing tumour thickness, ulceration of the primary tumour, and metastatic spread in the SN determined by histology as well as qRT-PCR. Quantitative analyses showed a logarithmic correlation between tumour burden and prognosis. However, as multivariate analyses reveal qRT-PCR was not superior compared to classical histology or immunohistology.

  8. Real-time deformation measurement using a transportable shearography system

    Science.gov (United States)

    Weijers, A. L.; van Brug, Hedser H.; Frankena, Hans J.

    1997-03-01

    A new system for deformation visualization has been developed, being a real time phase stepped shearing speckle interferometer. This system provides the possibility to measure quantitatively deformations of diffusely reflecting objects in an industrial environment. The main characteristics of this interferometer are its speed of operation and its reduced sensitivity to external disturbances. Apart from its semiconductor laser source, this system has a shoe-box size and is mounted on a tripod for easy handling during inspection. This paper describes the shearing speckle interferometry set-up, as it is developed at our laboratory and its potential for detecting defects.

  9. Towards real-time quantitative optical imaging for surgery

    Science.gov (United States)

    Gioux, Sylvain

    2017-07-01

    There is a pressing clinical need to provide image guidance during surgery. Currently, assessment of tissue that needs to be resected or avoided is performed subjectively leading to a large number of failures, patient morbidity and increased healthcare cost. Because near-infrared (NIR) optical imaging is safe, does not require contact, and can provide relatively deep information (several mm), it offers unparalleled capabilities for providing image guidance during surgery. In this work, we introduce a novel concept that enables the quantitative imaging of endogenous molecular information over large fields-of-view. Because this concept can be implemented in real-time, it is amenable to provide video-rate endogenous information during surgery.

  10. Quantitative real-time single particle analysis of virions

    Energy Technology Data Exchange (ETDEWEB)

    Heider, Susanne; Metzner, Christoph, E-mail: christoph.metzner@vetmeduni.ac.at

    2014-08-15

    Providing information about single virus particles has for a long time been mainly the domain of electron microscopy. More recently, technologies have been developed—or adapted from other fields, such as nanotechnology—to allow for the real-time quantification of physical virion particles, while supplying additional information such as particle diameter concomitantly. These technologies have progressed to the stage of commercialization increasing the speed of viral titer measurements from hours to minutes, thus providing a significant advantage for many aspects of virology research and biotechnology applications. Additional advantages lie in the broad spectrum of virus species that may be measured and the possibility to determine the ratio of infectious to total particles. A series of disadvantages remain associated with these technologies, such as a low specificity for viral particles. In this review we will discuss these technologies by comparing four systems for real-time single virus particle analysis and quantification. - Highlights: • We introduce four methods for virus particle-based quantification of viruses. • They allow for quantification of a wide range of samples in under an hour time. • The additional measurement of size and zeta potential is possible for some.

  11. Real-Time Measurements of Sediment Modification by Large Macrofauna

    Science.gov (United States)

    2003-09-30

    sediment acoustic properties? Our research has two thrusts: (1) the development of new technologies to measure, in real-time, organism movements and the...properties. As such, we are measuring rates of movement of large macrofauna in real-time via recently developed technologies and altering these...with PIT tags. Tags were injected into the coelom of the polychaetes and were glued on the shell of the clams. The adults were returned to the

  12. Real-time PCR quantitative assessment of hepatitis A virus ...

    African Journals Online (AJOL)

    We applied real-time RT-PCR (reverse transcription-polymerase chain reaction) to assess the incidence of hepatitis A virus, rotaviruses and enteroviruses in the Tyume River, an important water resource in the impoverished Eastern Cape Province of South Africa. Detection of noroviruses was done using conventional ...

  13. Statistical aspects of quantitative real-time PCR experiment design

    Czech Academy of Sciences Publication Activity Database

    Kitchen, R.R.; Kubista, Mikael; Tichopád, Aleš

    2010-01-01

    Roč. 50, č. 4 (2010), s. 231-236 ISSN 1046-2023 R&D Projects: GA AV ČR IAA500520809 Institutional research plan: CEZ:AV0Z50520701 Keywords : Real-time PCR * Experiment design * Nested analysis of variance Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.527, year: 2010

  14. System security assessment in real-time using synchrophasor measurements

    DEFF Research Database (Denmark)

    Jóhannsson, Hjörtur; Wache, Markus

    2013-01-01

    assessment and sheds light on ongoing research activities that focus on exploiting wide-area synchrophasor measurements for real-time security assessment of sustainable power systems. At last, an mathematical mapping enabling informative visualization of the system state in respect to aperiodic rotor angle...... measures to ensure stable and secure operation of the system are necessary. Time stamped synchrophasor measurements lay the foundation for development of new real-time applications for security and stability assessment. The paper provides overview of existing solutions for synchrophasor based security...

  15. Real-time measurement of mental workload: A feasibility study

    Science.gov (United States)

    Kramer, Arthur; Humphrey, Darryl; Sirevaag, Erik; Mecklinger, Axel

    1990-01-01

    The primary goal of the study was to explore the utility of event-related brain potentials (ERP) as real-time measures of workload. To this end, subjects performed two different tasks both separately and together. One task required that subjects monitor a bank of constantly changing gauges and detect critical deviations. Difficulty was varied by changing the predictability of the gauges. The second task was mental arithmetic. Difficulty was varied by requiring subjects to perform operations on either two or three columns of numbers. Two conditions that could easily be distinguished on the basis of performance measures were selected for the real-time evaluation of ERPs. A bootstrapping approach was adopted in which one thousand samples of n trials (n = 1, 3, 5 ...65) were classified using several measures of P300 and Slow Wave amplitude. Classification accuracies of 85 percent were achieved with 25 trials. Results are discussed in terms of potential enhancements for real-time recording.

  16. Real-time temperature field measurement based on acoustic tomography

    Science.gov (United States)

    Bao, Yong; Jia, Jiabin; Polydorides, Nick

    2017-07-01

    Acoustic tomography can be used to measure the temperature field from the time-of-flight (TOF). In order to capture real-time temperature field changes and accurately yield quantitative temperature images, two improvements to the conventional acoustic tomography system are studied: simultaneous acoustic transmission and TOF collection along multiple ray paths, and an offline iteration reconstruction algorithm. During system operation, all the acoustic transceivers send modulated and filtered wideband Kasami sequences simultaneously to facilitate fast and accurate TOF measurements using cross-correlation detection. For image reconstruction, the iteration process is separated and executed offline beforehand to shorten computation time for online temperature field reconstruction. The feasibility and effectiveness of the developed methods are validated in the simulation study. The simulation results demonstrate that the proposed method can reduce the processing time per frame from 160 ms to 20 ms, while the reconstruction error remains less than 5%. Hence, the proposed method has great potential in the measurement of rapid temperature change with good temporal and spatial resolution.

  17. Real time quantitative amplification detection on a microarray: towards high multiplex quantitative PCR.

    NARCIS (Netherlands)

    Pierik, A.; Moamfa, M; van Zelst, M.; Clout, D.; Stapert, H.; Dijksman, Johan Frederik; Broer, D.; Wimberger-Friedl, R.

    2012-01-01

    Quantitative real-time polymerase chain reaction (qrtPCR) is widely used as a research and diagnostic tool. Notwithstanding its many powerful features, the method is limited in the degree of multiplexing to about 6 due to spectral overlap of the available fluorophores. A new method is presented that

  18. Real-time measurement of soil stiffness during static compaction.

    Science.gov (United States)

    2009-01-01

    Is continuous sensing of soil properties during static pad foot roller compaction achievable? A new pad-based, rollerintegrated system for real-time measurement of the elastic modulus of fine- and mixed-grain soils is the goal of Development of So...

  19. Real-time color measurement using active illuminant

    Science.gov (United States)

    Tominaga, Shoji; Horiuchi, Takahiko; Yoshimura, Akihiko

    2010-01-01

    This paper proposes a method for real-time color measurement using active illuminant. A synchronous measurement system is constructed by combining a high-speed active spectral light source and a high-speed monochrome camera. The light source is a programmable spectral source which is capable of emitting arbitrary spectrum in high speed. This system is the essential advantage of capturing spectral images without using filters in high frame rates. The new method of real-time colorimetry is different from the traditional method based on the colorimeter or the spectrometers. We project the color-matching functions onto an object surface as spectral illuminants. Then we can obtain the CIE-XYZ tristimulus values directly from the camera outputs at every point on the surface. We describe the principle of our colorimetric technique based on projection of the color-matching functions and the procedure for realizing a real-time measurement system of a moving object. In an experiment, we examine the performance of real-time color measurement for a static object and a moving object.

  20. A Quantitative Approach to the Formal Verification of Real-Time Systems.

    Science.gov (United States)

    1996-09-01

    Computer Science A Quantitative Approach to the Formal Verification of Real-Time Systems Sergio Vale Aguiar Campos September 1996 CMU-CS-96-199...ptisiic raieaiSI v Diambimos Lboiamtad _^ A Quantitative Approach to the Formal Verification of Real-Time Systems Sergio Vale Aguiar Campos...implied, of NSF, the Semiconduc- tor Research Corporation, ARPA or the U.S. government. Keywords: real-time systems, formal verification , symbolic

  1. Development of a new real-time MCE-based quantitative analysis system

    Science.gov (United States)

    Zhang, Mingqiang; Sun, Fengrong; Yao, Gui-hua

    2009-10-01

    The quantitative analysis system for real time myocardial contrast echocardiography can measure the values of A(microvascular cross-sectional area or myocardial blood volume)and β(myocardial microbubble velocity), AÂ.β(myocardial blood flow), A-EER (endo-epi ratio of A ), β-EER and AÂ.β-EER from the signal intensity of real-time 2-D grayscale images and power Doppler images, draw the time-intensity curves to indicate the variation of the intensity of micro-bubbles scattering in subendocardial layer and subepicardial layer with the varying of myocardial segments, and estimate the hemodynamic parameters by nonlinear regression analysis. The system also conformed to the digital imaging and communications in medicine (DICOM) standard and could be integrated into the picture archiving and communication system (PACS). The examples of clinical study indicated the clinical effectiveness of the system and the reliability of the quantitative analysis techniques employed in the system.

  2. Real-time hostile attribution measurement and aggression in children.

    Science.gov (United States)

    Yaros, Anna; Lochman, John E; Rosenbaum, Jill; Jimenez-Camargo, Luis Alberto

    2014-01-01

    Hostile attributions are an important predictor of aggression in children, but few studies have measured hostile attributions as they occur in real-time. The current study uses an interactive video racing game to measure hostile attributions while children played against a presumed peer. A sample of 75 children, ages 10-13, used nonverbal and verbal procedures to respond to ambiguous provocation by their opponent. Hostile attributions were significantly positively related to parent-rated reactive aggression, when controlling for proactive aggression. Hostile attributions using a nonverbal response procedure were negatively related to proactive aggression, when controlling for reactive aggression. Results suggest hostile attributions in real-time occur quickly and simultaneously with social interaction, which differs from the deliberative, controlled appraisals measured with vignette-based instruments. The relation between real-time hostile attributions and reactive aggression could be accounted for by the impulsive response style that is characteristic of reactive aggression, whereas children exhibiting proactive aggression may be more deliberate and intentional in their responding, resulting in a negative relation with real-time hostile attributions. These findings can be used both to identify children at risk for aggression and to enhance preventive interventions. © 2014 Wiley Periodicals, Inc.

  3. A New Diagnostic Mechanism of Instruction: A Dynamic, Real-Time and Non-Interference Quantitative Measurement Technique for Adaptive E-Learning

    Science.gov (United States)

    Hsu, Pi-Shan; Chang, Te-Jeng; Wu, Ming-Hsiung

    2009-01-01

    The level of learners' expertise has been used as a metric and diagnostic mechanism of instruction. This metric influences mental effort directly according to the applications of cognitive load theory. Cognitive efficiency, an optimal measurement technique of expertise, was developed by Kalyuga and Sweller to replace instructional efficiency in…

  4. [Research progress of real-time quantitative PCR method for group A rotavirus detection].

    Science.gov (United States)

    Guo, Yan-Qing; Li, Dan-Di; Duan, Zhao-Jun

    2013-11-01

    Group A rotavirus is one of the most significant etiological agents which causes acute gastroenteritis among infants and young children worldwide. So far, several method which includes electron microscopy (EM), enzyme immunoassay (EIA), reverse transcription-polymerase chain reaction (RT-PCR)and Real-time Quantitative PCR has been established for the detection of rotavirus. Compared with other methods, Real-time quantitative PCR have advantages in specificity, sensitivity, genotyping and quantitative accuracy. This article shows a overview of the application of real-time quantitative PCR technique to detecte group A rotavirus.

  5. Real Time Measurement of Host Bioenergetics During Mycobacterium Tuberculosis Infection

    Science.gov (United States)

    2014-09-01

    1 AWARD NUMBER: W81XWH-13-1-0149 TITLE: Real Time Measurement of Host Bioenergetics During Mycobacterium Tuberculosis Infection...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The unique ability of Mycobacterium tuberculosis (Mtb) to persist in humans in a dormant, drug...Chapters: 1. Steyn AJC, Mai D, Saini V and Farhana A. Protein-protein interaction in the –omics era: Understanding Mycobacterium tuberculosis function. In

  6. Real-time evolvable pulse shaper for radiation measurements

    Energy Technology Data Exchange (ETDEWEB)

    Lanchares, Juan, E-mail: julandan@dacya.ucm.es [Facultad de Informática, Universidad Complutense de Madrid (UCM), C/Prof. José García Santesmases s/n, 28040 Madrid (Spain); Garnica, Oscar, E-mail: ogarnica@dacya.ucm.es [Facultad de Informática, Universidad Complutense de Madrid (UCM), C/Prof. José García Santesmases s/n, 28040 Madrid (Spain); Risco-Martín, José L., E-mail: jlrisco@dacya.ucm.es [Facultad de Informática, Universidad Complutense de Madrid (UCM), C/Prof. José García Santesmases s/n, 28040 Madrid (Spain); Ignacio Hidalgo, J., E-mail: hidalgo@dacya.ucm.es [Facultad de Informática, Universidad Complutense de Madrid (UCM), C/Prof. José García Santesmases s/n, 28040 Madrid (Spain); Regadío, Alberto, E-mail: alberto.regadio@insa.es [Área de Tecnologías Electrónicas, Instituto Nacional de Técnica Aeroespacial (INTA), 28850 Torrejón de Ardoz, Madrid (Spain)

    2013-11-01

    In the last two decades, recursive algorithms for real-time digital pulse shaping in pulse height measurements have been developed and published in number of articles and textbooks. All these algorithms try to synthesize in real time optimum or near optimum shapes in the presence of noise. Even though some of these shapers can be considered effective designs, some side effects like aging cannot be ignored. We may observe that after sensors degradation, the signal obtained is not valid. In this regard, we present in this paper a novel technique that, based on evolvable hardware concepts, is able to evolve the degenerated shaper into a new design with better performance than the original one under the new sensor features.

  7. Quantitative real-time monitoring of dryer effluent using fiber optic near-infrared spectroscopy.

    Science.gov (United States)

    Harris, S C; Walker, D S

    2000-09-01

    This paper describes a method for real-time quantitation of the solvents evaporating from a dryer. The vapor stream in the vacuum line of a dryer was monitored in real time using a fiber optic-coupled acousto-optic tunable filter near-infrared (AOTF-NIR) spectrometer. A balance was placed in the dryer, and mass readings were recorded for every scan of the AOTF-NIR. A partial least-squares (PLS) calibration was subsequently built based on change in mass over change in time for solvents typically used in a chemical manufacturing plant. Controlling software for the AOTF-NIR was developed. The software collects spectra, builds the PLS calibration model, and continuously fits subsequently collected spectra to the calibration, allowing the operator to follow the mass loss of solvent from the dryer. The results indicate that solvent loss can be monitored and quantitated in real time using NIR for the optimization of drying times. These time-based mass loss values have also been used to calculate "dynamic" vapor density values for the solvents. The values calculated are in agreement with values determined from the ideal gas law and could prove valuable as tools to measure temperature or pressure indirectly.

  8. Quantitative Detection of Respiratory Chlamydia pneumoniae Infection by Real-Time PCR

    OpenAIRE

    Kuoppa, Yvonne; Boman, Jens; Scott, Lena; Kumlin, Urban; Eriksson, Iréne; Allard, Annika

    2002-01-01

    Real-time PCR was evaluated as a quantitative diagnostic method for Chlamydia pneumoniae infection using different respiratory samples. Real-time PCR had efficiency equal to or better than that of nested touchdown PCR. This study confirmed sputum as the best sampling material to detect an ongoing C. pneumoniae infection.

  9. A quantitative and automatic echographic method for real-time localization of endovascular devices.

    Science.gov (United States)

    Conversano, Francesco; Casciaro, Ernesto; Franchini, Roberto; Lay-Ekuakille, Aimè; Casciaro, Sergio

    2011-10-01

    Current imaging methods for catheter position monitoring during minimally invasive surgery do not provide an effective support to surgeons, often resulting in the choice of more invasive procedures. This study was conducted to demonstrate the feasibility of non-ionizing monitoring of endovascular devices through embedded quantitative ultrasound (QUS) methods, providing catheter self-localization with respect to selected anatomical structures. QUS-based algorithms for real-time automatic tracking of device position were developed and validated on in vitro and ex vivo phantoms. A trans-esophageal ultrasound probe was adapted to simulate an endovascular device equipped with an intravascular ultrasound probe. B-mode images were acquired and processed in real time by means of a new algorithm for accurate measurement of device position. After off-line verification, automatic position calculation was found to be correct in 96% and 94% of computed frames in the in vitro and ex vivo phantoms, respectively. The average errors of distance measurements (bias ± 2SD) in a 41-step 10-cm-long parabolic pathway were 0.76 ± 3.75 mm or 0.52 ± 3.20 mm, depending on algorithm implementations. Our results showed the effectiveness of QUS-based tracking algorithms for real-time automatic calculation and display of endovascular system position. The method, validated for the case of an endoclamp balloon catheter, can be easily extended to most endovascular surgical systems.

  10. Real-Time Alpine Measurement System Using Wireless Sensor Networks.

    Science.gov (United States)

    Malek, Sami A; Avanzi, Francesco; Brun-Laguna, Keoma; Maurer, Tessa; Oroza, Carlos A; Hartsough, Peter C; Watteyne, Thomas; Glaser, Steven D

    2017-11-09

    Monitoring the snow pack is crucial for many stakeholders, whether for hydro-power optimization, water management or flood control. Traditional forecasting relies on regression methods, which often results in snow melt runoff predictions of low accuracy in non-average years. Existing ground-based real-time measurement systems do not cover enough physiographic variability and are mostly installed at low elevations. We present the hardware and software design of a state-of-the-art distributed Wireless Sensor Network (WSN)-based autonomous measurement system with real-time remote data transmission that gathers data of snow depth, air temperature, air relative humidity, soil moisture, soil temperature, and solar radiation in physiographically representative locations. Elevation, aspect, slope and vegetation are used to select network locations, and distribute sensors throughout a given network location, since they govern snow pack variability at various scales. Three WSNs were installed in the Sierra Nevada of Northern California throughout the North Fork of the Feather River, upstream of the Oroville dam and multiple powerhouses along the river. The WSNs gathered hydrologic variables and network health statistics throughout the 2017 water year, one of northern Sierra's wettest years on record. These networks leverage an ultra-low-power wireless technology to interconnect their components and offer recovery features, resilience to data loss due to weather and wildlife disturbances and real-time topological visualizations of the network health. Data show considerable spatial variability of snow depth, even within a 1 km 2 network location. Combined with existing systems, these WSNs can better detect precipitation timing and phase in, monitor sub-daily dynamics of infiltration and surface runoff during precipitation or snow melt, and inform hydro power managers about actual ablation and end-of-season date across the landscape.

  11. Real-Time Alpine Measurement System Using Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    Sami A. Malek

    2017-11-01

    Full Text Available Monitoring the snow pack is crucial for many stakeholders, whether for hydro-power optimization, water management or flood control. Traditional forecasting relies on regression methods, which often results in snow melt runoff predictions of low accuracy in non-average years. Existing ground-based real-time measurement systems do not cover enough physiographic variability and are mostly installed at low elevations. We present the hardware and software design of a state-of-the-art distributed Wireless Sensor Network (WSN-based autonomous measurement system with real-time remote data transmission that gathers data of snow depth, air temperature, air relative humidity, soil moisture, soil temperature, and solar radiation in physiographically representative locations. Elevation, aspect, slope and vegetation are used to select network locations, and distribute sensors throughout a given network location, since they govern snow pack variability at various scales. Three WSNs were installed in the Sierra Nevada of Northern California throughout the North Fork of the Feather River, upstream of the Oroville dam and multiple powerhouses along the river. The WSNs gathered hydrologic variables and network health statistics throughout the 2017 water year, one of northern Sierra’s wettest years on record. These networks leverage an ultra-low-power wireless technology to interconnect their components and offer recovery features, resilience to data loss due to weather and wildlife disturbances and real-time topological visualizations of the network health. Data show considerable spatial variability of snow depth, even within a 1 km 2 network location. Combined with existing systems, these WSNs can better detect precipitation timing and phase in, monitor sub-daily dynamics of infiltration and surface runoff during precipitation or snow melt, and inform hydro power managers about actual ablation and end-of-season date across the landscape.

  12. Novel bioluminescent quantitative detection of nucleic acid amplification in real-time.

    Science.gov (United States)

    Gandelman, Olga A; Church, Vicki L; Moore, Cathy A; Kiddle, Guy; Carne, Christopher A; Parmar, Surendra; Jalal, Hamid; Tisi, Laurence C; Murray, James A H

    2010-11-30

    The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal

  13. Novel bioluminescent quantitative detection of nucleic acid amplification in real-time.

    Directory of Open Access Journals (Sweden)

    Olga A Gandelman

    Full Text Available BACKGROUND: The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. PRINCIPAL FINDINGS: Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. CONCLUSIONS: The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a

  14. Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos

    Directory of Open Access Journals (Sweden)

    Van Zeveren Alex

    2005-12-01

    Full Text Available Abstract Background Real-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the succesful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published GAPD, ACTB or 18S rRNA have been used as a single reference gene without prior verification of their expression stability. Normalization of the data using unstable controls can result in erroneous conclusions, especially when only one reference gene is used. Results In this study the transcription levels of 8 commonly used reference genes (ACTB, GAPD, Histone H2A, TBP, HPRT1, SDHA, YWHAZ and 18S rRNA were determined at different preimplantation stages (2-cell, 8-cell, blastocyst and hatched blastocyst in order to select the most stable genes to normalize quantitative data within different preimplantation embryo stages. Conclusion Using the geNorm application YWHAZ, GAPD and SDHA were found to be the most stable genes across the examined embryonic stages, while the commonly used ACTB was shown to be highly regulated. We recommend the use of the geometric mean of those 3 reference genes as an accurate normalization factor, which allows small expression differences to be reliably measured.

  15. Detection of placenta elasticity modulus by quantitative real-time shear wave imaging.

    Science.gov (United States)

    Li, W J; Wei, Z T; Yan, R L; Zhang, Y L

    2012-01-01

    To explore the clinical values in detecting the placental elastic modulus using real-time quantitative shear wave elasticity imaging. A total of 30 women in the late pregnancy stage without complications and having normal, single pregnancies, as well as normal fetal growth, amniotic fluid index, and anterior placenta were selected. A real-time elasticity imaging shear wave ultrasonic diagnostic apparatus was used to randomly select regions of interest at the central and edge of the placenta. The elastography imaging mode was launched to measure the elasticity of the elastic modulus of these placental parts. A total of 15 measured values were obtained at the placental center and edge for each pregnancy case. Umbilical artery and uterine artery pulsatility index (PI) values for 18 cases were also randomly measured. The average value of 30 placental edges of the elastic modulus (n = 15) was (7.60 +/- 1.71) kPa. The average value of the 30 placental central elastic modulus (n = 15 ) was (7.84 +/- 1.68) kPa. No significant difference was observed between placenta central and edge elastic modulus. The PI mean value of umbilical artery in 18 cases was 0.94, whereas the average PI values of the uterine artery was 0.83. No linear correlation was found among the elastic modulus, the placental uterine artery PI values, and the umbilical artery PI values (p > 0.05). No difference between the placental center of normal pregnancies and the edge of the elastic modulus was detected. The elastic modulus of the placenta could be obtained in the best position. The placenta varied greatly between elastic modulus. No correlation was found between the placental elastic modulus, the uterine artery, and umbilical artery PI values. Real-time shear wave elasticity imaging technology can provide morphological evidence of placental function, which may emerge as a new clinical assessment approach.

  16. Accuracy of real time radiography burning rate measurement

    Science.gov (United States)

    Olaniyi, Bisola

    The design of a solid propellant rocket motor requires the determination of a propellant's burning-rate and its dependency upon environmental parameters. The requirement that the burning-rate be physically measured, establishes the need for methods and equipment to obtain such data. A literature review reveals that no measurement has provided the desired burning rate accuracy. In the current study, flash x-ray modeling and digitized film-density data were employed to predict motor-port area to length ratio. The pre-fired port-areas and base burning rate were within 2.5% and 1.2% of their known values, respectively. To verify the accuracy of the method, a continuous x-ray and a solid propellant rocket motor model (Plexiglas cylinder) were used. The solid propellant motor model was translated laterally through a real-time radiography system at different speeds simulating different burning rates. X-ray images were captured and the burning-rate was then determined. The measured burning rate was within 1.65% of the known values.

  17. Quantitative mitral valve modeling using real-time three-dimensional echocardiography: technique and repeatability.

    Science.gov (United States)

    Jassar, Arminder Singh; Brinster, Clayton J; Vergnat, Mathieu; Robb, J Daniel; Eperjesi, Thomas J; Pouch, Alison M; Cheung, Albert T; Weiss, Stuart J; Acker, Michael A; Gorman, Joseph H; Gorman, Robert C; Jackson, Benjamin M

    2011-01-01

    Real-time three-dimensional (3D) echocardiography has the ability to construct quantitative models of the mitral valve (MV). Imaging and modeling algorithms rely on operator interpretation of raw images and may be subject to observer-dependent variability. We describe a comprehensive analysis technique to generate high-resolution 3D MV models and examine interoperator and intraoperator repeatability in humans. Patients with normal MVs were imaged using intraoperative transesophageal real-time 3D echocardiography. The annulus and leaflets were manually segmented using a TomTec Echo-View workstation. The resultant annular and leaflet point cloud was used to generate fully quantitative 3D MV models using custom Matlab algorithms. Eight images were subjected to analysis by two independent observers. Two sequential images were acquired for 6 patients and analyzed by the same observer. Each pair of annular tracings was compared with respect to conventional variables and by calculating the mean absolute distance between paired renderings. To compare leaflets, MV models were aligned so as to minimize their sum of squares difference, and their mean absolute difference was measured. Mean absolute annular and leaflet distance was 2.4±0.8 and 0.6±0.2 mm for the interobserver and 1.5±0.6 and 0.5±0.2 mm for the intraobserver comparisons, respectively. There was less than 10% variation in annular variables between comparisons. These techniques generate high-resolution, quantitative 3D models of the MV and can be used consistently to image the human MV with very small interoperator and intraoperator variability. These data lay the framework for reliable and comprehensive noninvasive modeling of the normal and diseased MV. Copyright © 2011 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

  18. Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma

    DEFF Research Database (Denmark)

    Gjerdrum, Lise Mette; Sorensen, Boe Sandahl; Kjeldsen, Eigil

    2004-01-01

    We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods...... tumors as being amplified. Interestingly, all these scored 2+ with the HercepTest, but were negative using FISH. We believe that real-time quantitative PCR analysis of HER-2 DNA amplification following microdissection represents a useful supplementary or perhaps even an alternative technique...

  19. Real time measurements of methanol crossover in a DMFC

    Science.gov (United States)

    Han, Jiahua; Liu, Hongtan

    Methanol crossover is a serious problem in a direct methanol fuel cell (DMFC), which causes significant voltage loss and waste of fuel. Due to methanol crossover, most DMFCs must operate on a fuel with a very low methanol concentration; yet very low methanol concentration also causes a poor cell performance. Thus, it is very important to find the optimal operating conditions of methanol concentration and other operating parameters. In this research, methanol crossover rate in a DMFC is determined by measuring the carbon dioxide concentration at the cathode exit in real time. By measuring methanol crossover and cell performances at different inlet methanol concentrations and various operating conditions three types of characteristics are identified in the relationships between methanol crossover and cell current density. Further analysis of these relationships between methanol crossover and cell performances reveals the optimal methanol concentration and other operating parameters, at which the cell reaches optimal performance without incurring excessive methanol crossover. Furthermore, transient peaks of methanol crossover have been identified when the cell voltage suddenly changes. Analyses of these peaks show that they are caused by the hysteresis of methanol concentration at the interface between the anode catalyst layer and the membrane.

  20. A reusable electrochemical proximity assay for highly selective, real-time protein quantitation in biological matrices.

    Science.gov (United States)

    Hu, Jiaming; Yu, Yajiao; Brooks, Jessica C; Godwin, Leah A; Somasundaram, Subramaniam; Torabinejad, Ferdous; Kim, Joonyul; Shannon, Curtis; Easley, Christopher J

    2014-06-11

    Rapid and specific quantitation of a variety of proteins over a wide concentration range is highly desirable for biosensing at the point-of-care, in clinical laboratories, and in research settings. Our recently developed electrochemical proximity assay (ECPA) is a target-flexible, DNA-directed, direct-readout protein quantitation method with detection limits in the low femtomolar range, making it particularly amenable to point-of-care detection. However, consistent quantitation in more complex matrices is required at the point-of-care, and improvements in measurement speed are needed for clinical and research settings. Here, we address these concerns with a reusable ECPA, where a gentle regeneration of the surface DNA monolayer (used to capture the proximity complex) is achieved enzymatically through a novel combination of molecular biology and electrochemistry. Strategically placed uracils in the DNA sequence trigger selective cleavage of the backbone, releasing the assembled proximity complex. This allows repeated protein quantitation by square-wave voltammetry (SWV)-as quickly as 3 min between runs. The process can be repeated up to 19 times on a single electrode without loss of assay sensitivity, and currents are shown to be highly repeatable with similar calibrations using seven different electrodes. The utility of reusable ECPA is demonstrated through two important applications in complex matrices: (1) direct, quantitative monitoring of hormone secretion in real time from as few as five murine pancreatic islets and (2) standard addition experiments in unspiked serum for direct quantitation of insulin at clinically relevant levels. Results from both applications distinguish ECPA as an exceptional tool in protein quantitation.

  1. Real-time dopamine measurement in awake monkeys.

    Directory of Open Access Journals (Sweden)

    Erik W Schluter

    Full Text Available Fast-scan cyclic voltammetry (FSCV is often used to measure real-time dopamine (DA concentrations in awake, behaving rodents. Extending this technique to work in monkeys would provide a platform for advanced behavioral studies and a primate model for preclinical research. The present study demonstrates the feasibility of DA recordings in two awake monkeys (Macaca mulatta using a mixture of techniques adapted from rodent, primate and brain slice work. We developed a long carbon fiber electrode to operate in the larger primate brain. This electrode was lowered into the striatum each day using a recording chamber and a detachable micromanipulator system. A manipulator also moved one or more tungsten stimulating electrodes into either the nearby striatum or the ventral tegmental area/substantia nigra pars compacta (VTA/SNc. We developed an electrical stimulation controller to reduce artifacts during electrical stimulation. We also introduce a stimulation-based methodology for estimating distances between electrodes in the brain. Dopamine responses within the striatum were evoked by either stimulation of the striatum near the FSCV electrode, or stimulation within the VTA/SNc. Unexpected juice rewards also evoked dopamine responses in the ventral striatum. Thus, we demonstrate that robust dopamine responses can be recorded from awake, behaving primates with FSCV. In addition, we describe how a stimulation technique borrowed from the neuroprosthetics field can activate the distributed monkey midbrain dopamine system in a way that mimics rodent VTA stimulation.

  2. Comparison of Sensitivity and Quantitation between Microbead Dielectrophoresis-Based DNA Detection and Real-Time PCR

    Directory of Open Access Journals (Sweden)

    Michihiko Nakano

    2017-09-01

    Full Text Available In this study, we describe a microbead-based method using dielectrophoresis (DEP for the fast detection of DNA amplified by polymerase chain reaction (PCR. This electrical method measures the change in impedance caused by DEP-trapped microbeads to which biotinylated target DNA molecules are chemically attached. Using this method, measurements can be obtained within 20 min. Currently, real-time PCR is among the most sensitive methods available for the detection of target DNA, and is often used in the diagnosis of infectious diseases. We therefore compared the quantitation and sensitivity achieved by our method to those achieved with real-time PCR. We found that the microbead DEP-based method exhibited the same detection limit as real-time PCR, although its quantitative detection range was slightly narrower at 10–105 copies/reaction compared with 10–107 copies/reaction for real-time PCR. Whereas real-time PCR requires expensive and complex instruments, as well as expertise in primer design and experimental principles, our novel method is simple to use, inexpensive, and rapid. This method could potentially detect viral and other DNAs efficiently in combination with conventional PCR.

  3. Detection of Leishmania infantum DNA in conjunctival swabs of cats by quantitative real-time PCR.

    Science.gov (United States)

    Benassi, Julia Cristina; Benvenga, Graziella U; Ferreira, Helena Lage; Pereira, Vanessa F; Keid, Lara B; Soares, Rodrigo; Oliveira, Tricia Maria Ferreira de Sousa

    2017-06-01

    Although some studies have investigated the potential role of cats as a reservoir for Leishmania, their role in the epidemiology of visceral leishmaniasis (VL) is still poorly understood. Molecular diagnostic techniques are an important tool in VL diagnosis, and PCR shows high sensitivity and specificity for Leishmania spp. detection. Quantitative real-time PCR (qPCR) is a method that permits quantitative analysis of a large number of samples, resulting in more sensitive, accurate, and reproducible measurements of specific DNA present in the sample. This study compared real-time PCR (qPCR) and conventional PCR (cPCR) for detection of Leishmania spp. in blood and conjunctival swab (CS) samples of healthy cats from a non-endemic area in the state of São Paulo, Brazil. Of all CS samples, 1.85% (2/108) were positive for Leishmania spp. by both cPCR as qPCR (kappa index = 1), indicating excellent agreement between the two methods. The DNA from the two CS-cPCR- and CS-qPCR-positive samples was further tested with a PCR test amplifying the Leishmania spp. discriminative rRNA internal transcribed spacer 1 (ITS 1), of which one sample generated a 300-350-bp DNA fragment whose size varies according to the Leishmania species. Following sequencing, the fragment showed 100% similarity to a GenBank L. infantum sequence obtained from a cat in Italy. In conclusion, the association of qPCR and CS proved to be effective for detection of Leishmania in cats. Conjunctival swab samples were shown to be a practical and better alternative to blood samples and may be useful in the diagnosis and studies of feline leishmaniasis. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Quantitative real-time RT-PCR and chromogenic in situ hybridization

    DEFF Research Database (Denmark)

    Rosa, Fabíola E; Silveira, Sara M; Silveira, Cássia G T

    2009-01-01

    . METHODS: To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH. RESULTS: The concordance...

  5. Periodontal pathogens: a quantitative comparison of anaerobic culture and real-time PCR

    NARCIS (Netherlands)

    Boutaga, Khalil; van Winkelhoff, Arie Jan; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.

    2005-01-01

    Periodontitis is a multi-factorial chronic inflammatory and destructive disease of the tooth-supporting tissues. Quantitative anaerobic culture techniques have been used for microbial diagnosis of the different forms of the disease. The aim of this study was to compare real-time PCR with

  6. Real-time quantitative phase imaging based on transport of intensity equation with dual simultaneously recorded field of view.

    Science.gov (United States)

    Tian, Xiaolin; Yu, Wei; Meng, Xin; Sun, Aihui; Xue, Liang; Liu, Cheng; Wang, Shouyu

    2016-04-01

    Since quantitative phase distribution reflects both cellular shapes and conditions from another view, compared to traditional intensity observation, different quantitative phase microscopic methods are proposed for cellular detections. However, the transport of intensity equation-based approach not only presents phase, but also intensity, which attracts much attention. While classical transport of intensity equation needs multi-focal images which often cannot realize simultaneous phase measurement, in this Letter, to break through the limitation, a real-time quantitative phase imaging method using transport of intensity equation is proposed. Two identical CCD cameras are set at the binocular tubes to capture the same field of view but at different focal planes. With a double-frame algorithm assuming that the on-focal image is the average of over- and under-focal information, the proposed method is capable of calculating quantitative phase distributions of samples accurately and simultaneously indicating its potentialities in cellular real-time monitoring.

  7. Off-axis quantitative phase imaging processing using CUDA: toward real-time applications.

    Science.gov (United States)

    Pham, Hoa; Ding, Huafeng; Sobh, Nahil; Do, Minh; Patel, Sanjay; Popescu, Gabriel

    2011-07-01

    We demonstrate real time off-axis Quantitative Phase Imaging (QPI) using a phase reconstruction algorithm based on NVIDIA's CUDA programming model. The phase unwrapping component is based on Goldstein's algorithm. By mapping the process of extracting phase information and unwrapping to GPU, we are able to speed up the whole procedure by more than 18.8× with respect to CPU processing and ultimately achieve video rate for mega-pixel images. Our CUDA implementation also supports processing of multiple images simultaneously. This enables our imaging system to support high speed, high throughput, and real-time image acquisition and visualization.

  8. Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma

    DEFF Research Database (Denmark)

    Gjerdrum, Lise Mette; Sorensen, Boe Sandahl; Kjeldsen, Eigil

    2004-01-01

    We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods....... A single DCIS case was amplified in FISH, but not in IHC. Both HER-2 gene amplification and expression could be quantified in microdissected paraffin-embedded tumors using real-time PCR, DNA and RNA being successfully detected in 146 of 150 (97%) and 141 of 150 (94%) samples, respectively. PCR analysis...... for establishing HER-2 status in paraffin-embedded tumors....

  9. Evaluation of Various Real-Time Reverse Transcription Quantitative PCR Assays for Norovirus Detection.

    Science.gov (United States)

    Yoo, Ju Eun; Lee, Cheonghoon; Park, SungJun; Ko, GwangPyo

    2017-04-28

    Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for the sensitive and accurate detection of these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays, and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assays A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, and sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A ZEN internal quencher, which decreases nonspecific fluorescence during the PCR, was added to Assay D's probe, which further improved the assay performance. This study compared several detection assays for noroviruses, and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.

  10. Real-time Vehicle Reidentification System for Freeway Performance Measurements

    OpenAIRE

    Jeng, Shin-Ting

    2007-01-01

    Computational resources in the traffic operation field as well as the bandwidth of field communication links, are often quite limited. Accordingly, for real-time implementation of Advanced Transportation Management and Information Systems (ATMIS) strategies, such as vehicle reidentification, there is strong interest in development of field-based techniques and models that can perform satisfactorily while minimizing computational and communication requirements in the field. The ILD (Inductive ...

  11. Identification of Reference Genes for Real-Time Quantitative PCR Experiments in the Liverwort Marchantia polymorpha

    OpenAIRE

    Denis Saint-Marcoux; Hélène Proust; Liam Dolan; Langdale, Jane A

    2015-01-01

    Real-time quantitative polymerase chain reaction (qPCR) has become widely used as a method to compare gene transcript levels across different conditions. However, selection of suitable reference genes to normalize qPCR data is required for accurate transcript level analysis. Recently, Marchantia polymorpha has been adopted as a model for the study of liverwort development and land plant evolution. Identification of appropriate reference genes has therefore become a necessity for gene expressi...

  12. Estimation of Low Quantity Genes: A Hierarchical Model for Analyzing Censored Quantitative Real-Time PCR Data

    OpenAIRE

    Boyer, Tim C.; Tim Hanson; Singer, Randall S.

    2013-01-01

    Analysis of gene quantities measured by quantitative real-time PCR (qPCR) can be complicated by observations that are below the limit of quantification (LOQ) of the assay. A hierarchical model estimated using MCMC methods was developed to analyze qPCR data of genes with observations that fall below the LOQ (censored observations). Simulated datasets with moderate to very high levels of censoring were used to assess the performance of the model; model results were compared to approaches that r...

  13. Development of real-time PCR for detection and quantitation of Streptococcus parauberis.

    Science.gov (United States)

    Nguyen, T L; Lim, Y J; Kim, D-H; Austin, B

    2016-01-01

    Streptococcus parauberis is an increasing threat to aquaculture of olive flounder, Paralichthys olivaceus Temminck & Schlegel, in South Korea. We developed a real-time polymerase chain reaction (PCR) method using the TaqMan probe assay to detect and quantify S. parauberis by targeting the gyrB gene sequences, which are effective for molecular analysis of the genus Streptococcus. Our real-time PCR assay is capable of detecting 10 fg of genomic DNA per reaction. The intra- and interassay coefficient of variation (CV) values ranged from 0.42-1.95%, demonstrating that the assay has good reproducibility. There was not any cross-reactivity to Streptococcus iniae or to other streptococcal/lactococcal fish pathogens, such as S. agalactiae and Lactococcus garvieae, indicating that the assay is highly specific to S. parauberis. The results of the real-time PCR assay corresponded well to those of conventional culture assays for S. parauberis from inoculated tissue homogenates (r = 0.957; P < 0.05). Hence, this sensitive and specific real-time PCR is a valuable tool for diagnostic quantitation of S. parauberis in clinical samples. © 2014 John Wiley & Sons Ltd.

  14. Real-time PCR assay for rapid qualitative and quantitative detection of Entamoeba histolytica.

    Science.gov (United States)

    Orosz, Erika; Perkátai, Katalin; Kapusinszky, Beatrix; Farkas, Agnes; Kucsera, István

    2012-12-01

    Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

  15. Legionellosis and Lung Abscesses: Contribution of Legionella Quantitative Real-Time PCR to an Adapted Followup

    Directory of Open Access Journals (Sweden)

    G. Descours

    2013-01-01

    Full Text Available We report a case of severe Legionnaires' disease (LD complicated by a lung abscess in an immunocompetent patient who required ECMO therapy and thoracic surgery. The results of repeated Legionella quantitative real-time PCR performed on both sera and respiratory samples correlated with the LD severity and the poor clinical outcome. Moreover, the PCR allowed for the detection of Legionella DNA in the lung abscess specimen, which was negative when cultured for Legionella. This case report provides a logical basis for further investigations to examine whether the Legionella quantitative PCR could improve the assessment of LD severity and constitute a prognostic marker.

  16. PRIMAS: real-time image-based motion measurement system

    Science.gov (United States)

    Furnee, E. Hans

    1990-08-01

    The PRIMAS system derives from a long line of development at Delft University of Technology , originating from [1] with subsequent innovations such as strobed illumination (1974) of reflective markers, to obtain the simultaneous, equidistant, periodic sampling of all marker positions; real-time estimation of the marker centroids from the full, digitized, contours (1984) to retain the on-line data reduction, while enhancing the resolution; interfacing to industry-standard AT type personal computers, with modest disk requirements and no buffering, even for long data runs; 100 Hz, 0.1 ms integration time, electronically-shuttered TV cameras, to get an optimum marker contrast in high ambient or outdoor light conditions (1986). System specifications include a precision of typ. 1:18000 (X) for 2-D coordinate noise or repeatability. With the 100 Hz sample rate this implies an unprecedented spatio-temporal resolution [2]. This favors 3-D reconstruction, as well as a low noise propagation in the estimation of first and higher order derivatives, as are routinely required in biomechanics analysis. The latest feature is real-time marker identification by a software module within the data acquisition program. This option, for the not too complex situations, is feasible only by the data reduction inherent in on-line marker centroid processing. The 3-D calibration, reconstruction and further analytical and display programs are available in the ASYST 3.2 Scientific Language System. A source code option caters for customer extensions. The internal VME/VSB system bus allows the basic dual or quad camera 3-D systems to be readily expanded to larger configurations.

  17. Selection of Suitable Reference Genes for Quantitative Real-time PCR in Sapium sebiferum

    Directory of Open Access Journals (Sweden)

    Xue Chen

    2017-05-01

    Full Text Available Chinese tallow (Sapium sebiferum L. is a promising landscape and bioenergy plant. Measuring gene expression by quantitative real-time polymerase chain reaction (qRT-PCR can provide valuable information on gene function. Stably expressed reference genes for normalization are a prerequisite for ensuring the accuracy of the target gene expression level among different samples. However, the reference genes in Chinese tallow have not been systematically validated. In this study, 12 candidate reference genes (18S, GAPDH, UBQ, RPS15, SAND, TIP41, 60S, ACT7, PDF2, APT, TBP, and TUB were investigated with qRT-PCR in 18 samples, including those from different tissues, from plants treated with sucrose and cold stresses. The data were calculated with four common algorithms, geNorm, BestKeeper, NormFinder, and the delta cycle threshold (ΔCt. TIP41 and GAPDH were the most stable for the tissue-specific experiment, GAPDH and 60S for cold treatment, and GAPDH and UBQ for sucrose stresses, while the least stable genes were 60S, TIP41, and 18S respectively. The comprehensive results showed APT, GAPDH, and UBQ to be the top-ranked stable genes across all the samples. The stability of 60S was the lowest during all experiments. These selected reference genes were further validated by comparing the expression profiles of the chalcone synthase gene in Chinese tallow in different samples. The results will help to improve the accuracy of gene expression studies in Chinese tallow.

  18. A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl

    Science.gov (United States)

    Smith, Matthew M.; Schmutz, Joel A.; Apelgren, Chloe; Ramey, Andy M.

    2015-01-01

    Microscopic examination of blood smears can be effective at diagnosing and quantifying hematozoa infections. However, this method requires highly trained observers, is time consuming, and may be inaccurate for detection of infections at low levels of parasitemia. To develop a molecular methodology for identifying and quantifying Leucocytozoon parasite infection in wild waterfowl (Anseriformes), we designed a real-time, quantitative PCR protocol to amplify Leucocytozoon mitochondrial DNA using TaqMan fluorogenic probes and validated our methodology using blood samples collected from waterfowl in interior Alaska during late summer and autumn (n = 105). By comparing our qPCR results to those derived from a widely used nested PCR protocol, we determined that our assay showed high levels of sensitivity (91%) and specificity (100%) in detecting Leucocytozoon DNA from host blood samples. Additionally, results of a linear regression revealed significant correlation between the raw measure of parasitemia produced by our qPCR assay (Ct values) and numbers of parasites observed on blood smears (R2 = 0.694, P = 0.003), indicating that our assay can reliably determine the relative parasitemia levels among samples. This methodology provides a powerful new tool for studies assessing effects of haemosporidian infection in wild avian species.

  19. Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA

    Directory of Open Access Journals (Sweden)

    Ana Lisa do Vale Gomes

    2006-10-01

    Full Text Available This report describes the development of a SYBR Green I based real time polymerase chain reaction (PCR protocol for detection on the ABI Prism 7000 instrument. Primers targeting the gene encoding the SSU rRNA were designed to amplify with high specificity DNA from Schistosoma mansoni, in a real time quantitative PCR system. The limit of detection of parasite DNA for the system was 10 fg of purified genomic DNA, that means less than the equivalent to one parasite cell (genome ~580 fg DNA. The efficiency was 0.99 and the correlation coefficient (R² was 0.97. When different copy numbers of the target amplicon were used as standards, the assay could detect at least 10 copies of the specific target. The primers used were designed to amplify a 106 bp DNA fragment (Tm 83ºC. The assay was highly specific for S. mansoni, and did not recognize DNA from closely related non-schistosome trematodes. The real time PCR allowed for accurate quantification of S. mansoni DNA and no time-consuming post-PCR detection of amplification products by gel electrophoresis was required. The assay is potentially able to quantify S. mansoni DNA (and indirectly parasite burden in a number of samples, such as snail tissue, serum and feces from patients, and cercaria infested water. Thus, these PCR protocols have potential to be used as tools for monitoring of schistosome transmission and quantitative diagnosis of human infection.

  20. Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage.

    Science.gov (United States)

    McCulloch, Ryan S; Ashwell, Melissa S; O'Nan, Audrey T; Mente, Peter L

    2012-11-12

    Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes. Ten candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were: beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A (cyclophilin A), ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein-zeta polypeptide. The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were (most to least stable) succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, succinate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin. BestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species.

  1. Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage

    Directory of Open Access Journals (Sweden)

    McCulloch Ryan S

    2012-11-01

    Full Text Available Abstract Background Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes. Results Ten candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were: beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A (cyclophilin A, ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein—zeta polypeptide. The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were (most to least stable succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, succinate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin. Conclusions BestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species.

  2. Quantitative gene expression profiles in real time from expressed sequence tag databases.

    Science.gov (United States)

    Funari, Vincent A; Voevodski, Konstantin; Leyfer, Dimitry; Yerkes, Laura; Cramer, Donald; Tolan, Dean R

    2010-01-01

    An accumulation of expressed sequence tag (EST) data in the public domain and the availability of bioinformatic programs have made EST gene expression profiling a common practice. However, the utility and validity of using EST databases (e.g., dbEST) has been criticized, particularly for quantitative assessment of gene expression. Problems with EST sequencing errors, library construction, EST annotation, and multiple paralogs make generation of specific and sensitive qualitative arid quantitative expression profiles a concern. In addition, most EST-derived expression data exists in previously assembled databases. The Virtual Northern Blot (VNB) (http: //tlab.bu.edu/vnb.html) allows generation, evaluation, and optimization of expression profiles in real time, which is especially important for alternatively spliced, novel, or poorly characterized genes. Representative gene families with variable nucleotide sequence identity, tissue specificity, and levels of expression (bcl-xl, aldoA, and cyp2d9) are used to assess the quality of VNB's output. The profiles generated by VNB are more sensitive and specific than those constructed with ESTs listed in preindexed databases at UCSC and NCBI. Moreover, quantitative expression profiles produced by VNB are comparable to quantization obtained from Northern blots and qPCR. The VNB pipeline generates real-time gene expression profiles for single-gene queries that are both qualitatively and quantitatively reliable.

  3. Quantitative analysis of diet structure by real-time PCR, reveals different feeding patterns by two dominant grasshopper species.

    Science.gov (United States)

    Huang, Xunbing; Wu, Huihui; McNeill, Mark Richard; Qin, Xinghu; Ma, Jingchuan; Tu, Xiongbing; Cao, Guangchun; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

    2016-08-26

    Studies on grasshopper diets have historically employed a range of methodologies, each with certain advantages and disadvantages. For example, some methodologies are qualitative instead of quantitative. Others require long experimental periods or examine population-level effects, only. In this study, we used real-time PCR to examine diets of individual grasshoppers. The method has the advantage of being both fast and quantitative. Using two grasshopper species, Oedaleus asiaticus and Dasyhippus barbipes, we designed ITS primer sequences for their three main host plants, Stipa krylovii, Leymus chinensis and Cleistogenes squarrosa and used real-time PCR method to test diet structure both qualitatively and quantitatively. The lowest detection efficiency of the three grass species was ~80% with a strong correlation between actual and PCR-measured food intake. We found that Oedaleus asiaticus maintained an unchanged diet structure across grasslands with different grass communities. By comparison, Dasyhippus barbipes changed its diet structure. These results revealed why O. asiaticus distribution is mainly confined to Stipa-dominated grassland, and D. barbipes is more widely distributed across Inner Mongolia. Overall, real-time PCR was shown to be a useful tool for investigating grasshopper diets, which in turn offers some insight into grasshopper distributions and improved pest management.

  4. Selection and Validation of Reference Genes for Quantitative Real-time PCR in Gentiana macrophylla

    OpenAIRE

    He, Yihan; Yan, Hailing; Hua, Wenping; Huang, Yaya; Wang, Zhezhi

    2016-01-01

    Real time quantitative PCR (RT-qPCR or qPCR) has been extensively applied for analyzing gene expression because of its accuracy, sensitivity, and high throughput. However, the unsuitable choice of reference gene(s) can lead to a misinterpretation of results. We evaluated the stability of 10 candidates – five traditional housekeeping genes (UBC21, GAPC2, EF-1α4, UBQ10, and UBC10) and five novel genes (SAND1, FBOX, PTB1, ARP, and Expressed1) – using the transcriptome data of Gentiana macrophyll...

  5. The quantification of spermatozoa by real-time quantitative PCR, spectrophotometry, and spermatophore cap size.

    Science.gov (United States)

    Doyle, Jacqueline M; McCormick, Cory R; DeWoody, J Andrew

    2011-01-01

    Many animals, such as crustaceans, insects, and salamanders, package their sperm into spermatophores, and the number of spermatozoa contained in a spermatophore is relevant to studies of sexual selection and sperm competition. We used two molecular methods, real-time quantitative polymerase chain reaction (RT-qPCR) and spectrophotometry, to estimate sperm numbers from spermatophores. First, we designed gene-specific primers that produced a single amplicon in four species of ambystomatid salamanders. A standard curve generated from cloned amplicons revealed a strong positive relationship between template DNA quantity and cycle threshold, suggesting that RT-qPCR could be used to quantify sperm in a given sample. We then extracted DNA from multiple Ambystoma maculatum spermatophores, performed RT-qPCR on each sample, and estimated template copy numbers (i.e. sperm number) using the standard curve. Second, we used spectrophotometry to determine the number of sperm per spermatophore by measuring DNA concentration relative to the genome size. We documented a significant positive relationship between the estimates of sperm number based on RT-qPCR and those based on spectrophotometry. When these molecular estimates were compared to spermatophore cap size, which in principle could predict the number of sperm contained in the spermatophore, we also found a significant positive relationship between sperm number and spermatophore cap size. This linear model allows estimates of sperm number strictly from cap size, an approach which could greatly simplify the estimation of sperm number in future studies. These methods may help explain variation in fertilization success where sperm competition is mediated by sperm quantity. © 2010 Blackwell Publishing Ltd.

  6. Selection of reference genes for quantitative real-time PCR in bamboo (Phyllostachys edulis.

    Directory of Open Access Journals (Sweden)

    Chunjie Fan

    Full Text Available BACKGROUND: The Moso bamboo (Phyllostachys edulis is one of the most important forestry resources and plays essential ecological roles in southern China. A draft nuclear genome sequence is expected to be publicly available in the near future; an explosion of gene expression data related to the unique traits of Moso bamboo will undoubtedly follow. Reverse transcription quantitative real-time PCR ((RT-qPCR is a widely used method for gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. RESULT: In this study, 14 candidate reference genes were chosen, and their expression levels were assessed by (RT-qPCR in a set of six tissue samples (root, stem, mature stem, leaf, flower, and leaf sheath and at two developmental stages (before and after flowering in bamboo specimens obtained in three locations. The stability and suitability of the candidate reference genes were validated using the geNorm, NormFinder and BestKeeper programs. The results showed that TIP41 and NTB were suitable reference genes across all the tissues and at the different developmental stages examined in this study. While the expression of the NTB, TIP41 and UBQ were the mostly stable in different plant tissues samples, the expression of the TIP41, NTB and CAC were ranked the most stable in bamboo plants at various developmental stages. AP2-like gene was further assessed by using the reference genes TIP41 and NTB in comparison to ACT. Significant difference of the expression profile of AP2-like demonstrated the importance of choosing adequate reference genes in bamboo. CONCLUSION: TIP41 and NTB were found to be homogeneously expressed and were adequate for normalization purposes, showing equivalent transcript levels in different samples. They are therefore the recommended reference genes for measuring gene expression in P. edulis.

  7. Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae.

    Directory of Open Access Journals (Sweden)

    Meng Sun

    Full Text Available The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA, elongation factor 1 (EF1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ribosomal protein S13 (RPS13, ribosomal protein S20 (RPS20, tubulin (TUB, and β-actin (ACTB were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1 were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands. 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults. 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C. To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83 was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

  8. DNA-Based Sensor for Real-Time Measurement of the Enzymatic Activity of Human Topoisomerase I

    DEFF Research Database (Denmark)

    Marcussen, Lærke Bay; Jepsen, Morten Leth; Kristoffersen, Emil Laust

    2013-01-01

    Sensors capable of quantitative real-time measurements may present the easiest and most accurate way to study enzyme activities. Here we present a novel DNA-based sensor for specific and quantitative real-time measurement of the enzymatic activity of the essential human enzyme, topoisomerase I....... The basic design of the sensor relies on two DNA strands that hybridize to form a hairpin structure with a fluorophore-quencher pair. The quencher moiety is released from the sensor upon reaction with human topoisomerase I thus enabling real-time optical measurement of enzymatic activity. The sensor...... is specific for topoisomerase I even in raw cell extracts and presents a simple mean of following enzyme kinetics using standard laboratory equipment such as a qPCR machine or fluorimeter. Human topoisomerase I is a well-known target for the clinically used anti-cancer drugs of the camptothecin family...

  9. Real time quantitative phase microscopy based on single-shot transport of intensity equation (ssTIE) method

    Science.gov (United States)

    Yu, Wei; Tian, Xiaolin; He, Xiaoliang; Song, Xiaojun; Xue, Liang; Liu, Cheng; Wang, Shouyu

    2016-08-01

    Microscopy based on transport of intensity equation provides quantitative phase distributions which opens another perspective for cellular observations. However, it requires multi-focal image capturing while mechanical and electrical scanning limits its real time capacity in sample detections. Here, in order to break through this restriction, real time quantitative phase microscopy based on single-shot transport of the intensity equation method is proposed. A programmed phase mask is designed to realize simultaneous multi-focal image recording without any scanning; thus, phase distributions can be quantitatively retrieved in real time. It is believed the proposed method can be potentially applied in various biological and medical applications, especially for live cell imaging.

  10. Real time wave measurements and wave hindcasting in deep waters

    Digital Repository Service at National Institute of Oceanography (India)

    Anand, N.M.; Mandal, S.; SanilKumar, V.; Nayak, B.U.

    Deep water waves off Karwar (lat. 14~'45.1'N, long. 73~'34.8'E) at 75 m water depth pertaining to peak monsoon period have been measured using a Datawell waverider buoy. Measured wave data show that the significant wave height (Hs) predominantly...

  11. Real-time performance measurement system for automated teller machines

    NARCIS (Netherlands)

    Van Anholt, R. G.; Vis, I. F A

    2012-01-01

    Performance measurement systems have proven to facilitate process improvement in the past decades in various markets and environments. The objective of this paper is to design a performance measurement system to actively control, monitor and improve performance of automated teller machines. In our

  12. Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification

    Directory of Open Access Journals (Sweden)

    Kager Piet A

    2006-10-01

    Full Text Available Abstract Background Decisions concerning malaria treatment depend on species identification causing disease. Microscopy is most frequently used, but at low parasitaemia (Plasmodium antigen detection do often not allow for species discrimination as microscopy does, but also become insensitive at Methods This paper reports the development of a sensitive and specific real-time Quantitative Nucleic Acid Sequence Based Amplification (real-time QT-NASBA assays, based on the small-subunit 18S rRNA gene, to identify the four human Plasmodium species. Results The lower detection limit of the assay is 100 – 1000 molecules in vitro RNA for all species, which corresponds to 0.01 – 0.1 parasite per diagnostic sample (i.e. 50 μl of processed blood. The real-time QT-NASBA was further evaluated using 79 clinical samples from malaria patients: i.e. 11 Plasmodium. falciparum, 37 Plasmodium vivax, seven Plasmodium malariae, four Plasmodium ovale and 20 mixed infections. The initial diagnosis of 69 out of the 79 samples was confirmed with the developed real-time QT-NASBA. Re-analysis of seven available original slides resolved five mismatches. Three of those were initially identified as P. malariae mono-infection, but after re-reading the slides P. falciparum was found, confirming the real-time QT-NASBA result. The other two slides were of poor quality not allowing true species identification. The remaining five discordant results could not be explained by microscopy, but may be due to extreme low numbers of parasites present in the samples. In addition, 12 Plasmodium berghei isolates from mice and 20 blood samples from healthy donors did not show any reaction in the assay. Conclusion Real-time QT-NASBA is a very sensitive and specific technique with a detection limit of 0.1 Plasmodium parasite per diagnostic sample (50 μl of blood and can be used for the detection, identification and quantitative measurement of low parasitaemia of Plasmodium species, thus

  13. Real-time precision measuring device of tree diameter growth

    Science.gov (United States)

    Guo, Mingming; Chen, Aijun; Li, Dongsheng; Liu, Nan; Yao, Jingyuan

    2016-01-01

    DBH(diameter at breast height) is an important factor to reflect of the quality of plant growth, also an important parameter indispensable in forest resources inventory and forest carbon sink, the accurate measurement of DBH or not is directly related to the research of forest resources inventory and forest carbon sink. In this paper, the principle and the mathematical model of DBH measurement device were introduced, the fixture measuring device and the hardware circuit for this tree diameter were designed, the measurement software programs were compiled, and the precision measuring device of tree diameter growth was developed. Some experiments with Australia fir were conducted. Based on experiment data, the correlations among the DBH variation of Australian fir, the environment temperature, air humility and PAR(photosynthetically active radiation) were obtained. The effects of environmental parameters (environment temperature, air humility and PAR) on tree diameter were analyzed. Experimental results show that there is a positive correlation between DBH variation of Australian fir and environment temperature, a negative correlation between DBH variation of Australian fir and air humility , so is PAR.

  14. IN SITU Device for Real-Time Catalyst Deactivation Measurements

    Energy Technology Data Exchange (ETDEWEB)

    Fossil Energy Research

    2008-03-31

    SCR catalyst management has become an important operations and maintenance activity for coal-fired utility boilers in the United States. To facilitate this activity, a method to determine Catalyst Activity in situ is being developed. This report describes the methodology and presents the results of a two ozone season demonstration conducted at Alabama Power Company's Gorgas Unit 10 during the 2005 and 2006 ozone seasons. The results showed that the in situ measurements are in good agreement with the laboratory measurements and the technique has some advantages over the traditional laboratory method of determining Catalyst Activity and Reactor Potential. SCR Performance is determined by the overall Reactor Potential (the product of the Catalyst Activity and the available surface area per unit of flue gas). The in situ approach provides a direct measurement of Reactor Potential under actual operating conditions, whereas laboratory measurements of Catalyst Activity need to be coupled with estimates of catalyst pluggage and flue gas flowrate in order to assess Reactor Potential. The project also showed that the in situ activity results can easily be integrated into catalyst management software to aid in making informed catalyst decisions.

  15. Real-time measurement of perceptual qualities in conceptual design

    NARCIS (Netherlands)

    Bittermann, M.; Ciftcioglu, O.

    2006-01-01

    Implications of design decisions are hard to oversee for designers. This is the case in particular with respect to decisions, which influence perception related qualities of designs. Such qualities are for example visual openness, visual privacy, and spatial intimacy. They are difficult to measure

  16. Real-time diameter measurement using diffuse light

    Science.gov (United States)

    Luo, Xiaohe; Hui, Mei; Zhu, Qiudong; Wang, Shanshan

    2016-09-01

    A method for on-line rapid determination of the diameter of metallic cylinder is introduced in this paper. Under the radiation of diffuse light, there is a bright area close to the margin of metallic cylinder, and the method of this paper is based on the intensity distribution of the bright area. In this paper, with the radiation by a diffuse plane light with special shape, we present the relation expression of the distance between the peak point and the real edge of the cylinder and the distance between the diffuse light and the pinhole aperture of the camera. With the expression, the diameter of the cylinder to be measured can be calculated. In the experiments, monochromatic LED uniting with ground glass forms the diffuse light source, then the light irradiates the tested cylinder. After the cylinder, we use a lens with a front pinhole stop to choose the light into CMOS, then a computer is used to analyze images and export the measurement results. The measuring system using this method is very easily implemented, so it can realize the on-line rapid measurement. Experimental results are presented for six metallic cylinders with the diameter in 5 18mm range and roughness in Ra- 0.02um, and the precision reaches 3um.

  17. Microreactortechnology: Real-Time Flow Measurements in Organic Synthesis

    Directory of Open Access Journals (Sweden)

    Pieter J. Nieuwland

    2012-03-01

    Full Text Available With the commercial availability of integrated microreactor systems, the numbers of chemical processes that are performed nowadays in a continuous flow is growing rapidly. The control over mixing efficiency and homogeneous heating in these reactors allows industrial scale production that was often hampered by the use of large amounts of hazardous chemicals. Accurate actuation and in line measurements of the flows, to have a better control over the chemical reaction, is of added value for increasing reproducibility and a safe production.

  18. Real time PCR method for simultaneous detection, quantitation and differentiation of capripoxviruses.

    Science.gov (United States)

    Lamien, Charles Euloge; Lelenta, Mamadou; Goger, Wilfried; Silber, Roland; Tuppurainen, Eeva; Matijevic, Mirta; Luckins, Antony George; Diallo, Adama

    2011-01-01

    The genus Capripoxvirus (CaPV) comprises three members namely, sheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) affecting sheep, goats and cattle, respectively. CaPV infections produce similar symptoms in sheep and goats, and the three viruses cannot be distinguished serologically. Since there are conflicting opinions regarding the host specificity of CaPVs, particularly for goatpox and sheeppox viruses, the development of rapid genotyping tools will facilitate more accurate disease diagnosis and surveillance for better management of capripox outbreaks. This paper describes a species-specific, real time polymerase chain reaction (PCR), based on unique molecular markers that were found in the G-protein-coupled chemokine receptor (GPCR) gene sequences of CaPVs, that uses dual hybridization probes for their simultaneous detection, quantitation and genotyping. The assay can differentiate between CaPV strains based on differences in the melting point temperature (Tm) obtained after fluorescence melting curve analysis (FMCA). It is highly sensitive and presents low intra- and inter-run variation. This real time PCR assay will make a significant contribution to CaPV diagnosis and to the better understanding of the epidemiology of CaPVs by enabling rapid genotyping and gene-based classification of viral strains and unequivocal identification of isolates. Copyright © 2010 Elsevier B.V. All rights reserved.

  19. Improved radar data processing algorithms for quantitative rainfall estimation in real time.

    Science.gov (United States)

    Krämer, S; Verworn, H R

    2009-01-01

    This paper describes a new methodology to process C-band radar data for direct use as rainfall input to hydrologic and hydrodynamic models and in real time control of urban drainage systems. In contrast to the adjustment of radar data with the help of rain gauges, the new approach accounts for the microphysical properties of current rainfall. In a first step radar data are corrected for attenuation. This phenomenon has been identified as the main cause for the general underestimation of radar rainfall. Systematic variation of the attenuation coefficients within predefined bounds allows robust reflectivity profiling. Secondly, event specific R-Z relations are applied to the corrected radar reflectivity data in order to generate quantitative reliable radar rainfall estimates. The results of the methodology are validated by a network of 37 rain gauges located in the Emscher and Lippe river basins. Finally, the relevance of the correction methodology for radar rainfall forecasts is demonstrated. It has become clearly obvious, that the new methodology significantly improves the radar rainfall estimation and rainfall forecasts. The algorithms are applicable in real time.

  20. Radiation exposure in nuclear medicine: real-time measurement

    Energy Technology Data Exchange (ETDEWEB)

    Sylvain, Iara [Beaujon Hospital, Clichy (France). Dept. of Nuclear Medicine; E-mail: iara.sylvain@bjn.ap-hop-paris.fr; Bok, Bernard [Beaujon Hospital, Clichy (France). Dept. of Nuclear Medicine; X. Bichat University, Paris (France). Biophysics Lab.; E-mail: bernard.bok@bjn.ap-hop-paris.fr

    2002-09-01

    French regulations have introduced the use of electronic dosimeters for personnel monitoring of workers. In order to evaluate the exposure from diagnostic procedures to nuclear medicine staff, individual whole-body doses were measured daily with electronic (digital) personal dosimeters during 20 consecutive weeks and correlated with the work load of each day. Personal doses remained always below 20 mu Sv/d under normal working conditions. Radiation exposure levels were highest to tech staff, nurses and stretcher-bearers. The extrapolated annual cumulative doses for all staff remained less than 10% of the maximum legal limit for exposed workers (2 mSv/yr). Electronic dosimeters are not technically justified for routine survey of staff. The high sensitivity and immediate reading of electronic semiconductor dosimeters may become very useful for exposure control under risky working conditions. It may become an important help for optimising radiation protection. (author)

  1. Radiation exposure in nuclear medicine: real-time measurement

    Directory of Open Access Journals (Sweden)

    Iara Sylvain

    2002-09-01

    Full Text Available French regulations have introduced the use of electronic dosimeters for personal monitoring of workers. In order to evaluate the exposure from diagnostic procedures to nuclear medicine staff, individual whole-body doses were measured daily with electronic (digital personal dosimeters during 20 consecutive weeks and correlated with the work load of each day. Personal doses remained always below 20 µSv/d under normal working conditions. Radiation exposure levels were highest to tech staff, nurses and stretcher-bearers. The extrapolated annual cumulative doses for all staff remained less than 10 % of the maximum legal limit for exposed workers (2 mSv/yr. Electronic dosimeters are not technically justified for routine survey of staff. The high sensitivity and immediate reading of electronic semiconductor dosimeters may become very useful for exposure control under risky working conditions. It may become an important help for optimising radiation protection.A legislação francesa introduziu o uso de dosímetros eletrônicos para monitoração da exposição do trabalhador. Afim de avaliar a exposição do trabalhador proveniente de exames diagnósticos em medicina nuclear, doses individuais do corpo inteiro foram medidas diariamente com dosímetros eletrônicos (digitais durante 20 semanas consecutivas e correlatas com as atividades de trabalho de cada dia. As doses foram sempre inferiores à 20 µSv por dia em condições normais de trabalho. Os níveis de exposição de radiação mais elevados foram para os enfermeiros, manipuladores e maqueiros. A extrapolação da dose anual para todos os trabalhadores foi menos que 10 % do limite máximo legal para os trabalhadores expostos (2 mSv/ano. Dosímetros eletrônicos não são tecnicamente justificados para a o controle de rotina da exposição dos trabalhadores, mas a alta sensibilidade e a leitura imediata desses dosímetros podem vir a serem muito úteis para o controle da exposição em condi

  2. Investigations on abundance and activity of microbial sponge symbionts using quantitative real - time PCR

    DEFF Research Database (Denmark)

    Kumala, Lars; Hentschel, Ute; Bayer, Kristina

    Marine sponges are hosts to dense and diverse microbial consortia that are likely to play a key role in the metabolic processes of the host sponge due to their enormous abundance. Common symbioses between nitrogen transforming microorganisms and sponges indicate complex nitrogen cycling within...... the host. Of particular interest is determining the community structure and function of microbial symbionts in order to gain deeper insight into host-symbiont interactions. We investigated the abundance and activity of microbial symbionts in two Mediterranean sponge species using quantitative real-time PCR....... An absolute quantification of functional genes and transcripts in archaeal and bacterial symbionts was conducted to determine their involvement in nitrification and denitrification, comparing the low microbial abundance (LMA) sponge Dysidea avara with the high microbial abundance (HMA) representative Aplysina...

  3. A fluorescence-based quantitative real-time PCR assay for accurate Pocillopora damicornis species identification

    Science.gov (United States)

    Thomas, Luke; Stat, Michael; Evans, Richard D.; Kennington, W. Jason

    2016-09-01

    Pocillopora damicornis is one of the most extensively studied coral species globally, but high levels of phenotypic plasticity within the genus make species identification based on morphology alone unreliable. As a result, there is a compelling need to develop cheap and time-effective molecular techniques capable of accurately distinguishing P. damicornis from other congeneric species. Here, we develop a fluorescence-based quantitative real-time PCR (qPCR) assay to genotype a single nucleotide polymorphism that accurately distinguishes P. damicornis from other morphologically similar Pocillopora species. We trial the assay across colonies representing multiple Pocillopora species and then apply the assay to screen samples of Pocillopora spp. collected at regional scales along the coastline of Western Australia. This assay offers a cheap and time-effective alternative to Sanger sequencing and has broad applications including studies on gene flow, dispersal, recruitment and physiological thresholds of P. damicornis.

  4. Rapid detection of Ceratocystis platani inoculum by quantitative real-time PCR assay.

    Science.gov (United States)

    Luchi, Nicola; Ghelardini, Luisa; Belbahri, Lassaâd; Quartier, Marion; Santini, Alberto

    2013-09-01

    Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect a C. platani airborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani, detecting DNA concentrations as low as 1.2 × 10(-2) to 1.4 × 10(-2) pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect the C. platani inoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of a C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management.

  5. Selection and Validation of Reference Genes for Quantitative Real-time PCR in Gentiana macrophylla.

    Science.gov (United States)

    He, Yihan; Yan, Hailing; Hua, Wenping; Huang, Yaya; Wang, Zhezhi

    2016-01-01

    Real time quantitative PCR (RT-qPCR or qPCR) has been extensively applied for analyzing gene expression because of its accuracy, sensitivity, and high throughput. However, the unsuitable choice of reference gene(s) can lead to a misinterpretation of results. We evaluated the stability of 10 candidates - five traditional housekeeping genes (UBC21, GAPC2, EF-1α4, UBQ10, and UBC10) and five novel genes (SAND1, FBOX, PTB1, ARP, and Expressed1) - using the transcriptome data of Gentiana macrophylla. Common statistical algorithms ΔC t, GeNorm, NormFinder, and BestKeeper were run with samples collected from plants under various experimental conditions. For normalizing expression levels from tissues at different developmental stages, GAPC2 and UBC21 had the highest rankings. Both SAND1 and GAPC2 proved to be the optimal reference genes for roots from plants exposed to abiotic stresses while EF-1α4 and SAND1 were optimal when examining expression data from the leaves of stressed plants. Based on a comprehensive ranking of stability under different experimental conditions, we recommend that SAND1 and EF-1α4 are the most suitable overall. In this study, to find a suitable reference gene and its real-time PCR assay for G. macrophylla DNA content quantification, we evaluated three target genes including WRKY30, G10H, and SLS, through qualitative and absolute quantitative PCR with leaves under elicitors stressed experimental conditions. Arbitrary use of reference genes without previous evaluation can lead to a misinterpretation of the data. Our results will benefit future research on the expression of genes related to secoiridoid biosynthesis in this species under different experimental conditions.

  6. Quantitative analysis of the expression of ACAT genes in human tissues by real-time PCR.

    Science.gov (United States)

    Smith, Jeffery L; Rangaraj, Kavitha; Simpson, Robert; Maclean, Donald J; Nathanson, Les K; Stuart, Katherine A; Scott, Shaun P; Ramm, Grant A; de Jersey, John

    2004-04-01

    ACAT (also called sterol o-acyltransferase) catalyzes the esterification of cholesterol by reaction with long-chain acyl-CoA derivatives and plays a pivotal role in the regulation of cholesterol homeostasis. Although two human ACAT genes termed ACAT-1 and ACAT-2 have been reported, prior research on differential tissue expression is qualitative and incomplete. We have developed a quantitative multiplex assay for each ACAT isoform after RT treatment of total RNA using TaqMan real-time quantitative PCR normalized to beta-actin in the same reaction tube. This enabled us to calculate the relative abundance of transcripts in several human tissues as an ACAT-2/ACAT-1 ratio. In liver (n = 17), ACAT-1 transcripts were on average 9-fold (range, 1.7- to 167-fold) more abundant than ACAT-2, whereas in duodenal samples (n = 10), ACAT-2 transcripts were on average 3-fold (range, 0.39- to 12.2-fold) more abundant than ACAT-1. ACAT-2 was detected for the first time in peripheral blood mononuclear cells. Interesting differences in ACAT-2 mRNA expression were evident in subgroup analysis of samples from different sources. These results demonstrate quantitatively that ACAT-1 transcripts predominate in human liver and ACAT-2 transcripts predominate in human duodenum and support the notion that ACAT-2 has an important regulatory role in liver and intestine.

  7. Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform

    Science.gov (United States)

    Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

    2015-11-01

    Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (r

  8. Real-Time Measurement of Process Efficiency during Inertia Friction Welding (Preprint)

    Science.gov (United States)

    2017-12-20

    AFRL-RX-WP-JA-2017-0532 REAL-TIME MEASUREMENT OF PROCESS EFFICIENCY DURING INERTIA FRICTION WELDING (PREPRINT...4. TITLE AND SUBTITLE REAL-TIME MEASUREMENT OF PROCESS EFFICIENCY DURING INERTIA FRICTION WELDING (PREPRINT) 5a. CONTRACT NUMBER IN-HOUSE...200 words) Process efficiency is a crucial parameter for inertia friction welding (IFW) that is largely unknown at the present time. A new method

  9. Spatial filtering velocimetry for real-time out-of-plane displacement measurements

    DEFF Research Database (Denmark)

    Olesen, Anders Sig; Yura, H.T.; Jakobsen, Michael Linde

    2016-01-01

    We probe the dynamics of objective laser speckles as the axial distance between the object and the observation plane changes. With the purpose of measuring out-of-plane motion in real time, we apply optical spatial filtering velocimetry to the speckle dynamics. To achieve this, a rotationally sym...... element which implements the spatial filter and experimentally demonstrate the ability of the technology to obtain displacement measurements of a vibrating object in real-time....

  10. Integrating high-throughput pyrosequencing and quantitative real-time PCR to analyze complex microbial communities.

    Science.gov (United States)

    Zhang, Husen; Parameswaran, Prathap; Badalamenti, Jonathan; Rittmann, Bruce E; Krajmalnik-Brown, Rosa

    2011-01-01

    New high-throughput technologies continue to emerge for studying complex microbial communities. In particular, massively parallel pyrosequencing enables very high numbers of sequences, providing a more complete view of community structures and a more accurate inference of the functions than has been possible just a few years ago. In parallel, quantitative real-time PCR (QPCR) allows quantitative monitoring of specific community members over time, space, or different environmental conditions. In this review, we discuss the principles of these two methods and their complementary applications in studying microbial ecology in bioenvironmental systems. We explain parallel sequencing of amplicon libraries and using bar codes to differentiate multiple samples in a pyrosequencing run. We also describe best procedures and chemistries for QPCR amplifications and address advantages of applying automation to increase accuracy. We provide three examples in which we used pyrosequencing and QPCR together to define and quantify members of microbial communities: in the human large intestine, in a methanogenic digester whose sludge was made more bioavailable by a high-voltage pretreatment, and on the biofilm anode of a microbial electrolytic cell. We highlight our key findings in these systems and how both methods were used in concert to achieve those findings. Finally, we supply detailed methods for generating PCR amplicon libraries for pyrosequencing, pyrosequencing data analysis, QPCR methodology, instrumentation, and automation.

  11. Identification and validation of suitable housekeeping genes for normalizing quantitative real-time PCR assays in injured peripheral nerves.

    Science.gov (United States)

    Gambarotta, Giovanna; Ronchi, Giulia; Friard, Olivier; Galletta, Pantaleo; Perroteau, Isabelle; Geuna, Stefano

    2014-01-01

    Injury to the peripheral nerve induces dramatic changes in terms of cellular composition that are reflected on RNA quality and quantity, making messenger RNA expression analysis very complex. Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not suitable for quantitative real-time PCR normalization; moreover, the presence of pseudogenes in some of them impairs their use. To deal with this problem, we have developed a new method to identify new stable housekeeping genes based on publicly available microarray data on normal and injured nerves. Four new candidate stable genes were identified and validated by quantitative real-time PCR analysis on nerves during the different phases after nerve injury: nerve degeneration, regeneration and remyelination. The stability measure of these genes was calculated with both NormFinder and geNorm algorithms and compared with six commonly used housekeeping genes. This procedure allowed us to identify two new and highly stable genes that can be employed for normalizing injured peripheral nerve data: ANKRD27 and RICTOR. Besides providing a tool for peripheral nerve research, our study also describes a simple and cheap procedure that can be used to identify suitable housekeeping genes in other tissues and organs.

  12. Microscopy, culture, and quantitative real-time PCR examination confirm internalization of mycobacteria in plants.

    Science.gov (United States)

    Kaevska, M; Lvoncik, S; Slana, I; Kulich, P; Kralik, P

    2014-07-01

    The environment is a reservoir of nontuberculous mycobacteria and is considered a source of infection for animals and humans. Mycobacteria can persist in different types of environments for a relatively long time. We have studied their possible internalization into plant tissue through intact, as well as damaged, root systems of different types of plants grown in vitro and under field conditions. The substrate into which plants were seeded was previously contaminated with different strains of Mycobacterium avium (10(8) to 10(10) cells/g of soil) and feces from animals with paratuberculosis. We detected M. avium subsp. avium, hominissuis, and paratuberculosis in the stems and leaves of the plants by both culture and real-time quantitative PCR. The presence of mycobacteria in the plant tissues was confirmed by microscopy. The concentration of mycobacteria found inside plant tissue was several orders of magnitude lower (up to 10(4) cells/g of tissue) than the initial concentration of mycobacteria present in the culture medium or substrate. These findings led us to the hypothesis that plants may play a role in the spread and transmission of mycobacteria to other organisms in the environment. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  13. Comparison of high and low virulence serotypes of Actinobacillus pleuropneumoniae by quantitative real-time PCR

    DEFF Research Database (Denmark)

    Schou, Kirstine Klitgaard; Angen, Øystein; Boye, Mette

    of high virulence while serotype 6 strains are normally found to be less pathogenic. To gain an understanding of the differential virulence of serotype 2 and 6, the expression of a panel of Ap genes during infection of porcine epithelial lung cells (SJPL) were examined by quantitative real-time PCR (qPCR...... to be important for early establishment of the bacteria in the host were examined by qPCR. The genes examined were apfA, coding for a subunit of Type IV pili, kdsB coding for a gene involved in lippopolysacceride biosynthesis, and pgaB which is involved in biofilm formation, all three believed to be important...... with respect to host cells adhesion. Also included in the analysis were the capsular gene, cpxB, the RTX toxin genes apxII, and apxIV and the gene exbD, involved in binding of iron from host cells. Finally, three previously validated reference genes, glyA, pykA and tpiA were included for normalization of the qPCR...

  14. Development of Quantitative Real-time PCR Assays for Different Clades of “Candidatus Accumulibacter”

    Science.gov (United States)

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-05-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85-112% (R2 = 0.962-0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants.

  15. Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay.

    Science.gov (United States)

    Liu, Zongxiao; Teng, Yong; Liu, Hong; Jiang, Yulin; Xie, Xiayang; Li, Huifang; Lv, Jiangqiang; Gao, Longying; He, Junqiang; Shi, Xiujie; Tian, Feiyan; Yang, Jingshun; Xie, Congxin

    2008-04-01

    Spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are three important fish rhabdoviruses, causing serious Office International des Epizooties (OIE) classified diseases in wild and farmed fish. Here, a new multiplex real-time quantitative RT-PCR (mqRT-PCR) assay was developed for simultaneous detection, identification and quantification of these three rhabdoviruses. The sets of primers and probes were targeted to conserved regions of glycoprotein (G) gene of SVCV, nucleoprotein (N) gene of IHNV and G gene of VHSV and used to amplify. The sensitivity, specificity and interference test of mqRT-PCR assay was analyzed. It was shown that the detection levels of 100 copies of SVCV, 220 copies of IHNV and 140 copies of VHSV were achieved, and there was no non-specific amplification and cross-reactivity using RNA of pike fry rhabdovirus (PFRV), infectious pancreatic necrosis virus (IPNV) and grass carp reovirus (GCRV). A total of 80 clinical fish samples were tested using the mqRT-PCR assay and the results were confirmed by antigen-capture ELISA and cell culture assay. This assay has the potential to be used for both research applications and diagnosis.

  16. Reference gene selection for gene expression studies in lily using quantitative real-time PCR.

    Science.gov (United States)

    Zhang, M F; Liu, Q; Jia, G X

    2016-04-29

    Quantitative real-time polymerase chain reaction (qRT-PCR) is an important technology used to analyze gene-expression levels. Reference genes, which are assumed to be expressed consistently across various developmental stages and in different tissues, were selected for expression level analysis. Using digital gene expression technology, we selected nine reference genes (18S, EF, CYCOL, SAND, GAPDH, ACTIN, BHLH, TIP, and Clathrin) as candidate reference genes for further study. Using three different analysis methods (GeNorm, NormFinder, and BestKeeper), a total of 144 lily (Lilium x formolongi "Raizan 3") samples were analyzed. The samples were collected from four different tissues under various developmental stages. In addition, leaves treated with different plant hormones were collected and analyzed. The data showed that the stability of the nine reference genes differed among samples, but TIP, EF, Clathrin, and BHLH could be identified as the most stable genes overall. In addition, the relative expression level of LfFT in different lily tissues with the competence to flower was also analyzed to verify the selected reference genes. This study constitutes an important source for selecting reference genes when analyzing the expression patterns of flowering time and floral development regulation genes in lily cultivars.

  17. Identification of reference genes for real-time quantitative PCR experiments in the liverwort Marchantia polymorpha.

    Science.gov (United States)

    Saint-Marcoux, Denis; Proust, Hélène; Dolan, Liam; Langdale, Jane A

    2015-01-01

    Real-time quantitative polymerase chain reaction (qPCR) has become widely used as a method to compare gene transcript levels across different conditions. However, selection of suitable reference genes to normalize qPCR data is required for accurate transcript level analysis. Recently, Marchantia polymorpha has been adopted as a model for the study of liverwort development and land plant evolution. Identification of appropriate reference genes has therefore become a necessity for gene expression studies. In this study, transcript levels of eleven candidate reference genes have been analyzed across a range of biological contexts that encompass abiotic stress, hormone treatment and different developmental stages. The consistency of transcript levels was assessed using both geNorm and NormFinder algorithms, and a consensus ranking of the different candidate genes was then obtained. MpAPT and MpACT showed relatively constant transcript levels across all conditions tested whereas the transcript levels of other candidate genes were clearly influenced by experimental conditions. By analyzing transcript levels of phosphate and nitrate starvation reporter genes, we confirmed that MpAPT and MpACT are suitable reference genes in M. polymorpha and also demonstrated that normalization with an inappropriate gene can lead to erroneous analysis of qPCR data.

  18. Identification of reference genes for real-time quantitative PCR experiments in the liverwort Marchantia polymorpha.

    Directory of Open Access Journals (Sweden)

    Denis Saint-Marcoux

    Full Text Available Real-time quantitative polymerase chain reaction (qPCR has become widely used as a method to compare gene transcript levels across different conditions. However, selection of suitable reference genes to normalize qPCR data is required for accurate transcript level analysis. Recently, Marchantia polymorpha has been adopted as a model for the study of liverwort development and land plant evolution. Identification of appropriate reference genes has therefore become a necessity for gene expression studies. In this study, transcript levels of eleven candidate reference genes have been analyzed across a range of biological contexts that encompass abiotic stress, hormone treatment and different developmental stages. The consistency of transcript levels was assessed using both geNorm and NormFinder algorithms, and a consensus ranking of the different candidate genes was then obtained. MpAPT and MpACT showed relatively constant transcript levels across all conditions tested whereas the transcript levels of other candidate genes were clearly influenced by experimental conditions. By analyzing transcript levels of phosphate and nitrate starvation reporter genes, we confirmed that MpAPT and MpACT are suitable reference genes in M. polymorpha and also demonstrated that normalization with an inappropriate gene can lead to erroneous analysis of qPCR data.

  19. GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR

    Science.gov (United States)

    Gubelmann, Carine; Gattiker, Alexandre; Massouras, Andreas; Hens, Korneel; David, Fabrice; Decouttere, Frederik; Rougemont, Jacques; Deplancke, Bart

    2011-01-01

    The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/. Database URL: http://deplanckelab.epfl.ch. PMID:21917859

  20. Species identification of Cannabis sativa using real-time quantitative PCR (qPCR).

    Science.gov (United States)

    Johnson, Christopher E; Premasuthan, Amritha; Satkoski Trask, Jessica; Kanthaswamy, Sree

    2013-03-01

    Most narcotics-related cases in the United States involve Cannabis sativa. Material is typically identified based on the cystolithic hairs on the leaves and with chemical tests to identify of the presence of cannabinoids. Suspect seeds are germinated into a viable plant so that morphological and chemical tests can be conducted. Seed germination, however, causes undue analytical delays. DNA analyses that involve the chloroplast and nuclear genomes have been developed for identification of C. sativa materials, but they require several nanograms of template DNA. Using the trnL 3' exon-trnF intragenic spacer regions within the C. sativa chloroplast, we have developed a real-time quantitative PCR assay that is capable of identifying picogram amounts of chloroplast DNA for species determination of suspected C. sativa material. This assay provides forensic science laboratories with a quick and reliable method to identify an unknown sample as C. sativa. © 2013 American Academy of Forensic Sciences.

  1. Validation of reference genes for quantitative real-time PCR during latex regeneration in rubber tree.

    Science.gov (United States)

    Long, Xiangyu; He, Bin; Gao, Xinsheng; Qin, Yunxia; Yang, Jianghua; Fang, Yongjun; Qi, Jiyan; Tang, Chaorong

    2015-06-01

    In rubber tree, latex regeneration is one of the decisive factors influencing the rubber yield, although its molecular regulation is not well known. Quantitative real-time PCR (qPCR) is a popular and powerful tool used to understand the molecular mechanisms of latex regeneration. However, the suitable reference genes required for qPCR are not available to investigate the expressions of target genes during latex regeneration. In this study, 20 candidate reference genes were selected and evaluated for their expression stability across the samples during the process of latex regeneration. All reference genes showed a relatively wide range of the threshold cycle values, and their stability was validated by four different algorithms (comparative delta Ct method, Bestkeeper, NormFinder and GeNorm). Three softwares (comparative delta Ct method, NormFinder and GeNorm) exported similar results that identify UBC4, ADF, UBC2a, eIF2 and ADF4 as the top five suitable references, and 18S as the least suitable one. The application of the screened references would improve accuracy and reliability of gene expression analysis in latex regeneration experiments. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Selection of Reference Genes for Real-Time Quantitative PCR in Pinus massoniana Post Nematode Inoculation.

    Directory of Open Access Journals (Sweden)

    Yongcheng Wei

    Full Text Available Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P. massoniana post nematode inoculation in this study. Three statistical approaches such as geNorm, NormFinder and BestKeeper were selected to analyze the stability of candidate genes. The results indicated that U2af and β-TUB were the most stable reference genes. These two genes could be used for the normalization in most of the experiments of P. massoniana, while Histone and AK were the least stable ones. In addition, EF expressed at the lowest average Ct value was the most abundant candidate gene. As an important gene associated with defense mechanisms, ABC transporter was analyzed by qRT-PCR, and the results were used to confirm the reliability of two genes. The selected reference genes in the present study will be conducive to future gene expression normalized by qRT-PCR in P. massoniana.

  3. A method for accurate detection of genomic microdeletions using real-time quantitative PCR

    Directory of Open Access Journals (Sweden)

    Bassett Anne S

    2005-12-01

    Full Text Available Abstract Background Quantitative Polymerase Chain Reaction (qPCR is a well-established method for quantifying levels of gene expression, but has not been routinely applied to the detection of constitutional copy number alterations of human genomic DNA. Microdeletions or microduplications of the human genome are associated with a variety of genetic disorders. Although, clinical laboratories routinely use fluorescence in situ hybridization (FISH to identify such cryptic genomic alterations, there remains a significant number of individuals in which constitutional genomic imbalance is suspected, based on clinical parameters, but cannot be readily detected using current cytogenetic techniques. Results In this study, a novel application for real-time qPCR is presented that can be used to reproducibly detect chromosomal microdeletions and microduplications. This approach was applied to DNA from a series of patient samples and controls to validate genomic copy number alteration at cytoband 22q11. The study group comprised 12 patients with clinical symptoms of chromosome 22q11 deletion syndrome (22q11DS, 1 patient trisomic for 22q11 and 4 normal controls. 6 of the patients (group 1 had known hemizygous deletions, as detected by standard diagnostic FISH, whilst the remaining 6 patients (group 2 were classified as 22q11DS negative using the clinical FISH assay. Screening of the patients and controls with a set of 10 real time qPCR primers, spanning the 22q11.2-deleted region and flanking sequence, confirmed the FISH assay results for all patients with 100% concordance. Moreover, this qPCR enabled a refinement of the region of deletion at 22q11. Analysis of DNA from chromosome 22 trisomic sample demonstrated genomic duplication within 22q11. Conclusion In this paper we present a qPCR approach for the detection of chromosomal microdeletions and microduplications. The strategic use of in silico modelling for qPCR primer design to avoid regions of repetitive

  4. Evaluation of a quantitative real-time PCR for rapid detection of Riemerella Anatipestifer infection in birds.

    Science.gov (United States)

    Zhang, Qingshan; Wan, Chunhe; Li, Chenxi; Bai, Xiaofei; Liu, Ming; Liu, Siguo; Zhang, Yun

    2017-08-05

    To establish an accurate, rapid, and a quantifiable method for the detection of Riemerella anatipestifer infection, a widespread infectious disease in birds, we developed a TaqMan-based real-time PCR assay by using DtxR gene-specific primers and a TaqMan probe. The standard curve established with a linear correlation (R2) of 0.998 and efficiency of 99% between the Ct value and the logarithm of the plasmid copy number. The reproducibility and specificity of the real-time PCR assay were confirmed by using plasmids containing DtxR genes or DNAs extracted from well-known bacteria or viruses causing duck diseases. The real-time PCR assay was 100 times more sensitive than the conventional PCR. The results reveal that the established real-time PCR assay might be a useful method for diagnosis and quantitative detection of Riemerella anatipestifer in birds.

  5. Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze)

    Science.gov (United States)

    Quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used technique for measuring gene expression levels due to its simplicity, specificity, and sensitivity. Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a...

  6. Quantitative Real Time PCR approach to study gene expression profile during prenatal growth of skeletal muscle in pig of Duroc and Pietrain breeds

    Directory of Open Access Journals (Sweden)

    M. Cagnazzo

    2010-01-01

    Full Text Available The quantitative real time-PCR (QRT-PCR is a very sensitive method used to quantify mRNA level in gene expression analysis. Combining amplification, detection and quantification in a single step, allows a more accurate measurement compared to the traditional PCR end point analysis (Pfaffl, 2001; Bustin, 2002.

  7. Method and apparatus for real-time measurement of fuel gas compositions and heating values

    Science.gov (United States)

    Zelepouga, Serguei; Pratapas, John M.; Saveliev, Alexei V.; Jangale, Vilas V.

    2016-03-22

    An exemplary embodiment can be an apparatus for real-time, in situ measurement of gas compositions and heating values. The apparatus includes a near infrared sensor for measuring concentrations of hydrocarbons and carbon dioxide, a mid infrared sensor for measuring concentrations of carbon monoxide and a semiconductor based sensor for measuring concentrations of hydrogen gas. A data processor having a computer program for reducing the effects of cross-sensitivities of the sensors to components other than target components of the sensors is also included. Also provided are corresponding or associated methods for real-time, in situ determination of a composition and heating value of a fuel gas.

  8. Standardisation of data from real-time quantitative PCR methods – evaluation of outliers and comparison of calibration curves

    Directory of Open Access Journals (Sweden)

    Burns Malcolm J

    2005-12-01

    Full Text Available Abstract Background As real-time quantitative PCR (RT-QPCR is increasingly being relied upon for the enforcement of legislation and regulations dependent upon the trace detection of DNA, focus has increased on the quality issues related to the technique. Recent work has focused on the identification of factors that contribute towards significant measurement uncertainty in the real-time quantitative PCR technique, through investigation of the experimental design and operating procedure. However, measurement uncertainty contributions made during the data analysis procedure have not been studied in detail. This paper presents two additional approaches for standardising data analysis through the novel application of statistical methods to RT-QPCR, in order to minimise potential uncertainty in results. Results Experimental data was generated in order to develop the two aspects of data handling and analysis that can contribute towards measurement uncertainty in results. This paper describes preliminary aspects in standardising data through the application of statistical techniques to the area of RT-QPCR. The first aspect concerns the statistical identification and subsequent handling of outlying values arising from RT-QPCR, and discusses the implementation of ISO guidelines in relation to acceptance or rejection of outlying values. The second aspect relates to the development of an objective statistical test for the comparison of calibration curves. Conclusion The preliminary statistical tests for outlying values and comparisons between calibration curves can be applied using basic functions found in standard spreadsheet software. These two aspects emphasise that the comparability of results arising from RT-QPCR needs further refinement and development at the data-handling phase. The implementation of standardised approaches to data analysis should further help minimise variation due to subjective judgements. The aspects described in this paper will

  9. Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR

    Directory of Open Access Journals (Sweden)

    López-Revilla Rubén

    2010-05-01

    Full Text Available Abstract Background We have developed an ultrasensitive method based on conventional PCR preamplification followed by nested amplification through real time PCR (qPCR in the presence of the DNA intercalating agent EvaGreen. Results Amplification mixtures calibrated with a known number of pHV101 copies carrying a 645 base pair (bp-long insert of the human papillomavirus type 16 (HPV16 E6 oncogene were used to generate the E6-1 amplicon of 645 bp by conventional PCR and then the E6-2 amplicon of 237 bp by nested qPCR. Direct and nested qPCR mixtures for E6-2 amplification corresponding to 2.5 × 102-2.5 × 106 initial pHV101 copies had threshold cycle (Ct values in the ranges of 18.7-29.0 and 10.0-25.0, respectively. The Ct of qPCR mixtures prepared with 1/50 volumes of preamplified mixtures containing 50 ng of DNA of the SiHa cell line (derived from an invasive cervical cancer with one HPV16 genome per cell was 19.9. Thermal fluorescence extinction profiles of E6-2 amplicons generated from pHV101 and SiHa DNA were identical, with a peak at 85.5°C. Conclusions Our method based on conventional preamplification for 15 cycles increased 10,750 times the sensitivity of nested qPCR for the quantitation of the E6 viral oncogene and confirmed that the SiHa cell line contains one E6-HPV16 copy per cell.

  10. 1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer

    Directory of Open Access Journals (Sweden)

    Dráberová Lubica

    2011-04-01

    Full Text Available Abstract Background Quantitative real-time PCR (qPCR is becoming increasingly important for DNA genotyping and gene expression analysis. For continuous monitoring of the production of PCR amplicons DNA-intercalating dyes are widely used. Recently, we have introduced a new qPCR mix which showed improved amplification of medium-size genomic DNA fragments in the presence of DNA dye SYBR green I (SGI. In this study we tested whether the new PCR mix is also suitable for other DNA dyes used for qPCR and whether it can be applied for amplification of DNA fragments which are difficult to amplify. Results We found that several DNA dyes (SGI, SYTO-9, SYTO-13, SYTO-82, EvaGreen, LCGreen or ResoLight exhibited optimum qPCR performance in buffers of different salt composition. Fidelity assays demonstrated that the observed differences were not caused by changes in Taq DNA polymerase induced mutation frequencies in PCR mixes of different salt composition or containing different DNA dyes. In search for a PCR mix compatible with all the DNA dyes, and suitable for efficient amplification of difficult-to-amplify DNA templates, such as those in whole blood, of medium size and/or GC-rich, we found excellent performance of a PCR mix supplemented with 1 M 1,2-propanediol and 0.2 M trehalose (PT enhancer. These two additives together decreased DNA melting temperature and efficiently neutralized PCR inhibitors present in blood samples. They also made possible more efficient amplification of GC-rich templates than betaine and other previously described additives. Furthermore, amplification in the presence of PT enhancer increased the robustness and performance of routinely used qPCRs with short amplicons. Conclusions The combined data indicate that PCR mixes supplemented with PT enhancer are suitable for DNA amplification in the presence of various DNA dyes and for a variety of templates which otherwise can be amplified with difficulty.

  11. Real-time visualization and quantitation of vascular permeability in vivo: implications for drug delivery.

    Directory of Open Access Journals (Sweden)

    Desmond B S Pink

    Full Text Available The leaky, heterogeneous vasculature of human tumors prevents the even distribution of systemic drugs within cancer tissues. However, techniques for studying vascular delivery systems in vivo often require complex mammalian models and time-consuming, surgical protocols. The developing chicken embryo is a well-established model for human cancer that is easily accessible for tumor imaging. To assess this model for the in vivo analysis of tumor permeability, human tumors were grown on the chorioallantoic membrane (CAM, a thin vascular membrane which overlays the growing chick embryo. The real-time movement of small fluorescent dextrans through the tumor vasculature and surrounding tissues were used to measure vascular leak within tumor xenografts. Dextran extravasation within tumor sites was selectively enhanced an interleukin-2 (IL-2 peptide fragment or vascular endothelial growth factor (VEGF. VEGF treatment increased vascular leak in the tumor core relative to surrounding normal tissue and increased doxorubicin uptake in human tumor xenografts. This new system easily visualizes vascular permeability changes in vivo and suggests that vascular permeability may be manipulated to improve chemotherapeutic targeting to tumors.

  12. Real-time MSE measurements for current profile control on KSTARa)

    Science.gov (United States)

    De Bock, M. F. M.; Aussems, D.; Huijgen, R.; Scheffer, M.; Chung, J.

    2012-10-01

    To step up from current day fusion experiments to power producing fusion reactors, it is necessary to control long pulse, burning plasmas. Stability and confinement properties of tokamak fusion reactors are determined by the current or q profile. In order to control the q profile, it is necessary to measure it in real-time. A real-time motional Stark effect diagnostic is being developed at Korean Superconducting Tokamak for Advanced Research for this purpose. This paper focuses on 3 topics important for real-time measurements: minimize the use of ad hoc parameters, minimize external influences and a robust and fast analysis algorithm. Specifically, we have looked into extracting the retardance of the photo-elastic modulators from the signal itself, minimizing the influence of overlapping beam spectra by optimizing the optical filter design and a multi-channel, multiharmonic phase locking algorithm.

  13. A novel quantitative real-time polymerase chain reaction method for detecting toxigenic Pasteurella multocida in nasal swabs from swine.

    Science.gov (United States)

    Scherrer, Simone; Frei, Daniel; Wittenbrink, Max Michael

    2016-12-01

    Progressive atrophic rhinitis (PAR) in pigs is caused by toxigenic Pasteurella multocida. In Switzerland, PAR is monitored by selective culture of nasal swabs and subsequent polymerase chain reaction (PCR) screening of bacterial colonies for the P. multocida toxA gene. A panel of 203 nasal swabs from a recent PAR outbreak were used to evaluate a novel quantitative real-time PCR for toxigenic P. multocida in porcine nasal swabs. In comparison to the conventional PCR with a limit of detection of 100 genome equivalents per PCR reaction, the real-time PCR had a limit of detection of 10 genome equivalents. The real-time PCR detected toxA-positive P. multocida in 101 samples (49.8%), whereas the conventional PCR was less sensitive with 90 toxA-positive samples (44.3%). In comparison to the real-time PCR, 5.4% of the toxA-positive samples revealed unevaluable results by conventional PCR. The approach of culture-coupled toxA PCR for the monitoring of PAR in pigs is substantially improved by a novel quantitative real-time PCR.

  14. Development of quantitative real-time PCR for detection and enumeration of Enterobacteriaceae.

    Science.gov (United States)

    Takahashi, Hajime; Saito, Rumi; Miya, Satoko; Tanaka, Yuichiro; Miyamura, Natsumi; Kuda, Takashi; Kimura, Bon

    2017-04-04

    The family Enterobacteriaceae, members of which are widely distributed in the environment, includes many important human pathogens. In this study, a rapid real-time PCR method targeting rplP, coding for L16 protein, a component of the ribosome large subunit, was developed for enumerating Enterobacteriaceae strains, and its efficiency was evaluated using naturally contaminated food products. The rplP-targeted real-time PCR amplified Enterobacteriaceae species with Ct values of 14.0-22.8, whereas the Ct values for non-Enterobacteriaceae species were >30, indicating the specificity of this method for the Enterobacteriaceae. Using a calibration curve of Ct=-3.025 (log CFU/g)+37.35, which was calculated from individual plots of the cell numbers in different concentrations of 5 Enterobacteriaceae species, the rplP-targeted real-time PCR was applied to 51 food samples. A real-time PCR and culture methods was obtained in a majority of the food samples (81.8%), with good correlation (r2=0.8285). This study demonstrated that the rplP-targeted real-time PCR method could detect and enumerate Enterobacteriaceae species in foods rapidly and accurately, and therefore, it can be used for the microbiological risk analysis of foods. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

    Science.gov (United States)

    There is a growing interest in the application of human-associated fecal sourceidentification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data q...

  16. A human fecal contamination index for ranking impaired recreational watersusing the HF183 quantitative real-time PCR method

    Science.gov (United States)

    Human fecal pollution of surface water remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for recreational water quality risk managem...

  17. EVALUATION OF A RAPID, QUANTITATIVE REAL-TIME PCR METHOD FOR ENUMERATION OF PATHOGENIC CANDIDA CELLS IN WATER

    Science.gov (United States)

    Quantitative Real-Time PCR (QRT-PCR) technology, incorporating fluorigenic 5' nuclease (TaqMan?) chemistry, was developed for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C....

  18. Quantitation of RHD by real-time polymerase chain reaction for determination of RHD zygosity and RHD mosaicism/chimerism

    DEFF Research Database (Denmark)

    Krog, Grethe Risum; Clausen, Frederik Banch; Dziegiel, Morten Hanefeld

    2007-01-01

    Determination of RHD zygosity of the spouse is crucial in preconception counseling of families with history of hemolytic disease of the fetus and newborn caused by anti-D. RHD zygosity can be determined by quantitative real-time polymerase chain reaction (PCR) basically by determining RHD dosage...

  19. Decay Of Bacterial Pathogens, Fecal Indicators, And Real-Time Quantitative PCR Genetic Markers In Manure-Amended Soils

    Science.gov (United States)

    This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria (FIB), and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manure-amended agricultural soils. Known concentrations of transformed green...

  20. Decay Of Bacterial Pathogen, Fecal Indicators, And Real-Time Quantitative PCR Genetic Markers In Manure Amended Soils

    Science.gov (United States)

    This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria, and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manre-amended agricultural soils. Known concentrations of transformed green fluore...

  1. Quantitative detection of the free-living amoeba Hartmannella vermiformis in surface water by using real-time PCR

    NARCIS (Netherlands)

    Kuiper, M.W.; Valster, R.M.; Wullings, B.A.; Boonstra, H.; Smidt, H.; Kooij, van der D.

    2006-01-01

    A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic

  2. Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples

    Science.gov (United States)

    Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster are considered to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. In response, the United States Environmental Protectio...

  3. A Real-Time Wireless Sweat Rate Measurement System for Physical Activity Monitoring.

    Science.gov (United States)

    Brueck, Andrew; Iftekhar, Tashfin; Stannard, Alicja B; Yelamarthi, Kumar; Kaya, Tolga

    2018-02-10

    There has been significant research on the physiology of sweat in the past decade, with one of the main interests being the development of a real-time hydration monitor that utilizes sweat. The contents of sweat have been known for decades; sweat provides significant information on the physiological condition of the human body. However, it is important to know the sweat rate as well, as sweat rate alters the concentration of the sweat constituents, and ultimately affects the accuracy of hydration detection. Towards this goal, a calorimetric based flow-rate detection system was built and tested to determine sweat rate in real time. The proposed sweat rate monitoring system has been validated through both controlled lab experiments (syringe pump) and human trials. An Internet of Things (IoT) platform was embedded, with the sensor using a Simblee board and Raspberry Pi. The overall prototype is capable of sending sweat rate information in real time to either a smartphone or directly to the cloud. Based on a proven theoretical concept, our overall system implementation features a pioneer device that can truly measure the rate of sweat in real time, which was tested and validated on human subjects. Our realization of the real-time sweat rate watch is capable of detecting sweat rates as low as 0.15 µL/min/cm², with an average error in accuracy of 18% compared to manual sweat rate readings.

  4. Improved process control through real-time measurement of mineral content

    Energy Technology Data Exchange (ETDEWEB)

    Turler, Daniel; Karaca, Murat; Davis, William B.; Giauque, Robert D.; Hopkins, Deborah

    2001-11-02

    In a highly collaborative research and development project with mining and university partners, sensors and data-analysis tools are being developed for rock-mass characterization and real-time measurement of mineral content. Determining mineralogy prior to mucking in an open-pit mine is important for routing the material to the appropriate processing stream. A possible alternative to lab assay of dust and cuttings obtained from drill holes is continuous on-line sampling and real-time x-ray fluorescence (XRF) spectroscopy. Results presented demonstrate that statistical analyses combined with XRF data can be employed to identify minerals and, possibly, different rock types. The objective is to create a detailed three-dimensional mineralogical map in real time that would improve downstream process efficiency.

  5. Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications

    OpenAIRE

    Rutledge, R. G.

    2004-01-01

    Quantitative real-time PCR has revolutionized many aspects of genetic research, biomedical diagnostics and pathogen detection. Nevertheless, the full potential of this technology has yet to be realized, primarily due to the limitations of the threshold-based methodologies that are currently used for quantitative analysis. Prone to errors caused by variations in reaction preparation and amplification conditions, these approaches necessitate construction of standard curves for each target seque...

  6. Ruggedized downhole tool for real-time measurements and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Hess, Ryan Falcone; Lindblom, Scott C.; Yelton, William G.; Limmer, Steven J.; Boyle, Timothy J.; Cieslewski, Grzegorz

    2018-01-09

    The present invention relates to ruggedized downhole tools and sensors, as well as uses thereof. In particular, these tools can operate under extreme conditions and, therefore, allow for real-time measurements in geothermal reservoirs or other potentially harsh environments. One exemplary sensor includes a ruggedized ion selective electrode (ISE) for detecting tracer concentrations in real-time. In one embodiment, the ISE includes a solid, non-conductive potting material and an ion selective material, which are disposed in a temperature-resistant electrode body. Other electrode configurations, tools, and methods are also described.

  7. On-Orbit Quantitative Real-Time Gene Expression Analysis Using the Wetlab-2 System

    Science.gov (United States)

    Parra, Macarena; Jung, Jimmy; Almeida, Eduardo; Boone, Travis; Tran, Luan; Schonfeld, Julie

    2015-01-01

    NASA Ames Research Center's WetLab-2 Project enables on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The resulting RNA is dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The project selected the Cepheid SmartCycler (TradeMark), a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, because of its advantages including rugged modular design, low power consumption, rapid thermal ramp times and four-color multiplex detection. Single tube multiplex assays can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to conduct qRT-PCR and generate results on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also eliminate the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples and provide on-orbit gene expression benchmarking prior to sample return. Finally, the system can also be used for analysis of

  8. Site selective real-time measurements of atmospheric N2O isotopomers by laser spectroscopy

    Directory of Open Access Journals (Sweden)

    W. A. Brand

    2012-07-01

    Full Text Available We describe the first high precision real-time analysis of the N2O site-specific isotopic composition at ambient mixing ratios. Our technique is based on mid-infrared quantum cascade laser absorption spectroscopy (QCLAS combined with an automated preconcentration unit. The QCLAS allows for simultaneous and specific analysis of the three main stable N2O isotopic species, 14N15N16O, 15N14N16O, 14N14N16O, and the respective site-specific relative isotope ratio differences δ15Nα and δ 15Nβ. Continuous, stand-alone operation is achieved by using liquid nitrogen free N2O preconcentration, a quasi-room-temperature quantum cascade laser (QCL, quantitative sample transfer to the QCLAS and an optimized calibration algorithm. The N2O site-specific isotopic composition (δ15Nα and δ15Nβ can be analysed with a long-term precision of 0.2‰. The potential of this analytical tool is illustrated by continuous N2O isotopomer measurements above a grassland plot over a three week period, which allowed identification of microbial source and sink processes.

  9. Using Biometric Measurement in Real-Time as a Sympathetic System in Computer Games

    Science.gov (United States)

    Charij, Stephanie; Oikonomou, Andreas

    2013-01-01

    With the increasing potential for gaming hardware and peripherals to support biometrics, their application within the games industry for software and design should be considered. This paper assesses the ability to use a form of biometric measurement, heart rate, in real-time to improve the challenge and enjoyment of a game by catering it to…

  10. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data

    NARCIS (Netherlands)

    Ramakers, Christian; Ruijter, Jan M.; Deprez, Ronald H. Lekanne; Moorman, Antoon F. M.

    2003-01-01

    Quantification of mRNAs using real-time polymerase chain reaction (PCR) by monitoring the product formation with the fluorescent dye SYBR Green I is being extensively used in neurosciences, developmental biology, and medical diagnostics. Most PCR data analysis procedures assume that the PCR

  11. Estimating marginal properties of quantitative real-time PCR data using nonlinear mixed models

    DEFF Research Database (Denmark)

    Gerhard, Daniel; Bremer, Melanie; Ritz, Christian

    2014-01-01

    A unified modeling framework based on a set of nonlinear mixed models is proposed for flexible modeling of gene expression in real-time PCR experiments. Focus is on estimating the marginal or population-based derived parameters: cycle thresholds and ΔΔc(t), but retaining the conditional mixed mod...

  12. Development of a real-time quantitative assay for detection of Epstein-Barr virus

    NARCIS (Netherlands)

    H.G.M. Niesters (Bert); E. Fries; K.C. Wolthers (Katja); A.D.M.E. Osterhaus (Albert); J.J. Cornelissen (Jan); J.W.J. van Esser (Joost)

    2000-01-01

    textabstractWith the use of real-time PCR, we developed and evaluated a rapid, sensitive, specific, and reproducible method for the detection of Epstein-Barr virus (EBV) DNA in plasma samples. This method allowed us to screen plasma and serum samples over a range between 100 and

  13. Avian influenza virus detection and quantitation by real-time RT-PCR

    Science.gov (United States)

    Real-time RT-PCR (rRT-PCR) has been used for avian influenza virus (AIV) detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of rRT-PCR are: high sensitivity, high specificity, rapid time-to-result, scalability, cost, and its inherentl...

  14. A Quantitative Real-Time PCR Approach for Assessing Campylobacter jejuni and Campylobacter coli Colonization in Broiler Herds.

    Science.gov (United States)

    Haas, Katrin; Overesch, Gudrun; Kuhnert, Peter

    2017-04-01

    Human campylobacteriosis is a major public health concern in developed countries, with Campylobacter jejuni and Campylobacter coli from poultry recognized as the main source of human infection. Identification of Campylobacter-positive broiler herds before slaughter is essential for implementing measures to avoid carryover of pathogens via the slaughter process into the food chain. However, appropriate methods that have been validated for testing poultry flocks antemortem are lacking for Campylobacter. A quantitative real-time PCR (qPCR) that allows simultaneous detection and quantification of C. jejuni and C. coli was adapted and optimized to be applied on boot socks. The adjusted qPCR serves as an easy, sensitive, and quantitative method for Campylobacter detection in poultry flocks antemortem by analysis of boot socks. An adequate correlation was found between qPCR and culture, as well as between boot socks and cecal samples, which are regarded as the "gold standard." Therefore, boot sock sampling followed by qPCR analysis provides a reliable and simple method for assessing Campylobacter load within a flock prior to slaughter. The approach allows categorization of broiler herds into negative, low, moderate, or high Campylobacter colonization. Based on the results of this new approach, risk assessment models, such as evaluating the possible effect of sorting flocks before slaughter, can be easily implemented. Similarly, targeted identification of highly colonized flocks for improvement of biosecurity measures at the farm level will become feasible, presenting an opportunity to increase food safety.

  15. Real-time 3D measurement based on structured light illumination considering camera lens distortion

    Science.gov (United States)

    Feng, Shijie; Chen, Qian; Zuo, Chao; Sun, Jiasong; Yu, ShiLing

    2014-12-01

    Optical three-dimensional (3-D) profilometry is gaining increasing attention for its simplicity, flexibility, high accuracy, and non-contact nature. Recent advances in imaging sensors and digital projection technology further its progress in high-speed, real-time applications, enabling 3-D shapes reconstruction of moving objects and dynamic scenes. In traditional 3-D measurement system where the processing time is not a key factor, camera lens distortion correction is performed directly. However, for the time-critical high-speed applications, the time-consuming correction algorithm is inappropriate to be performed directly during the real-time process. To cope with this issue, here we present a novel high-speed real-time 3-D coordinates measuring technique based on fringe projection with the consideration of the camera lens distortion. A pixel mapping relation between a distorted image and a corrected one is pre-determined and stored in computer memory for real-time fringe correction. And a method of lookup table (LUT) is introduced as well for fast data processing. Our experimental results reveal that the measurement error of the in-plane coordinates has been reduced by one order of magnitude and the accuracy of the out-plane coordinate been tripled after the distortions being eliminated. Moreover, owing to the merit of the LUT, the 3-D reconstruction can be achieved at 92.34 frames per second.

  16. Real time observation and automated measurement of red blood cells agglutination inside a passive microfluidic biochip containing embedded reagents.

    Science.gov (United States)

    Huet, Maxime; Cubizolles, Myriam; Buhot, Arnaud

    2017-07-15

    The process of agglutination is commonly used for the detection of biomarkers like proteins or viruses. The multiple bindings between micrometer sized particles, either latex beads or red blood cells (RBCs), create aggregates that are easily detectable and give qualitative information about the presence of the biomarkers. In most cases, the detection is made by simple naked-eye observation of agglutinates without any access to the kinetics of agglutination. In this study, we address the development of a real-time time observation of RBCs agglutination. Using ABO blood typing as a proof-of-concept, we developed i) an integrated biological protocol suitable for further use as point-of-care (POC) analysis and ii) two dedicated image processing algorithms for the real-time and quantitative measurement of agglutination. Anti-A or anti-B typing reagents were dried inside the microchannel of a passive microfluidic chip designed to enhance capillary flow. A blood drop deposit at the tip of the biochip established a simple biological protocol. In situ agglutination of autologous RBCs was achieved by means of embedded reagents and real time agglutination process was monitored by video recording. Using a training set of 24 experiments, two real-time indicators based on correlation and variance of gray levels were optimized and then further confirmed on a validation set. 100% correct discrimination between positive and negative agglutinations was performed within less than 2min by measuring real-time evolution of both correlation and variance indicators. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. A Bayesian method for calculating real-time quantitative PCR calibration curves using absolute plasmid DNA standards

    Directory of Open Access Journals (Sweden)

    Haugland Richard A

    2008-02-01

    Full Text Available Abstract Background In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignored in calibration calculations and in some cases impossible to characterize. A flexible statistical method that can account for uncertainty between plasmid and genomic DNA targets, replicate testing, and experiment-to-experiment variability is needed to estimate calibration curve parameters such as intercept and slope. Here we report the use of a Bayesian approach to generate calibration curves for the enumeration of target DNA from genomic DNA samples using absolute plasmid DNA standards. Results Instead of the two traditional methods (classical and inverse, a Monte Carlo Markov Chain (MCMC estimation was used to generate single, master, and modified calibration curves. The mean and the percentiles of the posterior distribution were used as point and interval estimates of unknown parameters such as intercepts, slopes and DNA concentrations. The software WinBUGS was used to perform all simulations and to generate the posterior distributions of all the unknown parameters of interest. Conclusion The Bayesian approach defined in this study allowed for the estimation of DNA concentrations from environmental samples using absolute standard curves generated by real-time qPCR. The approach accounted for uncertainty from multiple sources such as experiment-to-experiment variation, variability between replicate measurements, as well as uncertainty introduced when employing calibration curves generated from absolute plasmid DNA standards.

  18. Using Quantitative Real-Time PCR to Detect MicroRNA Expression Profile During Embryonic Stem Cell Differentiation.

    Science.gov (United States)

    Pan, Xiaoping; Murashov, Alexander K; Stellwag, Edmund J; Zhang, Baohong

    2017-01-01

    Quantitative real-time PCR (qRT-PCR) is a reliable method to determine and monitor microRNA (miRNA) expression profiles in different cells, tissues, and organisms. Although there are several different strategies in performing qRT-PCR to determine miRNA expression, all of them have two steps in common: reverse transcription for obtaining cDNA from mature miRNA sequencing and standard real-time PCR for amplification of cDNA. This chapter demonstrates the application of quantitative real-time PCR for determining miRNA expression profiles during mouse embryonic stem cell differentiation. In this method, a mature miRNA sequence is first reverse transcribed into a long cDNA with a 40-50 nt miRNA-specific stem-loop primer; then, a standard real-time PCR reaction is performed for determining miRNA expression using a forward miRNA-specific primer and a universal reverse primer.

  19. System-specific periodicity in quantitative real-time polymerase chain reaction data questions threshold-based quantitation.

    Science.gov (United States)

    Spiess, Andrej-Nikolai; Rödiger, Stefan; Burdukiewicz, Michał; Volksdorf, Thomas; Tellinghuisen, Joel

    2016-12-13

    Real-time quantitative polymerase chain reaction (qPCR) data are found to display periodic patterns in the fluorescence intensity as a function of sample number for fixed cycle number. This behavior is seen for technical replicate datasets recorded on several different commercial instruments; it occurs in the baseline region and typically increases with increasing cycle number in the growth and plateau regions. Autocorrelation analysis reveals periodicities of 12 for 96-well systems and 24 for a 384-well system, indicating a correlation with block architecture. Passive dye experiments show that the effect may be from optical detector bias. Importantly, the signal periodicity manifests as periodicity in quantification cycle (Cq) values when these are estimated by the widely applied fixed threshold approach, but not when scale-insensitive markers like first- and second-derivative maxima are used. Accordingly, any scale variability in the growth curves will lead to bias in constant-threshold-based Cqs, making it mandatory that workers should either use scale-insensitive Cqs or normalize their growth curves to constant amplitude before applying the constant threshold method.

  20. Field instruments for real time in-situ crude oil concentration measurements

    Energy Technology Data Exchange (ETDEWEB)

    Fuller, C.B.; Bonner, J.S.; Page, C.A.; Arrambide, G. [Texas A and M Univ., Corpus Christi, TX (United States). Conrad Blucher Inst. for Surveying and Science; Sterling, M.C.Jr.; Ojo, T.O. [Texas A and M Univ., College Station, TX (United States). Dept. of Civil Engineering

    2003-07-01

    Accidental oil spills, contaminant release during resuspension, storms, and harmful algal blooms are all episodic events that can effect coastal margins. It is important to quantitatively describe water and ecological quality evolution and predict the impact to these areas by such events, but traditional sampling methods miss environmental activity during cyclical events. This paper presents a new sampling approach that involves continuous, real-time in-situ monitoring to provide data for development of comprehensive modeling protocols. It gives spill response coordinators greater assurance in making decisions using the latest visualization tools which are based on a good understanding of the physical processes at work in pulsed events. Five sensors for rapid monitoring of crude oil concentrations in aquatic systems were described. The in-situ and ex-situ sensors can measure plume transport and estimate polycyclic aromatic hydrocarbon exposure concentrations to assess risk of toxicity. A brief description and evaluation of the following 5 sensors was provided: the LISST-100 by Sequoia Instrument, a submersible multi-angle laser scattering instrument; the AU-10 field fluorometer by Turner Designs, an ex-situ single wavelength fluorometer; the Flashlamp by WET Labs Inc., an in-situ single wavelength fluorometer; and, the ECO-FL3 and SAFire by WET Labs Inc., two in-situ multiple wavelength fluorometers. These instruments were used to analyze crude oil emissions of various concentrations. All of the instruments followed a linear response within the tested concentration range. At the lowest concentrations the LISST-100 was not as effective as the fluorometers because of limited particle volume for scatter. For the AU-10 field fluorometer, the highest concentrations tested were above the measurement range of the instrument. 6 refs., 5 figs.

  1. An inductive sensor for real-time measurement of plantar normal and shear forces distribution.

    Science.gov (United States)

    Du, Li; Zhu, Xiaoliang; Zhe, Jiang

    2015-05-01

    The objective of this paper is to demonstrate a multiplexed inductive force sensor for simultaneously measuring normal force and shear forces on a foot. The sensor measures the normal force and shear forces by monitoring the inductance changes of three planar sensing coils. Resonance frequency division multiplexing was applied to signals from the multiple sensing coils, making it feasible to simultaneously measure the three forces (normal force, shear forces in x- and y-axis) on a foot using only one set of measurement electronics with high sensitivity and resolution. The testing results of the prototype sensor have shown that the sensor is capable of measuring normal force ranging from 0 to 800 N and shear forces ranging from 0 to 130 N in real time. With its high resolution, high sensitivity, and the capability of monitoring forces at different positions of a foot simultaneously, this sensor can be potentially used for real-time measurement of plantar normal force and shear forces distribution on diabetes patient's foot. Real-time monitoring of the normal force and shear forces on diabetes patient's foot can provide useful information for physicians and diabetes patients to take actions in preventing foot ulceration.

  2. Real-time locating and speed measurement of fibre fuse using optical frequency-domain reflectometry.

    Science.gov (United States)

    Jiang, Shoulin; Ma, Lin; Fan, Xinyu; Wang, Bin; He, Zuyuan

    2016-05-05

    We propose and experimentally demonstrate real-time locating and speed measurement of fibre fuse by analysing the Doppler shift of reflected light using optical frequency-domain reflectometry (OFDR). Our method can detect the start of a fibre fuse within 200 ms which is equivalent to a propagation distance of about 10 cm in standard single-mode fibre. We successfully measured instantaneous speed of propagating fibre fuses and observed their subtle fluctuation owing to the laser power instability. The resolution achieved for speed measurement in our demonstration is 1 × 10(-3) m/s. We studied the fibre fuse propagation speed dependence on the launched power in different fibres. Our method is promising for both real time fibre fuse monitoring and future studies on its propagation and termination.

  3. Validation of Reference Genes for Real-Time Quantitative PCR (qPCR Analysis of Avibacterium paragallinarum.

    Directory of Open Access Journals (Sweden)

    Shuxiang Wen

    Full Text Available Real-time quantitative reverse transcription PCR (qRT-PCR offers a robust method for measurement of gene expression levels. Selection of reliable reference gene(s for gene expression study is conducive to reduce variations derived from different amounts of RNA and cDNA, the efficiency of the reverse transcriptase or polymerase enzymes. Until now reference genes identified for other members of the family Pasteurellaceae have not been validated for Avibacterium paragallinarum. The aim of this study was to validate nine reference genes of serovars A, B, and C strains of A. paragallinarum in different growth phase by qRT-PCR. Three of the most widely used statistical algorithms, geNorm, NormFinder and ΔCT method were used to evaluate the expression stability of reference genes. Data analyzed by overall rankings showed that in exponential and stationary phase of serovar A, the most stable reference genes were gyrA and atpD respectively; in exponential and stationary phase of serovar B, the most stable reference genes were atpD and recN respectively; in exponential and stationary phase of serovar C, the most stable reference genes were rpoB and recN respectively. This study provides recommendations for stable endogenous control genes for use in further studies involving measurement of gene expression levels.

  4. Effects of DNA extraction and purification methods on real-time quantitative PCR analysis of Roundup Ready soybean.

    Science.gov (United States)

    Demeke, Tigst; Ratnayaka, Indira; Phan, Anh

    2009-01-01

    The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

  5. Real-time measurements and their effects on state estimation of distribution power system

    DEFF Research Database (Denmark)

    Han, Xue; You, Shi; Thordarson, Fannar

    2013-01-01

    This paper aims at analyzing the potential value of using different real-time metering and measuring instruments applied in the low voltage distribution networks for state-estimation. An algorithm is presented to evaluate different combinations of metering data using a tailored state estimator....... It is followed by a case study based on the proposed algorithm. A real distribution grid feeder with different types of meters installed either in the cabinets or at the customer side is selected for simulation and analysis. Standard load templates are used to initiate the state estimation. The deviations...... between the estimated values (voltage and injected power) and the measurements are applied to evaluate the accuracy of the estimated grid states. Eventually, some suggestions are provided for the distribution grid operators on placing the real-time meters in the distribution grid....

  6. 3D real-time measurement system of seam with laser

    Science.gov (United States)

    Huang, Min-shuang; Huang, Jun-fen

    2014-02-01

    3-D Real-time Measurement System of seam outline based on Moiré Projection is proposed and designed. The system is composed of LD, grating, CCD, video A/D, FPGA, DSP and an output interface. The principle and hardware makeup of high-speed and real-time image processing circuit based on a Digital Signal Processor (DSP) and a Field Programmable Gate Array (FPGA) are introduced. Noise generation mechanism in poor welding field conditions is analyzed when Moiré stripes are projected on a welding workpiece surface. Median filter is adopted to smooth the acquired original laser image of seam, and then measurement results of a 3-D outline image of weld groove are provided.

  7. Rapid assessment of Cascadia tsunamis from real-time PANGA GPS crustal deformation measurements

    Science.gov (United States)

    Melbourne, T. I.; Santillan, M.; Miner, A.; Webb, F.

    2008-12-01

    Cascadia's natural hazards include earthquakes, tsunamis, volcanic eruptions, landslides, and tectonic subsidence along its coasts and inland waterways exacerbated by sea-level rise. The Pacific Northwest Geodetic Array, now comprised of nearly 200 continuous GPS receivers, has been deployed over the last two decades to focus exclusively on mitigating these hazards. In addition, over 150 receivers of the EarthScope Plate Boundary Observatory have also been installed in Cascadia, thus comprising a combined network of over 350 instruments. Of the 200 PANGA stations, nearly 140 are high-rate, real-time telemetered receivers mounted on CWU-built, tectonics-grade monuments. These stations straddle active crustal faults, volcanoes and landslides, they span the megathrust forearc and tsunamigenic regions along the Pacific coast, and they monitor ageing man-made structures such as dams, levees and elevated freeways. All data from this array, currently at over 140 stations, is streamed in real-time into CWU where it is archived and processed with JPL's GIPSY software. In 2005 PANGA received support from NASA, NSF and the USGS to implement real-time processing in support of mitigating Cascadia's natural hazards. We have implemented Trimble Navigation's proprietary RTK software and network monitoring software on all 140 stations, and specific parameter estimation routines on a subset of these stations. Pending available funding, we are also working to implement processing of this data with the RTGipsy software, which produces position time series within a global, not local, reference frame. We are currently writing applications that will facilitate rapid recovery during and after a large seismic event, tsunami, or volcanic eruption. These applications are focused on: - Inverting GPS deformation measurements for earthquake fault location, size, and slip distribution; - Using slip distributions to predict tsunami magnitude and run-up estimates; - Real-time monitoring of

  8. Selection of appropriate reference genes for quantitative real-time PCR in Oxytropis ochrocephala Bunge using transcriptome datasets under abiotic stress treatments

    OpenAIRE

    huihui ezhuang; Yanping eFu; Wei eHe; Lin eWang; Yahui eWei

    2015-01-01

    Background: Oxytropis ochrocephala Bunge, an indigenous locoweed species in China, poses great threats to livestock on grasslands. There is a need for further genetic study in the plants per se, for understanding the basis of its acclimation mechanism in various unfavorable environmental conditions and to implement effective control measures. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method for gene expression analysis. To facil...

  9. Non-Contact Circuit for Real-Time Electric and Magnetic Field Measurements

    Science.gov (United States)

    2015-10-01

    s FFT length. Our noise floor is shaped as expected for D-dot sensor, including visible effects from both 1/f noise and our 1-kHz low- pass filter...referred noise spectral density GOTS Government-off-the-shelf IC integrated circuit IIDGWN independently distributed Gaussian white noise LED light...ARL-TR-7507 ● OCT 2015 US Army Research Laboratory Non-Contact Circuit for Real-Time Electric and Magnetic Field Measurements

  10. Real-Time Measurement of Host Bioenergetics During Mycobacterium Tuberculosis Infection

    Science.gov (United States)

    2015-05-01

    AWARD NUMBER: W81XWH-13-1-0149 TITLE: “Real-Time Measurement of Host Bioenergetics During Mycobacterium Tuberculosis Infection...successfully adapted metabolic flux analysis using a Seahorse XF96 metabolic flux analyzer to study Mycobacterium tuberculosis energy metabolism in an... Mycobacterium tuberculosis function. In: Systems Biology of Tuberculosis . Editors: J McFadden, D Beste and A Kierzek. 2013. Springer, New York, NY. 2

  11. Noncontact ECG recording system with real time capacitance measurement for motion artifact reduction.

    Science.gov (United States)

    Torfs, Tom; Chen, Yun-Hsuan; Kim, Hyejung; Yazicioglu, Refet Firat

    2014-10-01

    A system for noncontact ECG recording is proposed that measures the real time electrode-body capacitance concurrently with the ECG as a reference signal for motion artifact reduction. Simultaneous recordings of these two signals from the human body in the presence of electrode motion artifacts are shown and an adaptive least-mean-squares (LMS) filtering algorithm run on these signals is demonstrated to be able to reduce the severity of certain types of electrode motion artifacts.

  12. Real-time betatron tune measurement in the accelerator ramp at COSY-Jülich

    CERN Document Server

    Dietrich, J

    2000-01-01

    A new real-time method for betatron tune measurements at COSY was developed and tested from the early 1997. A bandlimited broadband noise source was used for beam excitation, the transversal beam position oscillation was bunch-synchronous sampled and digitized with a high resolution ADC. The Fourier transform of the acquired data represents immediately the betatron tune. After the first promising experiments an automatic tunemeter was constructed. The tunemeter is used as routine diagnostic tool since end of 1998.

  13. Real-time three-dimensional counting and shape measurement of RBCs using digital holographic cytometry

    Science.gov (United States)

    Funamizu, Hideki; Sonoda, Kotaro; Goto, Ryoji; Aizu, Yoshihisa

    2017-04-01

    Digital holography is a useful technique for recording and reconstruction of the complex amplitude of an optical field. In this technique, an interference pattern of two waves is detected by an image sensor, and digital holograms are acquired in computer. The wavefront is reconstructed by a numerical calculation. In this study, we present the real-time threedimensional counting and shape measurement of RBCs using flow cytometry with digital holographic microscopy.

  14. Real-Time Dynamic Impact Strain Deformation Measurements of Transparent Poly(urethane urea) Materials

    Science.gov (United States)

    2010-09-01

    strain hardening, which is validated by the real-time 3D strain evolution measurements via digital photogrammetry . These efforts are a part of an... photogrammetry 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT UU 18. NUMBER OF PAGES 24 19a. NAME OF RESPONSIBLE PERSON Jian H...shields or lenses, structure adhesives, foams, composite structures, and films for structural retrofit. Transparent poly(urethane urea) (PUU) materials

  15. Development of a Capacitive Ice Sensor to Measure Ice Growth in Real Time

    OpenAIRE

    Zhi, Xiang; Cho, Hyo Chang; Wang, Bo; Ahn, Cheol Hee; Moon, Hyeong Soon; Go, Jeung Sang

    2015-01-01

    This paper presents the development of the capacitive sensor to measure the growth of ice on a fuel pipe surface in real time. The ice sensor consists of pairs of electrodes to detect the change in capacitance and a thermocouple temperature sensor to examine the ice formation situation. In addition, an environmental chamber was specially designed to control the humidity and temperature to simulate the ice formation conditions. From the humidity, a water film is formed on the ice sensor, which...

  16. Design and Validation of Real-Time PCR: Quantitative Diagnosis of Common Leishmania Species in Iran.

    Science.gov (United States)

    Fekri Soofi Abadi, Maryam; Dabiri, Shahriar; Fotouhi Ardakani, Reza; Fani Malaki, Lina; Amirpoor Rostami, Sahar; Ziasistani, Mahsa; Dabiri, Donya

    2016-07-01

    Design and validation of Real-time PCR on the protected gene region ITS2 to quantify the parasite load in common leishmania (L) species. Probe and primer were designed from the ITS2 region between the rRNA genes with minimum gene variation in three common leishmania species followed by a Real-time PCR using the Taq man probe method in the form of absolute quantification. A series of different concentrations of leishmania were analyzed. After the purified PCR product was successfully placed in a PTG19-T plasmid vector, specialized ITS2 region was cloned in this plasmid. In the last phase, the cloned gene was transferred to the Ecoli.Top10F bacteria. The standard plasmid was provided in 10(7) to 10(1) copies/rxn concentrations. The specification and clinical sensitivity of the data was analyzed using inter and intra scales. The probe and primer were designed using three species, including L. infantum, L. major, and L.tropica. Seven concentrations of purified parasite in culture media showed that the selected region for quantifying the parasite is suitable. Clinical and analytical specificity and sensitivity were both 100%, respectively. The Taq man method for the ITS2 region in leishmania is one the most sensitive diagnostic test for identifying the parasite load and is suggested as a tool for fast identification and quantification of species.

  17. Effect of ionizing radiation on the quantitative detection of Salmonella using real-time PCR

    Science.gov (United States)

    Lim, Sangyong; Jung, Jinwoo; Kim, Minjeong; Ryu, Sangryeol; Kim, Dongho

    2008-09-01

    Food irradiation is an economically viable technology for inactivating foodborne pathogens, but irradiation can mask pathogens in unhygienically prepared food. The aim of this study was to investigate the effect of irradiation treatment on the detection of Salmonella using real-time PCR. Three commercially available kits were tested, of which the InstaGene Matrix procedure was most effective in preparing template DNA from Salmonella exposed to radiation in broth culture. The minimum level of detection by real-time PCR combined with InstaGene Matrix was 3 log units of Salmonella per milliliter. However, when pure cultures of Salmonella were irradiated at 3 and 5 kGy, the cycle threshold ( CT) increased 1-1.5-fold compared to irradiation at 0 and 1 kGy. This indicated that irradiation treatment may result in an underestimation of bacterial counts due to radiation-induced DNA lesions. We also compared CT values in inoculated chicken homogenates before and after irradiation, which in this model caused a 1.3-3.3-fold underestimation of bacterial counts with respect to irradiation dose.

  18. Development of a real-time resistance measurement for Vibrio parahaemolyticus detection by the lecithin-dependent hemolysin gene.

    Directory of Open Access Journals (Sweden)

    Guiming Xiang

    Full Text Available The marine bacterium Vibrio parahaemolyticus (V. parahaemolyticus causes gastroenteritis in humans via the ingestion of raw or undercooked contaminated seafood, and early diagnosis and prompt treatment are important for the prevention of V. parahaemolyticus-related diseases. In this study, a real-time resistance measurement based on loop-mediated isothermal amplification (LAMP, electrochemical ion bonding (Crystal violet and Mg(2+, real-time monitoring, and derivative analysis was developed. V. parahaemolyticus DNA was first amplified by LAMP, and the products (DNA and pyrophosphate represented two types of negative ions that could combine with a positive dye (Crystal violet and positive ions (Mg(2+ to increase the resistance of the reaction liquid. This resistance was measured in real-time using a specially designed resistance electrode, thus permitting the quantitative detection of V. parahaemolyticus. The results were obtained in 1-2 hours, with a minimum bacterial density of 10 CFU.mL(-1 and high levels of accuracy (97%, sensitivity (96.08%, and specificity (97.96% when compared to cultivation methods. Therefore, this simple and rapid method has a potential application in the detection of V. parahaemolyticus on a gene chip or in point-of-care testing.

  19. Real-time measurement of dust in the workplace using video exposure monitoring: Farming to pharmaceuticals

    Science.gov (United States)

    Walsh, P. T.; Forth, A. R.; Clark, R. D. R.; Dowker, K. P.; Thorpe, A.

    2009-02-01

    Real-time, photometric, portable dust monitors have been employed for video exposure monitoring (VEM) to measure and highlight dust levels generated by work activities, illustrate dust control techniques, and demonstrate good practice. Two workplaces, presenting different challenges for measurement, were used to illustrate the capabilities of VEM: (a) poultry farming activities and (b) powder transfer operations in a pharmaceutical company. For the poultry farm work, the real-time monitors were calibrated with respect to the respirable and inhalable dust concentrations using cyclone and IOM reference samplers respectively. Different rankings of exposure for typical activities were found on the small farm studied here compared to previous exposure measurements at larger poultry farms: these were mainly attributed to the different scales of operation. Large variations in the ratios of respirable, inhalable and real-time monitor TWA concentrations of poultry farm dust for various activities were found. This has implications for the calibration of light-scattering dust monitors with respect to inhalable dust concentration. In the pharmaceutical application, the effectiveness of a curtain barrier for dust control when dispensing powder in a downflow booth was rapidly demonstrated.

  20. Real-time vision-based traffic flow measurements and incident detection

    Science.gov (United States)

    Fishbain, Barak; Ideses, Ianir; Mahalel, David; Yaroslavsky, Leonid

    2009-02-01

    Visual surveillance for traffic systems requires short processing time, low processing cost and high reliability. Under those requirements, image processing technologies offer a variety of systems and methods for Intelligence Transportation Systems (ITS) as a platform for traffic Automatic Incident Detection (AID). There exist two classes of AID methods mainly studied: one is based on inductive loops, radars, infrared sonar and microwave detectors and the other is based on video images. The first class of methods suffers from drawbacks in that they are expensive to install and maintain and they are unable to detect slow or stationary vehicles. Video sensors, on the other hand, offer a relatively low installation cost with little traffic disruption during maintenance. Furthermore, they provide wide area monitoring allowing analysis of traffic flows and turning movements, speed measurement, multiple-point vehicle counts, vehicle classification and highway state assessment, based on precise scene motion analysis. This paper suggests the utilization of traffic models for real-time vision-based traffic analysis and automatic incident detection. First, the traffic flow variables, are introduced. Then, it is described how those variables can be measured from traffic video streams in real-time. Having the traffic variables measured, a robust automatic incident detection scheme is suggested. The results presented here, show a great potential for integration of traffic flow models into video based intelligent transportation systems. The system real time performance is achieved by utilizing multi-core technology using standard parallelization algorithms and libraries (OpenMP, IPP).

  1. Fully integrated whole blood testing by real-time absorption measurement on a centrifugal platform.

    Science.gov (United States)

    Steigert, J; Grumann, M; Brenner, T; Riegger, L; Harter, J; Zengerle, R; Ducrée, J

    2006-08-01

    We present a novel microfluidic concept to enable a fast colorimetric alcohol assay from a single droplet of whole blood. The reduced turn-around time of 150 seconds is, on the one hand, achieved by a full process integration including metering, mixing with reagents, and sedimentation of cellular constituents. On the other hand, our novel total internal reflection (TIR) scheme allows to monitor the increase of the absorbance values in real-time. Thus, the saturation values can be predicted accurately based on an extrapolation of real-time measurements acquired during a 100 second initial period of rotation. Additionally, we present a metering structure to define nanolitre sample volumes at a coefficient of variation (CV) below 5%.

  2. Real-Time Stability Margin Measurements for X-38 Robustness Analysis

    Science.gov (United States)

    Bosworth, John T.; Stachowiak, Susan J.

    2005-01-01

    A method has been developed for real-time stability margin measurement calculations. The method relies on a tailored-forced excitation targeted to a specific frequency range. Computation of the frequency response is matched to the specific frequencies contained in the excitation. A recursive Fourier transformation is used to make the method compatible with real-time calculation. The method was incorporated into the X-38 nonlinear simulation and applied to an X-38 robustness test. X-38 stability margins were calculated for different variations in aerodynamic and mass properties over the vehicle flight trajectory. The new method showed results comparable to more traditional stability analysis techniques, and at the same time, this new method provided coverage that is more complete and increased efficiency.

  3. A digital approach for real time high-rate high-resolution radiation measurements

    Energy Technology Data Exchange (ETDEWEB)

    Gerardi, G.; Abbene, L., E-mail: leonardo.abbene@unipa.it

    2014-12-21

    Modern spectrometers are currently developed by using digital pulse processing (DPP) systems, showing several advantages over traditional analog electronics. The aim of this work is to present digital strategies, in a time domain, for the development of real time high-rate high-resolution spectrometers. We propose a digital method, based on the single delay line (SDL) shaping technique, able to perform multi-parameter analysis with high performance even at high photon counting rates. A robust pulse shape and height analysis (PSHA), applied on single isolated time windows of the detector output waveforms, is presented. The potentialities of the proposed strategy are highlighted through both theoretical and experimental approaches. To strengthen our approach, the implementation of the method on a real-time system together with some experimental results are presented. X-ray spectra measurements with a semiconductor detector are performed both at low and high photon counting rates (up to 1.1 Mcps)

  4. Development of a Capacitive Ice Sensor to Measure Ice Growth in Real Time

    Directory of Open Access Journals (Sweden)

    Xiang Zhi

    2015-03-01

    Full Text Available This paper presents the development of the capacitive sensor to measure the growth of ice on a fuel pipe surface in real time. The ice sensor consists of pairs of electrodes to detect the change in capacitance and a thermocouple temperature sensor to examine the ice formation situation. In addition, an environmental chamber was specially designed to control the humidity and temperature to simulate the ice formation conditions. From the humidity, a water film is formed on the ice sensor, which results in an increase in capacitance. Ice nucleation occurs, followed by the rapid formation of frost ice that decreases the capacitance suddenly. The capacitance is saturated. The developed ice sensor explains the ice growth providing information about the icing temperature in real time.

  5. Development of a capacitive ice sensor to measure ice growth in real time.

    Science.gov (United States)

    Zhi, Xiang; Cho, Hyo Chang; Wang, Bo; Ahn, Cheol Hee; Moon, Hyeong Soon; Go, Jeung Sang

    2015-03-19

    This paper presents the development of the capacitive sensor to measure the growth of ice on a fuel pipe surface in real time. The ice sensor consists of pairs of electrodes to detect the change in capacitance and a thermocouple temperature sensor to examine the ice formation situation. In addition, an environmental chamber was specially designed to control the humidity and temperature to simulate the ice formation conditions. From the humidity, a water film is formed on the ice sensor, which results in an increase in capacitance. Ice nucleation occurs, followed by the rapid formation of frost ice that decreases the capacitance suddenly. The capacitance is saturated. The developed ice sensor explains the ice growth providing information about the icing temperature in real time.

  6. Quantitative analysis of herpes virus sequences from normal tissue and fibropapillomas of marine turtles with real-time PCR

    Science.gov (United States)

    Quackenbush, S.L.; Casey, R.N.; Murcek, R.J.; Paul, T.A.; Work, Thierry M.; Limpus, C.J.; Chaves, A.; duToit, L.; Perez, J.V.; Aguirre, A.A.; Spraker, T.R.; Horrocks, J.A.; Vermeer, L.A.; Balazs, G.S.; Casey, J.W.

    2001-01-01

    Quantitative real-time PCR has been used to measure fibropapilloma-associated turtle herpesvirus (FPTHV) pol DNA loads in fibropapillomas, fibromas, and uninvolved tissues of green, loggerhead, and olive ridley turtles from Hawaii, Florida, Costa Rica, Australia, Mexico, and the West Indies. The viral DNA loads from tumors obtained from terminal animals were relatively homogenous (range 2a??20 copies/cell), whereas DNA copy numbers from biopsied tumors and skin of otherwise healthy turtles displayed a wide variation (range 0.001a??170 copies/cell) and may reflect the stage of tumor development. FPTHV DNA loads in tumors were 2.5a??4.5 logs higher than in uninvolved skin from the same animal regardless of geographic location, further implying a role for FPTHV in the etiology of fibropapillomatosis. Although FPTHV pol sequences amplified from tumors are highly related to each other, single signature amino acid substitutions distinguish the Australia/Hawaii, Mexico/Costa Rica, and Florida/Caribbean groups.

  7. Linear Methods for Analysis and Quality Control of Relative Expression Ratios from Quantitative Real-Time Polymerase Chain Reaction Experiments

    Directory of Open Access Journals (Sweden)

    Robert B. Page

    2011-01-01

    Full Text Available Relative expression quantitative real-time polymerase chain reaction (RT-qPCR experiments are a common means of estimating transcript abundances across biological groups and experimental treatments. One of the most frequently used expression measures that results from such experiments is the relative expression ratio (RE, which describes expression in experimental samples (i.e., RNA isolated from organisms, tissues, and/or cells that were exposed to one or more experimental or nonbaseline condition in terms of fold change relative to calibrator samples (i.e., RNA isolated from organisms, tissues, and/or cells that were exposed to a control or baseline condition. Over the past decade, several models of RE have been proposed, and it is now clear that endogenous reference gene stability and amplification efficiency must be assessed in order to ensure that estimates of RE are valid. In this review, we summarize key issues associated with estimating RE from cycle threshold data. In addition, we describe several methods based on linear modeling that enable researchers to estimate model parameters and conduct quality control procedures that assess whether model assumptions have been violated.

  8. Establishment of a Real-Time, Quantitative, and Reproducible Mouse Model of Staphylococcus Osteomyelitis Using Bioluminescence Imaging

    Science.gov (United States)

    Funao, Haruki; Nagai, Shigenori; Sasaki, Aya; Hoshikawa, Tomoyuki; Aizawa, Mamoru; Okada, Yasunori; Chiba, Kazuhiro; Koyasu, Shigeo; Toyama, Yoshiaki; Matsumoto, Morio

    2012-01-01

    Osteomyelitis remains a serious problem in the orthopedic field. There are only a few animal models in which the quantity and distribution of bacteria can be reproducibly traced. Here, we established a real-time quantitative mouse model of osteomyelitis using bioluminescence imaging (BLI) without sacrificing the animals. A bioluminescent strain of Staphylococcus aureus was inoculated into the femurs of mice. The bacterial photon intensity (PI) was then sequentially measured by BLI. Serological and histological analyses of the mice were performed. The mean PI peaked at 3 days, and stable signals were maintained for over 3 months after inoculation. The serum levels of interleukin-6, interleukin-1β, and C-reactive protein were significantly higher in the infected mice than in the control mice on day 7. The serum monocyte chemotactic protein 1 level was also significantly higher in the infected group at 12 h than in the control group. A significantly higher proportion of granulocytes was detected in the peripheral blood of the infected group after day 7. Additionally, both acute and chronic histological manifestations were observed in the infected group. This model is useful for elucidating the pathophysiology of both acute and chronic osteomyelitis and to assess the effects of novel antibiotics or antibacterial implants. PMID:22104103

  9. Quantitative real-time PCR (qPCR) assay for human-dog-cat species identification and nuclear DNA quantification.

    Science.gov (United States)

    Kanthaswamy, S; Premasuthan, A; Ng, J; Satkoski, J; Goyal, V

    2012-03-01

    In the United States, human forensic evidence collected from crime scenes is usually comingled with biomaterial of canine and feline origins. Knowledge of the concentration of nuclear DNA extracted from a crime scene biological sample and the species from which the sample originated is essential for DNA profiling. The ability to accurately detect and quantify target DNA in mixed-species samples is crucial when target DNA may be overwhelmed by non-target DNA. We have designed and evaluated a species-specific (human, dog and cat) nuclear DNA identification assay based on the TaqMan(®) quantitative real-time PCR (qPCR) technology that can simultaneously detect and measure minute quantities of DNA specific to either humans, dogs and/or cats. The fluorogenic triplex assay employs primers and hydrolysis probes that target the human TH01 locus as well as the dog and cat Melanocortin 1 Receptor (MC1R) sequences in a species-specific manner. We also demonstrate that the assay is a highly sensitive, reliable and robust method for identifying and quantifying mixed-species templates of human-dog-cat origin with as little as 0.4 pg of human and cat nuclear DNA, respectively, and 4.0 pg of dog nuclear DNA. Published by Elsevier Ireland Ltd.

  10. Dynamic fiber Bragg grating strain sensor interrogation with real-time measurement

    Science.gov (United States)

    Park, Jinwoo; Kwon, Yong Seok; Ko, Myeong Ock; Jeon, Min Yong

    2017-11-01

    We demonstrate a 1550 nm band resonance Fourier-domain mode-locked (FDML) fiber laser with fiber Bragg grating (FBG) array. Using the FDML fiber laser, we successfully demonstrate real-time monitoring of dynamic FBG strain sensor interrogation for structural health monitoring. The resonance FDML fiber laser consists of six multiplexed FBGs, which are arranged in series with delay fiber lengths. It is operated by driving the fiber Fabry-Perot tunable filter (FFP-TF) with a sinusoidal waveform at a frequency corresponding to the round-trip time of the laser cavity. Each FBG forms a laser cavity independently in the FDML fiber laser because the light travels different length for each FBG. The very closely positioned two FBGs in a pair are operated simultaneously with a frequency in the FDML fiber laser. The spatial positions of the sensing pair can be distinguished from the variation of the applied frequency to the FFP-TF. One of the FBGs in the pair is used as a reference signal and the other one is fixed on the piezoelectric transducer stack to apply the dynamic strain. We successfully achieve real-time measurement of the abrupt change of the frequencies applied to the FBG without any signal processing delay. The real-time monitoring system is displayed simultaneously on the monitor for the variation of the two peaks, the modulation interval of the two peaks, and their fast Fourier transform spectrum. The frequency resolution of the dynamic variation could reach up to 0.5 Hz for 2 s integration time. It depends on the integration time to measure the dynamic variation. We believe that the real-time monitoring system will have a potential application for structural health monitoring.

  11. Quantitative 'real-time' imaging of multi-phase flow in ceramic monoliths.

    Science.gov (United States)

    Sederman, A J; Mantle, M D; Gladden, L F

    2003-01-01

    An extension of the RARE technique has been developed which acquires multiple images from a single radio-frequency excitation. This pulse sequence has been used to image, in real-time, gas flow through stagnant liquid within parallel-channel ceramic monoliths. From these images, gas-phase volume fractions, and distributions of gas bubble length and velocity as a function of gas flow rate (50-300 cm3 min(-1)) and channel size (300 and 400 channels per square inch, cpsi) are obtained directly. Increasing the gas flow rate increased the number of large bubbles and the average bubble velocity. A bimodal distribution in the bubble velocities was observed for flow within the larger channel size (300 cpsi) in contrast to a broad unimodal distribution characterizing two-phase flow within the smaller channel size (400 cpsi).

  12. Application of neural network for real-time measurement of electrical resistivity in cold crucible

    Science.gov (United States)

    Votava, Pavel; Poznyak, Igor

    2017-08-01

    The article describes use of an Induction furnace with cold crucible as a tool for real-time measurement of a melted material electrical resistivity. The measurement is based on an inverse problem solution of a 2D mathematical model, possibly implementable in a microcontroller or a FPGA in a form of a neural network. The 2D mathematical model results has been provided as a training set for the neural network. At the end, the implementation results are discussed together with uncertainty of measurement, which is done by the neural network implementation itself.

  13. Quantitative Detection of Clostridium perfringens in Broiler Chickens by Real-Time PCR Targeting the Alpha-Toxin Gene

    DEFF Research Database (Denmark)

    Abildgaard, Lone; Engberg, Ricarda M.; Schramm, Andreas

    2006-01-01

    QUANTITATIVE DETECTION OF CLOSTRIDIUM PERFRINGENS IN BROILER CHICKENS BY REAL-TIME PCR TARGETING THE ALPHA-TOXIN GENE L. Abildgaard 1, R.M. Engberg 1, A. Schramm 2, O. Højberg 1 1 Danish Institute of Agricultural Sciences, Department of Animal Health, Welfare and Nutrition, Tjele, Denmark; 2...... University of Aarhus, Institute of Biological Sciences, Department of Microbiology, Aarhus, Denmark Necrotic enteritis is a severe gastrointestinal disease in broiler chickens caused by C. perfringens producing α-toxin (phospholipase C). The incidence of necrotic enteritis in broilers has been reduced...... by antibiotics (ionophores) presently used to prevent parasitic coccidiosis. From 2012 the European Union has banned these anticoccidials as feed additives, wherefore alternatives are needed to suppress C. perfringens and/or α-toxin production. A real-time PCR primer-probe set targeting the α-toxin gene...

  14. Global real-time dose measurements using the Automated Radiation Measurements for Aerospace Safety (ARMAS) system

    Science.gov (United States)

    Tobiska, W. Kent; Bouwer, D.; Smart, D.; Shea, M.; Bailey, J.; Didkovsky, L.; Judge, K.; Garrett, H.; Atwell, W.; Gersey, B.; Wilkins, R.; Rice, D.; Schunk, R.; Bell, D.; Mertens, C.; Xu, X.; Wiltberger, M.; Wiley, S.; Teets, E.; Jones, B.; Hong, S.; Yoon, K.

    2016-11-01

    The Automated Radiation Measurements for Aerospace Safety (ARMAS) program has successfully deployed a fleet of six instruments measuring the ambient radiation environment at commercial aircraft altitudes. ARMAS transmits real-time data to the ground and provides quality, tissue-relevant ambient dose equivalent rates with 5 min latency for dose rates on 213 flights up to 17.3 km (56,700 ft). We show five cases from different aircraft; the source particles are dominated by galactic cosmic rays but include particle fluxes for minor radiation periods and geomagnetically disturbed conditions. The measurements from 2013 to 2016 do not cover a period of time to quantify galactic cosmic rays' dependence on solar cycle variation and their effect on aviation radiation. However, we report on small radiation "clouds" in specific magnetic latitude regions and note that active geomagnetic, variable space weather conditions may sufficiently modify the magnetospheric magnetic field that can enhance the radiation environment, particularly at high altitudes and middle to high latitudes. When there is no significant space weather, high-latitude flights produce a dose rate analogous to a chest X-ray every 12.5 h, every 25 h for midlatitudes, and every 100 h for equatorial latitudes at typical commercial flight altitudes of 37,000 ft ( 11 km). The dose rate doubles every 2 km altitude increase, suggesting a radiation event management strategy for pilots or air traffic control; i.e., where event-driven radiation regions can be identified, they can be treated like volcanic ash clouds to achieve radiation safety goals with slightly lower flight altitudes or more equatorial flight paths.

  15. Shelf Life of Food Products: From Open Labeling to Real-Time Measurements.

    Science.gov (United States)

    Corradini, Maria G

    2018-01-12

    The labels currently used on food and beverage products only provide consumers with a rough guide to their expected shelf lives because they assume that a product only experiences a limited range of predefined handling and storage conditions. These static labels do not take into consideration conditions that might shorten a product's shelf life (such as temperature abuse), which can lead to problems associated with food safety and waste. Advances in shelf-life estimation have the potential to improve the safety, reliability, and sustainability of the food supply. Selection of appropriate kinetic models and data-analysis techniques are essential to predict shelf life, to account for variability in environmental conditions, and to allow real-time monitoring. Novel analytical tools to determine safety and quality attributes in situ coupled with modern tracking technologies and appropriate predictive tools have the potential to provide accurate estimations of the remaining shelf life of a food product in real time. This review summarizes the necessary steps to attain a transition from open labeling to real-time shelf-life measurements. Expected final online publication date for the Annual Review of Food Science and Technology Volume 9 is March 25, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  16. Real-time heart rate measurement for multi-people using compressive tracking

    Science.gov (United States)

    Liu, Lingling; Zhao, Yuejin; Liu, Ming; Kong, Lingqin; Dong, Liquan; Ma, Feilong; Pang, Zongguang; Cai, Zhi; Zhang, Yachu; Hua, Peng; Yuan, Ruifeng

    2017-09-01

    The rise of aging population has created a demand for inexpensive, unobtrusive, automated health care solutions. Image PhotoPlethysmoGraphy(IPPG) aids in the development of these solutions by allowing for the extraction of physiological signals from video data. However, the main deficiencies of the recent IPPG methods are non-automated, non-real-time and susceptible to motion artifacts(MA). In this paper, a real-time heart rate(HR) detection method for multiple subjects simultaneously was proposed and realized using the open computer vision(openCV) library, which consists of getting multiple subjects' facial video automatically through a Webcam, detecting the region of interest (ROI) in the video, reducing the false detection rate by our improved Adaboost algorithm, reducing the MA by our improved compress tracking(CT) algorithm, wavelet noise-suppression algorithm for denoising and multi-threads for higher detection speed. For comparison, HR was measured simultaneously using a medical pulse oximetry device for every subject during all sessions. Experimental results on a data set of 30 subjects show that the max average absolute error of heart rate estimation is less than 8 beats per minute (BPM), and the processing speed of every frame has almost reached real-time: the experiments with video recordings of ten subjects under the condition of the pixel resolution of 600× 800 pixels show that the average HR detection time of 10 subjects was about 17 frames per second (fps).

  17. Development and Test of an Infrastructure Free Real-Time Water Level Measurement System

    Science.gov (United States)

    Breuer, E. R.; Heitsenrether, R.; Hensley, W., III; Krug, W.; Wolcott, D.

    2016-02-01

    NOAA's Center for Operational Oceanographic Products and Services (CO-OPS) is responsible for developing and maintaining the National Water Level Observation Network (NWLON). NWLON consists of over 200 long term observatories that provide near real-time, 6 minute average, water level observations from locations throughout all U.S. coasts. CO-OPS continually analyzes state-of-the-art and emerging technologies to identify potential improvements in data quality and operating efficiency. NOAA, recognizing the changing conditions, anticipates a critical need for real time oceanographic and meteorological observations where traditional approaches are less feasible. CO-OPS is working on the design, development and testing of a real-time tidal measurement system, "The Hermit," for use in coastal regions. The latest prototype has recently completed a successful 3 month field test deployment in the St Andrews Sound region of Georgia, a location where relatively few long term water level records have been collected to date. The test location provided unique challenges such as having a very limited coastal infrastructure and experiencing a 7-8 foot tidal range. The Hermit consists of a bottom mounted pressure/conductivity/temperature sensor (Seabird SBE 26+) and a surface communications buoy which are linked via acoustic modems (Link Quest). The surface buoy relays data back to the CO-OPS database in near-real time using an Iridium satellite based communication system. Additionally, the buoy includes an AirMar all-in-one meteorological sensor. In addition to The Hermit deployment, three test GPS bench marks and a tide staff were installed on a nearby coastline to vertically reference water level measurements. During this deployment, The Hermit successfully provided near real-time measurements of bottom pressure, water conductivity and temperature, wind speed and direction, air temperature, and barometric pressure over the 3 month deployment. During the test period, several

  18. Measuring the wavefront distortion of a phased-array laser radar by using a real-time optoelectronic measurement system

    Science.gov (United States)

    Zheng, Chunyan; Wu, Jian

    2009-11-01

    A real-time optoelectronic measurement system is proposed to measure the wavefront distortions of scanning beams of a phased-array laser radar. This measurement system includes electric control rotating and translating platforms and a cyclic radial shearing interferometer(CRSI). CRSI is an effective interferometry to mesure the laser wavefront. A inversion algorithm is used to precisely reconstruct wavefront phase distribution from interferograms generated by the CRSI. An actual experiment of laser wavefront distortion measurement is implemented successfully. The experimental results show that this optoelectromic measurement system can measure laser wavefront distortion of a phased-array laser radar in accuracy and in real time.

  19. Real-time spatiotemporal measurement of ultrafast fields from multimode optical fibers

    Science.gov (United States)

    Guang, Zhe; Rhodes, Michelle; Zhu, Ping; Trebino, Rick

    2017-02-01

    Ultrashort pulses emerging from multimode optical fibers are spatiotemporally complex—the multiple fiber modes have different spatial shapes and different propagation velocities and dispersions inside fibers. To measure the complete spatiotemporal field from multimode fibers in real time, we propose and demonstrate a technique for the complete measurement of these pulses using a simple pulse characterization technique, called Spatially and Temporally Resolved Intensity and Phase Evaluation Device: Full Information from a Single Hologram (STRIPED FISH). It yields the complete electric field vs. space and time from multiple digital holograms, simultaneously recorded at different frequencies on a single camera frame.

  20. GPU-assisted high-resolution, real-time 3-D shape measurement

    Science.gov (United States)

    Zhang, Song; Royer, Dale; Yau, Shing-Tung

    2006-10-01

    This paper describes a Graphics Processing Unit (GPU)-assisted real-time three-dimensional shape measurement system. Our experiments demonstrated that the absolute coordinates calculation and rendering speed of a GPU is more than four times faster than that of a dual CPU workstation with the same graphics card. By implementing the GPU into our system, we realized simultaneous absolute coordinate acquisition, reconstruction and display at 30 frames per second with a resolution of approximately 266K points per frame. Moreover, a 2+1 phase-shifting algorithm was employed to alleviate the measurement error caused by motion. Applications of the system include medical imaging, manufacturing, entertainment, and security.

  1. Laser 3-D measuring system and real-time visual feedback for teaching and correcting breathing

    Science.gov (United States)

    Povšič, Klemen; Fležar, Matjaž; Možina, Janez; Jezeršek, Matija

    2012-03-01

    We present a novel method for real-time 3-D body-shape measurement during breathing based on the laser multiple-line triangulation principle. The laser projector illuminates the measured surface with a pattern of 33 equally inclined light planes. Simultaneously, the camera records the distorted light pattern from a different viewpoint. The acquired images are transferred to a personal computer, where the 3-D surface reconstruction, shape analysis, and display are performed in real time. The measured surface displacements are displayed with a color palette, which enables visual feedback to the patient while breathing is being taught. The measuring range is approximately 400×600×500 mm in width, height, and depth, respectively, and the accuracy of the calibrated apparatus is +/-0.7 mm. The system was evaluated by means of its capability to distinguish between different breathing patterns. The accuracy of the measured volumes of chest-wall deformation during breathing was verified using standard methods of volume measurements. The results show that the presented 3-D measuring system with visual feedback has great potential as a diagnostic and training assistance tool when monitoring and evaluating the breathing pattern, because it offers a simple and effective method of graphical communication with the patient.

  2. Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR

    Science.gov (United States)

    Bushon, R.N.; Kephart, C.M.; Koltun, G.F.; Francy, D.S.; Schaefer, F. W.; Lindquist, H.D. Alan

    2010-01-01

    Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms. ?? 2010 The Society for Applied Microbiology.

  3. A real-time PCR quantitative detection assay for Pseudomonas savastanoi pv. nerii in Nerium oleander

    Directory of Open Access Journals (Sweden)

    P. Bella

    2009-01-01

    Full Text Available A real-time PCR assay based on TaqMan chemistry was developed for the detection of Pseudomonas savastanoi pathovars that cause bacterial knot disease on different plant species. Primers and probe sequences were based on the iaaL gene coding for (indole-3-acetyl-L-lysine synthetase and previously used in conventional PCR tests. Assay specificity was tested with an extended range of strains of P. savastanoi from eight hosts, with 13 other Pseudomonas spp., and with other microorganisms naturally occurring on or in oleander plants. A pure culture cell suspension was quantifi ed over a seven log concentration range (108 to 102 cfu ml-1 . Different protocols were developed for the detection and quantifi cation of P. savastanoi pv. nerii from symptomatic and asymptomatic oleander plants. A 24-h bacterial enrichment step either on PVF-1 or OKA-M broth improved the sensitivity of the assay, making it suitable to screen planting material for latent infections.

  4. Application of fiber Bragg grating sensors to real-time strain measurement of cryogenic tanks

    Science.gov (United States)

    Takeda, Nobuo; Mizutani, Tadahito; Hayashi, Kentaro; Okabe, Yoji

    2003-08-01

    Although many researches of strain measurement using fiber Bragg grating (FBG) sensors were conducted, there were few applications of FBG sensors to spacecraft in operation. It is very significant to develop an onboard system for the real-time strain measurement during the flight operation. In the present research, the real-time strain measurement of a composite liquid hydrogen (LH2) tank, which consisted of CFRP and aluminum liner, was attempted. Adhesive property of the FBG sensors was investigated first of all. As a result, UV coated FBG sensors and polyurethane adhesive were adopted. Then, reflection spectra from FBG sensors were measured through the tensile test at liquid helium (LHe) temperature. Since the center wavelength shifted in proportion to the applied strain, the FBG sensor was suitable as a precise strain sensor even at LHe temperature. Next, the development of an onboard FBG demodulator was discussed. This onboard demodulator was designed for weight saving to be mounted on a reusable rocket vehicle test (RVT) operated by the Institute of Space and Astronautical Science (ISAS). FBG sensors were bonded on the surface of the composite LH2 tank for the RVT. Then, strain measurement using the onboard demodulator was conducted through the cryogenic pressure test of the tank and compared with the result measured using the optical spectrum analyzer (OSA).

  5. Real-time separation of non-stationary sound fields with pressure and particle acceleration measurements.

    Science.gov (United States)

    Bi, Chuan-Xing; Geng, Lin; Zhang, Xiao-Zheng

    2014-06-01

    To extract the desired non-stationary sound field generated by a target source in the presence of disturbing sources, a real-time sound field separation method with pressure and particle acceleration measurements is proposed. In this method, the pressure and particle acceleration signals at a time instant are first measured on one measurement plane, where the particle acceleration is obtained by the finite difference approximation with the aid of an auxiliary measurement plane; then, the desired pressure signal generated by the target source at the same time instant can be extracted in a timely manner, by a simple superposition of the measured pressure and the convolution between the measured particle acceleration and the derived impulse response function. Thereby, the proposed method possesses a significant feature of real-time separation of non-stationary sound fields, which provides the potential to in situ analyze the radiation characteristics of a non-stationary source. The proposed method was examined through numerical simulation and experiment. Results demonstrated that the proposed method can not only extract the desired time-evolving pressure signal generated by the target source at any space point, but can also obtain the desired spatial distribution of the pressure field generated by the target source at any time instant.

  6. Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

    Directory of Open Access Journals (Sweden)

    Peng Xia

    2009-01-01

    Full Text Available Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf mitochondrial (mtDNA and nuclear (nDNA DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

  7. Real-time quantitative PCR for assessment of antiviral drug effects against Epstein-Barr virus replication and EBV late mRNA expression.

    Science.gov (United States)

    Ballout, Mirvat; Germi, Raphaële; Fafi-Kremer, Samira; Guimet, Josette; Barguès, Gerard; Seigneurin, Jean-Marie; Morand, Patrice

    2007-07-01

    This study assesses the ability of quantitative real-time PCR to measure the effects of virus DNA polymerase inhibitors on EBV DNA and late mRNAs syntheses in EBV-producing cell lines. In-house real-time quantitative PCRs were used to measure EBV DNA (thymidine kinase) and mRNAs (BLLF1 gene/gp350/220, BVRF2 gene/protease) in P3HR-1 and B95-8 cells induced for EBV production by PMA and exposed to ganciclovir, cidofovir and foscarnet. The calculated 50% effective concentrations (EC(50)) for viral DNA replication inhibition in P3HR-1 cells after 7 days of drug exposure were 0.28+/-0.06, 0.29+/-0.01 and 13.6+/-0.17 microg/mL for ganciclovir, cidofovir and foscarnet, respectively. The EC(50) for B95-8 cells were 0.44+/-0.02, 0.70+/-0.06 and 46.8+/-0.5 microg/mL, respectively. The quantitation of the late viral mRNAs showed a decrease of 79-89% in the mRNA amount after 4 days of antiviral treatment. Nevertheless, a substantial amount of mRNA still remained detectable after drug exposure. The real-time PCR is an improvement in the attempt to simplify EBV DNA-quantitation for antiviral assays. The quantitation of late mRNA does not appear as more informative than DNA quantitation for the assessment of the DNA polymerase inhibitor activity, but it may be useful to assess the antiviral activity of drugs acting by another mechanism.

  8. Comparison of real-time PCR quantitative analysis of the cytomegalovirus DNA level using LightCycler 2.0 and LightCycler 480 instruments.

    Science.gov (United States)

    Przybylski, Maciej; Dzieciatkowski, Tomasz; Les, Katarzyna; Mucha, Maja Anna; Wroblewska, Marta; Mlynarczyk, Grazyna

    2012-11-01

    Cytomegalovirus infection is a frequent complication after transplantation. It is commonly believed that the level of CMV viraemia, usually measured with real-time PCR, is directly correlated with the risk of developing a serious cytomegaloviral disease. The aim was the comparison of results obtained with a commercial test for the quantitative diagnostics of CMV infections, using LightCycler 2.0 and LightCycler 480 real-time PCR systems. Study comprised 97 samples of nucleic acids isolated from serum, in which the CMV DNA was detected during routine tests. Measurement in both analysers was performed simultaneously for paired serum samples. Comparison and of the results obtained with two types of thermocyclers included regression analysis, Spearman correlation, Friedman test and Bland-Altman analysis (pmeasurements between instruments (pinstruments. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

  9. Automated extraction and quantitation of oncogenic HPV genotypes from cervical samples by a real-time PCR-based system.

    Science.gov (United States)

    Broccolo, Francesco; Cocuzza, Clementina E

    2008-03-01

    Accurate laboratory assays for the diagnosis of persistent oncogenic HPV infection are being recognized increasingly as essential for clinical management of women with cervical precancerous lesions. HPV viral load has been suggested to be a surrogate marker of persistent infection. Four independent real-time quantitative TaqMan PCR assays were developed for: HPV-16, -31, -18 and/or -45 and -33 and/or -52, -58, -67. The assays had a wide dynamic range of detection and a high degree of accuracy, repeatability and reproducibility. In order to minimize material and hands-on time, automated nucleic acid extraction was performed using a 96-well plate format integrated into a robotic liquid handler workstation. The performance of the TaqMan assays for HPV identification was assessed by comparing results with those obtained by means of PCR using consensus primers (GP5+/GP6+) and sequencing (296 samples) and INNO-LiPA analysis (31 samples). Good agreement was found generally between results obtained by real-time PCR assays and GP(+)-PCR system (kappa statistic=0.91). In conclusion, this study describes four newly developed real-time PCR assays that provide a reliable and high-throughput method for detection of not only HPV DNA but also HPV activity of the most common oncogenic HPV types in cervical specimens.

  10. Development and validation of a real-time quantitative PCR assay to detect Xanthomonas axonopodis pv. allii from onion seed.

    Science.gov (United States)

    Robène, Isabelle; Perret, Marion; Jouen, Emmanuel; Escalon, Aline; Maillot, Marie-Véronique; Chabirand, Aude; Moreau, Aurélie; Laurent, Annie; Chiroleu, Frédéric; Pruvost, Olivier

    2015-07-01

    Bacterial blight of onion is an emerging disease threatening world onion production. The causal agent Xanthomonas axonopodis pv. allii is seed transmitted and a reliable and sensitive tool is needed to monitor seed exchanges. A triplex quantitative real-time PCR assay was developed targeting two X. axonopodis pv. allii-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Amplification of at least one marker indicates the presence of the bacterium in seed extracts. This real-time PCR assay detected all the 79 X. axonopodis pv. allii strains tested and excluded 85.2% of the 135 non-target strains and particularly all 39 saprophytic and pathogenic bacteria associated with onion. Cross-reactions were mainly obtained for strains assigned to nine phylogenetically related X. axonopodis pathovars. The cycle cut-off was estimated statistically at 36.3 considering a risk of false positive of 1%. The limit of detection obtained in at least 95% of the time (LOD 95%) was 5×10(3) CFU/g (colony forming unit/g). The sensitivity threshold was found to be 1 infected seed in 32,790 seeds. This real-time PCR assay should be useful for preventing the long-distance spread of X. axonopodis pv. allii via contaminated seed lots and determining the epidemiology of the bacterium. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose

    DEFF Research Database (Denmark)

    Cruys-Bagger, Nicolaj; Guilin, Ren; Tatsumi, Hirosuke

    2012-01-01

    An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi-crystalline and amorphous......) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH-biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the β-anomer of cello-oligosaccharides and the approach were validated against HPLC....... It is suggested that quantitative, real-time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose...

  12. A highly sensitive quantitative real-time pcr assay for determination of mutant jak2 exon 12 allele burden

    DEFF Research Database (Denmark)

    Kjær, L.; Riley, C.H.; Westman, M.

    2012-01-01

    Mutations in the Janus kinase 2 (JAK2) gene have become an important identifier for the Philadelphia-chromosome negative chronic myeloproliferative neoplasms. In contrast to the JAK2V617F mutation, the large number of JAK2 exon 12 mutations has challenged the development of quantitative assays. We...... present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel...... tool for quantitative monitoring of the mutant allele burden and accordingly also for determining the impact of treatment with interferon-α-2, shown to induce molecular remission in JAK2V617F-positive patients, which may be a future treatment option for JAK2 exon 12-positive patients as well....

  13. Real-time label-free measurement of HIV-1 protease activity by nanopore analysis.

    Science.gov (United States)

    Wang, Liang; Han, Yujing; Zhou, Shuo; Guan, Xiyun

    2014-12-15

    A label-free method for the measurement of the activity of HIV-1 protease is developed by real-time monitoring of the cleavage of a peptide substrate by HIV-1 protease in a nanopore. The method is rapid and sensitive: picomolar concentrations of HIV-1 protease could be detected in ~10 min. Simulated clinical samples are analyzed, and the activity of HIV-1 protease could be accurately detected. Our developed nanopore sensor design strategy should find useful applications in the development of stochastic sensors for other proteases of medical, pharmaceutical, and biological importance. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Real-time PCR assays for the quantitation of rDNA from apricot and other plant species in marzipan.

    Science.gov (United States)

    Haase, Ilka; Brüning, Philipp; Matissek, Reinhard; Fischer, Markus

    2013-04-10

    Marzipan or marzipan raw paste is a typical German sweet which is consumed directly or is used as an ingredient in the bakery industry/confectionery (e.g., in stollen) and as filling for chocolate candies. Almonds (blanched and pealed) and sugar are the only ingredients for marzipan production according to German food guidelines. Especially for the confectionery industry, the use of persipan, which contains apricot or peach kernels instead of almonds, is preferred due to its stronger aroma. In most of the companies, both raw pastes are produced, in most cases on the same production line, running the risk of an unintended cross contamination. Additionally, due to high almond market values, dilutions of marzipan with cheaper seeds may occur. Especially in the case of apricot and almond, the close relationship of both species is a challenge for the analysis. DNA based methods for the qualitative detection of apricot, peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea in marzipan have recently been published. In this study, different quantitation strategies on the basis of real-time PCR have been evaluated and a relative quantitation method with a reference amplification product was shown to give the best results. As the real-time PCR is based on the high copy rDNA-cluster, even contaminations <1% can be reliably quantitated.

  15. Chemisorption of iodine-125 to gold nanoparticles allows for real-time quantitation and potential use in nanomedicine

    Science.gov (United States)

    Walsh, Adrian A.

    2017-04-01

    Gold nanoparticles have been available for many years as a research tool in the life sciences due to their electron density and optical properties. New applications are continually being developed, particularly in nanomedicine. One drawback is the need for an easy, real-time quantitation method for gold nanoparticles so that the effects observed in in vitro cell toxicity assays and cell uptake studies can be interpreted quantitatively in terms of nanoparticle loading. One potential method of quantifying gold nanoparticles in real time is by chemisorption of iodine-125, a gamma emitter, to the nanoparticles. This paper revisits the labelling of gold nanoparticles with iodine-125, first described 30 years ago and never fully exploited since. We explore the chemical properties and usefulness in quantifying bio-functionalised gold nanoparticle binding in a quick and simple manner. The gold particles were labelled specifically and quantitatively simply by mixing the two items. The nature of the labelling is chemisorption and is robust, remaining bound over several weeks in a variety of cell culture media. Chemisorption was confirmed as potassium iodide can remove the label whereas sodium chloride and many other buffers had no effect. Particles precoated in polymers or proteins can be labelled just as efficiently allowing for post-labelling experiments in situ rather than using radioactive gold atoms in the production process. We also demonstrate that interparticle exchange of I-125 between different size particles does not appear to take place confirming the affinity of the binding.

  16. Chemisorption of iodine-125 to gold nanoparticles allows for real-time quantitation and potential use in nanomedicine

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Adrian A, E-mail: a.walsh@nanobiosols.com [Liverpool Science Park, Nano Biosols Ltd (United Kingdom)

    2017-04-15

    Gold nanoparticles have been available for many years as a research tool in the life sciences due to their electron density and optical properties. New applications are continually being developed, particularly in nanomedicine. One drawback is the need for an easy, real-time quantitation method for gold nanoparticles so that the effects observed in in vitro cell toxicity assays and cell uptake studies can be interpreted quantitatively in terms of nanoparticle loading. One potential method of quantifying gold nanoparticles in real time is by chemisorption of iodine-125, a gamma emitter, to the nanoparticles. This paper revisits the labelling of gold nanoparticles with iodine-125, first described 30 years ago and never fully exploited since. We explore the chemical properties and usefulness in quantifying bio-functionalised gold nanoparticle binding in a quick and simple manner. The gold particles were labelled specifically and quantitatively simply by mixing the two items. The nature of the labelling is chemisorption and is robust, remaining bound over several weeks in a variety of cell culture media. Chemisorption was confirmed as potassium iodide can remove the label whereas sodium chloride and many other buffers had no effect. Particles precoated in polymers or proteins can be labelled just as efficiently allowing for post-labelling experiments in situ rather than using radioactive gold atoms in the production process. We also demonstrate that interparticle exchange of I-125 between different size particles does not appear to take place confirming the affinity of the binding.

  17. Real-Time Measurements of Aft Dome Insulation Erosion on Space Shuttle Reusable Solid Rocket Motor

    Science.gov (United States)

    McWhorter, Bruce; Ewing, Mark; Albrechtsen, Kevin; Noble, Todd; Longaker, Matt

    2004-01-01

    Real-time erosion of aft dome internal insulation was measured with internal instrumentation on a static test of a lengthened version of the Space Shuffle Reusable Solid Rocket Motor (RSRM). This effort marks the first time that real-time aft dome insulation erosion (Le., erosion due to the combined effects of thermochemical ablation and mechanical abrasion) was measured in this kind of large motor static test [designated as Engineering Test Motor number 3 (ETM3)I. This paper presents data plots of the erosion depth versus time. The data indicates general erosion versus time behavior that is in contrast to what would be expected from earlier analyses. Engineers have long known that the thermal environment in the aft dome is severe and that the resulting aft dome insulation erosion is significant. Models of aft dome erosion involve a two-step process of computational fluid dynamics (CFD) modeling and material ablation modeling. This modeling effort is complex. The time- dependent effects are difficult to verify with only prefire and postfire insulation measurements. Nozzle vectoring, slag accumulation, and changing boundary conditions will affect the time dependence of aft dome erosion. Further study of this data and continued measurements on future motors will increase our understanding of the aft dome flow and erosion environment.

  18. Real-time measurements of endogenous carbon monoxide production in isolated pig lungs.

    Science.gov (United States)

    Maignan, Maxime; Briot, Raphael; Romanini, Daniel; Gennai, Stephane; Hazane-Puch, Florence; Brouta, Angelique; Debaty, Guillaume; Ventrillard, Irene

    2014-04-01

    Ischemia-reperfusion injuries are a critical determinant of lung transplantation success. The endogenous production of carbon monoxide (CO) is triggered by ischemia-reperfusion injuries. Our aim was, therefore, to assess the feasibility of exhaled CO measurements during the ex vivo evaluation of lungs submitted to ischemia-reperfusion injuries. Five pigs were euthanized and their lungs removed after pneumoplegia. After cold storage (30 min, 4°C), the lungs were connected to an extracorporeal membrane oxygenation circuit, slowly warmed-up, and ventilated. At the end of a 45-min steady state, CO measurements were performed by optical-feedback cavity-enhanced absorption spectroscopy, a specific laser-based technique for noninvasive and real-time low gas concentration measurements. Exhaled CO concentration from isolated lungs reached 0.45±0.19  ppmv and was above CO concentration in ambient air and in medical gas. CO variations peaked during the expiratory phase. Changes in CO concentration in ambient air did not alter CO concentrations in isolated lungs. Exhaled CO level was also found to be uncorrelated to heme oxygenase (HO-1) gene expression. These results confirm the feasibility of accurate and real-time CO measurement in isolated lungs. The presented technology could help establishing the exhaled CO concentration as a biomarker of ischemia-reperfusion injury in ex vivo lung perfusion.

  19. A real-time measurement system for long-life flood monitoring and warning applications.

    Science.gov (United States)

    Marin-Perez, Rafael; García-Pintado, Javier; Gómez, Antonio Skarmeta

    2012-01-01

    A flood warning system incorporates telemetered rainfall and flow/water level data measured at various locations in the catchment area. Real-time accurate data collection is required for this use, and sensor networks improve the system capabilities. However, existing sensor nodes struggle to satisfy the hydrological requirements in terms of autonomy, sensor hardware compatibility, reliability and long-range communication. We describe the design and development of a real-time measurement system for flood monitoring, and its deployment in a flash-flood prone 650 km(2) semiarid watershed in Southern Spain. A developed low-power and long-range communication device, so-called DatalogV1, provides automatic data gathering and reliable transmission. DatalogV1 incorporates self-monitoring for adapting measurement schedules for consumption management and to capture events of interest. Two tests are used to assess the success of the development. The results show an autonomous and robust monitoring system for long-term collection of water level data in many sparse locations during flood events.

  20. A Real-Time Measurement System for Long-Life Flood Monitoring and Warning Applications

    Directory of Open Access Journals (Sweden)

    Antonio Skarmeta Gómez

    2012-03-01

    Full Text Available A flood warning system incorporates telemetered rainfall and flow/water level data measured at various locations in the catchment area. Real-time accurate data collection is required for this use, and sensor networks improve the system capabilities. However, existing sensor nodes struggle to satisfy the hydrological requirements in terms of autonomy, sensor hardware compatibility, reliability and long-range communication. We describe the design and development of a real-time measurement system for flood monitoring, and its deployment in a flash-flood prone 650 km2 semiarid watershed in Southern Spain. A developed low-power and long-range communication device, so-called DatalogV1, provides automatic data gathering and reliable transmission. DatalogV1 incorporates self-monitoring for adapting measurement schedules for consumption management and to capture events of interest. Two tests are used to assess the success of the development. The results show an autonomous and robust monitoring system for long-term collection of water level data inmany sparse locations during flood events.

  1. Preliminary results of real-time in-vitro electronic speckle pattern interferometry (ESPI) measurements in otolaryngology

    Science.gov (United States)

    Conerty, Michelle D.; Castracane, James; Cacace, Anthony T.; Parnes, Steven M.; Gardner, Glendon M.; Miller, Mitchell B.

    1995-05-01

    Electronic Speckle Pattern Interferometry (ESPI) is a nondestructive optical evaluation technique that is capable of determining surface and subsurface integrity through the quantitative evaluation of static or vibratory motion. By utilizing state of the art developments in the areas of lasers, fiber optics and solid state detector technology, this technique has become applicable in medical research and diagnostics. Based on initial support from NIDCD and continued support from InterScience, Inc., we have been developing a range of instruments for improved diagnostic evaluation in otolaryngological applications based on the technique of ESPI. These compact fiber optic instruments are capable of making real time interferometric measurements of the target tissue. Ongoing development of image post- processing software is currently capable of extracting the desired quantitative results from the acquired interferometric images. The goal of the research is to develop a fully automated system in which the image processing and quantification will be performed in hardware in near real-time. Subsurface details of both the tympanic membrane and vocal cord dynamics could speed the diagnosis of otosclerosis, laryngeal tumors, and aid in the evaluation of surgical procedures.

  2. Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods.

    Science.gov (United States)

    Shanks, Orin C; Kelty, Catherine A; Oshiro, Robin; Haugland, Richard A; Madi, Tania; Brooks, Lauren; Field, Katharine G; Sivaganesan, Mano

    2016-05-01

    There is growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data quality across laboratories. Data quality is typically determined through a series of specifications that ensure good experimental practice and the absence of bias in the results due to DNA isolation and amplification interferences. However, there is currently a lack of consensus on how best to evaluate and interpret human fecal source identification qPCR experiments. This is, in part, due to the lack of standardized protocols and information on interlaboratory variability under conditions for data acceptance. The aim of this study is to provide users and reviewers with a complete series of conditions for data acceptance derived from a multiple laboratory data set using standardized procedures. To establish these benchmarks, data from HF183/BacR287 and HumM2 human-associated qPCR methods were generated across 14 laboratories. Each laboratory followed a standardized protocol utilizing the same lot of reference DNA materials, DNA isolation kits, amplification reagents, and test samples to generate comparable data. After removal of outliers, a nested analysis of variance (ANOVA) was used to establish proficiency metrics that include lab-to-lab, replicate testing within a lab, and random error for amplification inhibition and sample processing controls. Other data acceptance measurements included extraneous DNA contamination assessments (no-template and extraction blank controls) and calibration model performance (correlation coefficient, amplification efficiency, and lower limit of quantification). To demonstrate the implementation of the proposed standardized protocols and data acceptance criteria, comparable data from two additional laboratories were reviewed. The data acceptance criteria

  3. Estimation of low quantity genes: a hierarchical model for analyzing censored quantitative real-time PCR data.

    Science.gov (United States)

    Boyer, Tim C; Hanson, Tim; Singer, Randall S

    2013-01-01

    Analysis of gene quantities measured by quantitative real-time PCR (qPCR) can be complicated by observations that are below the limit of quantification (LOQ) of the assay. A hierarchical model estimated using MCMC methods was developed to analyze qPCR data of genes with observations that fall below the LOQ (censored observations). Simulated datasets with moderate to very high levels of censoring were used to assess the performance of the model; model results were compared to approaches that replace censored observations with a value on the log scale approximating zero or with values ranging from one to the LOQ of ten gene copies. The model was also compared to a Tobit regression model. Finally, all approaches for handling censored observations were evaluated with DNA extracted from samples that were spiked with known quantities of the antibiotic resistance gene tetL. For the simulated datasets, the model outperformed substitution of all values from 1-10 under all censoring scenarios in terms of bias, mean square error, and coverage of 95% confidence intervals for regression parameters. The model performed as well or better than substitution of a value approximating zero under two censoring scenarios (approximately 57% and 79% censored values). The model also performed as well or better than Tobit regression in two of three censoring scenarios (approximately 79% and 93% censored values). Under the levels of censoring present in the three scenarios of this study, substitution of any values greater than 0 produced the least accurate results. When applied to data produced from spiked samples, the model produced the lowest mean square error of the three approaches. This model provides a good alternative for analyzing large amounts of left-censored qPCR data when the goal is estimation of population parameters. The flexibility of this approach can accommodate complex study designs such as longitudinal studies.

  4. Estimation of low quantity genes: a hierarchical model for analyzing censored quantitative real-time PCR data.

    Directory of Open Access Journals (Sweden)

    Tim C Boyer

    Full Text Available Analysis of gene quantities measured by quantitative real-time PCR (qPCR can be complicated by observations that are below the limit of quantification (LOQ of the assay. A hierarchical model estimated using MCMC methods was developed to analyze qPCR data of genes with observations that fall below the LOQ (censored observations. Simulated datasets with moderate to very high levels of censoring were used to assess the performance of the model; model results were compared to approaches that replace censored observations with a value on the log scale approximating zero or with values ranging from one to the LOQ of ten gene copies. The model was also compared to a Tobit regression model. Finally, all approaches for handling censored observations were evaluated with DNA extracted from samples that were spiked with known quantities of the antibiotic resistance gene tetL. For the simulated datasets, the model outperformed substitution of all values from 1-10 under all censoring scenarios in terms of bias, mean square error, and coverage of 95% confidence intervals for regression parameters. The model performed as well or better than substitution of a value approximating zero under two censoring scenarios (approximately 57% and 79% censored values. The model also performed as well or better than Tobit regression in two of three censoring scenarios (approximately 79% and 93% censored values. Under the levels of censoring present in the three scenarios of this study, substitution of any values greater than 0 produced the least accurate results. When applied to data produced from spiked samples, the model produced the lowest mean square error of the three approaches. This model provides a good alternative for analyzing large amounts of left-censored qPCR data when the goal is estimation of population parameters. The flexibility of this approach can accommodate complex study designs such as longitudinal studies.

  5. Predictors of mortality in autoimmune disease patients with concurrent cytomegalovirus infections detected by quantitative real-time PCR.

    Science.gov (United States)

    Lee, Kyoung Yong; Yoo, Byung-Woo; Ahn, Sung Soo; Bae, William Han; Lee, Hyukmin; Jung, Seung Min; Lee, Sang-Won; Park, Yong-Beom; Song, Jason Jungsik

    2017-01-01

    Cytomegaloviruses (CMV) can have a significant impact on the prognosis of immunocompromised patients. Unlike in the transplantation and AIDS fields, only a few studies on CMV infections have been published in the field of autoimmunity. In this study, we examined the clinical outcomes of CMV infections in patients with autoimmune diseases at a single tertiary medical institution. A retrospective study was performed to identify the mortality risk factors associated with CMV infections in patients with autoimmune diseases. We reviewed the medical records of patients with autoimmune diseases who were diagnosed with CMV infections using real-time quantitative polymerase chain reaction between December 2005 and March 2016. Clinical and laboratory parameters as well as treatment outcomes were analyzed. Seventy-three CMV infected patients were separated into survivors and non-survivors. Non-survivors had significantly higher median CMV-DNA copy numbers than survivors (95,500 vs 6,700 copies/mL, p = 0.005) and demonstrated significantly more frequent incidents of CMV pneumonitis (69.2 vs 36.2%, p = 0.007). After adjusting for multiple confounding covariates, the log CMV-DNA copies/mL (hazard ratio, 1.48; 95% confidence interval, 1.14-1.92; p = 0.003) and the presence of concurrent infections (hazard ratio, 22.00; 95% confidence interval, 2.75-175.97, p = 0.004) were identified as independent mortality risk factors. Furthermore, patients with high CMV copy numbers (> 60,000 copies/mL) had higher in-hospital mortality than those with low CMV copy numbers (p autoimmune diseases. Therefore, serial measurements of CMV-DNA copy numbers and close observation for signs of other infections are recommended for patients with autoimmune diseases who have concurrent CMV infection.

  6. Monitoring and simulating real-time electric power system operation with phasor measurements

    Energy Technology Data Exchange (ETDEWEB)

    Phadke, A.G. [Virginia Polytechnic Inst. and State Univ., Blacksburg, VA (United States); Thorp, J.S. [American Electric Power Corp. (United States)

    1995-01-01

    In this research project, two important results have been achieved. The concept of generator axis load flow has been developed more fully, and has been tested through simulations on the 39-bus system (with 10 generators). Generator axis load flow is a load flow calculation which views the entire network from a few retained buses such as the internal nodes of the generators. As these nodes can be indirectly monitored in real time through phasor measurements of generator terminal quantities, it becomes possible to track and predict the behavior of the entire network from these few observation points. This is extremely valuable in the task of predicting network instability in real time. The task of instability prediction of a multi-machine power system is one of the most difficult analytical exercises. We investigated two of the most promising approaches: the extended equal area method, and the transient energy function method. Although both of these methods work well in many instances, we have shown that in other cases, the predictions made by the two methods are incorrect. The failure of the methods can be traced to their inability to deal with the behavior of the system after the first turning point of the motor swing curves. Instead of using these methods, we propose the direct integration of the machine swing equations following the start of a disturbance. Coupled with the generator aids load flow developed above, and using the high speed computers available now, we show that for systems of significant size (39 bus system), accurate predictions through direct computation are possible. The report also includes results on computational efficiency of the method of faster-than-real-time integration using machine equations and the generator aids load flow. It is anticipated that this technique will be useful in most practical applications in power system control centers of the future.

  7. Real-time brain activity measurement and signal processing system using highly sensitive MI sensor

    Directory of Open Access Journals (Sweden)

    Kewang Wang

    2017-05-01

    Full Text Available Superconducting Quantum Interference Devices (SQUIDs are the most used sensor to detect the extremely weak magnetic field of brain. However, the sensor heads need to be kept at very low temperature to maintain superconductivity, and that makes the devices large-scale and inconvenient. In order to measure brain activity in normal environment, we had constructed a measurement system based on highly sensitive Magneto-Impedance (MI sensor, and reported the study of measuring Auditory Evoked Field (AEF brain waves. In this study, the system was improved, and the sensor signals can be processed in real-time to monitor brain activity. We use this system to measure the alpha rhythm in the occipital region and the Event-Related Field (ERF P300 in the frontal, the parietal and both the temporal regions.

  8. Real-time brain activity measurement and signal processing system using highly sensitive MI sensor

    Science.gov (United States)

    Wang, Kewang; Cai, Changmei; Yamamoto, Michiharu; Uchiyama, Tsuyoshi

    2017-05-01

    Superconducting Quantum Interference Devices (SQUIDs) are the most used sensor to detect the extremely weak magnetic field of brain. However, the sensor heads need to be kept at very low temperature to maintain superconductivity, and that makes the devices large-scale and inconvenient. In order to measure brain activity in normal environment, we had constructed a measurement system based on highly sensitive Magneto-Impedance (MI) sensor, and reported the study of measuring Auditory Evoked Field (AEF) brain waves. In this study, the system was improved, and the sensor signals can be processed in real-time to monitor brain activity. We use this system to measure the alpha rhythm in the occipital region and the Event-Related Field (ERF) P300 in the frontal, the parietal and both the temporal regions.

  9. Using Indirect Turbulence Measurements for Real-Time Parameter Estimation in Turbulent Air

    Science.gov (United States)

    Martos, Borja; Morelli, Eugene A.

    2012-01-01

    The use of indirect turbulence measurements for real-time estimation of parameters in a linear longitudinal dynamics model in atmospheric turbulence was studied. It is shown that measuring the atmospheric turbulence makes it possible to treat the turbulence as a measured explanatory variable in the parameter estimation problem. Commercial off-the-shelf sensors were researched and evaluated, then compared to air data booms. Sources of colored noise in the explanatory variables resulting from typical turbulence measurement techniques were identified and studied. A major source of colored noise in the explanatory variables was identified as frequency dependent upwash and time delay. The resulting upwash and time delay corrections were analyzed and compared to previous time shift dynamic modeling research. Simulation data as well as flight test data in atmospheric turbulence were used to verify the time delay behavior. Recommendations are given for follow on flight research and instrumentation.

  10. Real-Time Measurement of Material Elastic Properties in a High Gamma Irradiation Environment

    Energy Technology Data Exchange (ETDEWEB)

    Ken Telschow; Rob Schley; Dave Cottle

    2006-05-01

    This paper describes the first noncontact elastic vibration measurements of an object in a high gamma radiation field. Using a laser-coupled resonant ultrasound technique, the vibration modes of an Inconel hollow capped cylinder were measured as the gamma radiation field was increased to 104 Gy/h. This measurement technique allowed shifts in the resonant frequency of the sample’s vibration modes to be tracked over a 170-h period. The vibration mode frequencies changed in a manner consistent with the temperature dependence of the elastic stiffness coefficients of the material. These results demonstrate the efficacy of the laser approach for real-time resonant ultrasound measurements in this severely hostile nuclear environment.

  11. Strain ratio measurement of femoral cartilage by real-time elastosonography: preliminary results

    Energy Technology Data Exchange (ETDEWEB)

    Ipek, Ali; Unal, Ozlem; Kartal, Merve Gulbiz; Arslan, Halil [Yildirim Beyazit University, Department of Radiology, Faculty of Medicine, Ataturk Training and Research Hospital, Ankara (Turkey); Isik, Cetin; Bozkurt, Murat [Yildirim Beyazit University, Department of Orthopedics, Faculty of Medicine, Ataturk Training and Research Hospital, Ankara (Turkey)

    2015-04-01

    The purpose of this study was to evaluate strain ratio measurement of femoral cartilage using real-time elastosonography. Twenty-five patients with femoral cartilage pathology on MRI (study group) were prospectively compared with 25 subjects with normal findings on MRI (control group) using real-time elastosonography. Strain ratio measurements of pathologic and normal cartilage were performed and compared, both within the study group and between the two groups. Elastosonography colour-scale coding showed a colour change from blue to red in pathologic cartilage and only blue colour-coding in normal cartilage. In the study group, the median strain ratio was higher in pathologic cartilage areas compared to normal areas (median, 1.49 [interquartile range, 0.80-2.53] vs. median, 0.01 [interquartile range, 0.01-0.01], p < 0.001, respectively). The median strain ratio of the control group was 0.01 (interquartile range, 0.01-0.01), and there was no significant difference compared to normal areas of the study group. There was, however, a significant difference between the control group cartilage and pathologic cartilage of the study group (p < 0.001). Elastosonography may be an effective, easily accessible, and relatively simple tool to demonstrate pathologic cartilage and to differentiate it from normal cartilage in the absence of advanced imaging facility such as MRI. (orig.)

  12. Real-time reference-based dynamic phase retrieval algorithm for optical measurement.

    Science.gov (United States)

    Wang, Tianyi; Kai, Li; Kemao, Qian

    2017-09-20

    To study dynamic behaviors of a phenomenon, measuring the evolving field of a specimen/material/structure is required. Optical interferometry, as a full-field, non-contact, and highly sensitive optical measurement technique, has been applied, where the evolving field is represented as dynamic phase distribution. A dynamic phase retrieval algorithm, called least-squares with 3 unknowns (LS3U), which estimates the phase change between each two consecutive patterns by a least-squares fitting method and denoises the phase change by a windowed Fourier filtering (WFF) algorithm, has been shown to be a simple yet effective algorithm. However, LS3U is computationally expensive, restricting its potential application in real-time dynamic phase retrieval systems. In this paper, a real-time LS3U algorithm powered by GPU parallel computing is proposed, with which frame rates of up to 64.5 frames per second (fps) and 131.8 fps are achieved on NVIDIA's GTX 680 and GTX 1080 graphics cards, respectively.

  13. Chrominance to dimension: a real-time method for measuring the size of single gold nanoparticles.

    Science.gov (United States)

    Jing, Chao; Gu, Zhen; Ying, Yi-Lun; Li, Da-Wei; Zhang, Lei; Long, Yi-Tao

    2012-05-15

    Noble metal nanoparticles have excellent optical and chemical properties and are widely used in optics, sensors, and biomedicines. The inherent characteristics of metal nanoparticles, particularly their size, play important roles in their applications. The ability to readily measure the size of single nanomaterials on-site is crucial to the rapid development of single-particle sensors. In this study, we developed a facile and real-time method for estimating the diameter of single gold nanoparticles (GNPs) that range from 35 to 110 nm in diameter; this technique uses the chrominance of the GNP's plasmon resonance scattering light that is captured by a dark-field microscope (DFM). The RGB (three primary colors, red, green, and blue) chrominance information from the dark-field image can be directly converted into the diameters of the GNPs using the relationship between the particle size and the scattering light peak wavelength; this conversion was carried out using Matlab program based on an RGB-To-Wavelength (RTW) process. This approach is more convenient, less time-consuming, and enables observation under arbitrary conditions compared to the scanning electron microscopy (SEM) technique. The differences between the diameters of the GNPs that were calculated using this method and those that were measured using SEM were less than 5 nm. The RTW method has also been applied in the monitoring of the refractive index of the media surrounding the GNPs, and their dynamic acting within cells in real-time.

  14. An Alternative to Classical Real-time Magnetic Field Measurements using a Magnet Model

    CERN Document Server

    Caspers, Friedhelm; Lewis, J; Lindroos, M; Salvermoser, T

    1997-01-01

    Longitudinal and transverse beam control in circular accelerators depends critically on a reliable real-time knowledge of the magnetic bending field. Traditionally this is achieved with a long-measurement coil placed in a reference magnet. In the CERN PS Booster, such a measurement generates a 1 Gauss step-size train with an absolute precision of 0.1%. Modern magnet control can be done with a precision of 0.01%. Consequently, a synthesised magnetic field train based on a reliable magnet model could potentially yield a 10 times better result. The PSB will become a part of the injector chain for the future Large Hadron Collider (LHC). Therefore the main power supply of the PSB has been upgraded to full cycle control. This has made it possible to follow the entire magnetic cycle with a refined model, and to synthesise a real-time magnetic field train from a newly developed programmable pulse generator. We will discuss the general design concepts and the first results.

  15. Toward Real-Time Automated Detection of Turns during Gait Using Wearable Inertial Measurement Units

    Directory of Open Access Journals (Sweden)

    Domen Novak

    2014-10-01

    Full Text Available Previous studies have presented algorithms for detection of turns during gait using wearable sensors, but those algorithms were not built for real-time use. This paper therefore investigates the optimal approach for real-time detection of planned turns during gait using wearable inertial measurement units. Several different sensor positions (head, back and legs and three different detection criteria (orientation, angular velocity and both are compared with regard to their ability to correctly detect turn onset. Furthermore, the different sensor positions are compared with regard to their ability to predict the turn direction and amplitude. The evaluation was performed on ten healthy subjects who performed left/right turns at three amplitudes (22, 45 and 90 degrees. Results showed that turn onset can be most accurately detected with sensors on the back and using a combination of orientation and angular velocity. The same setup also gives the best prediction of turn direction and amplitude. Preliminary measurements with a single amputee were also performed and highlighted important differences such as slower turning that need to be taken into account.

  16. A robust adaptive denoising framework for real-time artifact removal in scalp EEG measurements

    Science.gov (United States)

    Kilicarslan, Atilla; Grossman, Robert G.; Contreras-Vidal, Jose Luis

    2016-04-01

    Objective. Non-invasive measurement of human neural activity based on the scalp electroencephalogram (EEG) allows for the development of biomedical devices that interface with the nervous system for scientific, diagnostic, therapeutic, or restorative purposes. However, EEG recordings are often considered as prone to physiological and non-physiological artifacts of different types and frequency characteristics. Among them, ocular artifacts and signal drifts represent major sources of EEG contamination, particularly in real-time closed-loop brain-machine interface (BMI) applications, which require effective handling of these artifacts across sessions and in natural settings. Approach. We extend the usage of a robust adaptive noise cancelling (ANC) scheme ({H}∞ filtering) for removal of eye blinks, eye motions, amplitude drifts and recording biases simultaneously. We also characterize the volume conduction, by estimating the signal propagation levels across all EEG scalp recording areas due to ocular artifact generators. We find that the amplitude and spatial distribution of ocular artifacts vary greatly depending on the electrode location. Therefore, fixed filtering parameters for all recording areas would naturally hinder the true overall performance of an ANC scheme for artifact removal. We treat each electrode as a separate sub-system to be filtered, and without the loss of generality, they are assumed to be uncorrelated and uncoupled. Main results. Our results show over 95-99.9% correlation between the raw and processed signals at non-ocular artifact regions, and depending on the contamination profile, 40-70% correlation when ocular artifacts are dominant. We also compare our results with the offline independent component analysis and artifact subspace reconstruction methods, and show that some local quantities are handled better by our sample-adaptive real-time framework. Decoding performance is also compared with multi-day experimental data from 2 subjects

  17. Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites

    NARCIS (Netherlands)

    van der Meide, Wendy; Guerra, Jorge; Schoone, Gerard; Farenhorst, Marit; Coelho, Leila; Faber, William; Peekel, Inge; Schallig, Henk

    2008-01-01

    DNA or RNA amplification methods for detection of Leishmania parasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays

  18. Embryonation of Ostertagia ostertagi eggs affects the outcome of real-time quantitative PCR

    DEFF Research Database (Denmark)

    Drag, Markus; Höglund, Johan; Nejsum, Peter

    The aims of this study were to assess how the development of Ostertagia ostertagi eggs into first-stage larva (L1) affects the copy numbers of the Internal Transcribed Spacer region 2 (ITS2) of the ribosomal DNA; and based on these results, to suggest optimal storage conditions for faecal samples...... the outcome of qPCR analysis for the quantitative determination of O. ostertagi eggs in cattle faeces. Cold storage at 4°C for up to 3 days or anaerobicvacuum packing at 25°C for up to 336 h will entail no undesirable effects on ITS2 copies....

  19. A Low Cost Automated Weather Station for Real Time Local Measurements

    Directory of Open Access Journals (Sweden)

    M. Abdul-Niby

    2017-06-01

    Full Text Available In this paper, we present an automated weather station for real time and local measurements, based on an embedded system that continuously measures several weather factors such as temperature, humidity, barometric pressure, wind speed, wind direction, and rainfall. This weather station consists of two parts which are located indoor and outdoor and connected together wirelessly. The outdoor weather station measures the current temperature, humidity, barometric pressure, wind speed, wind direction and recent rain amount. The indoor station displays the outdoor reading as well as the temperature and humidity for the room it is located in, on a graphic liquid crystal display. In addition, this weather information can be accessed from any place through an iOS and Android application called Blynk.

  20. Modeling real-time PCR kinetics: Richards reparametrized equation for quantitative estimation of European hake (Merluccius merluccius).

    Science.gov (United States)

    Sánchez, Ana; Vázquez, José A; Quinteiro, Javier; Sotelo, Carmen G

    2013-04-10

    Real-time PCR is the most sensitive method for detection and precise quantification of specific DNA sequences, but it is not usually applied as a quantitative method in seafood. In general, benchmark techniques, mainly cycle threshold (Ct), are the routine method for quantitative estimations, but they are not the most precise approaches for a standard assay. In the present work, amplification data from European hake (Merluccius merluccius) DNA samples were accurately modeled by three sigmoid reparametrized equations, where the lag phase parameter (λc) from the Richards equation with four parameters was demonstrated to be the perfect substitute for Ct for PCR quantification. The concentrations of primers and probes were subsequently optimized by means of that selected kinetic parameter. Finally, the linear correlation among DNA concentration and λc was also confirmed.

  1. Real-time PCR and microscopy: are the two methods measuring the same unit of arbuscular mycorrhizal fungal abundance?

    Science.gov (United States)

    Gamper, Hannes A; Young, J Peter W; Jones, David L; Hodge, Angela

    2008-05-01

    To enable quantification of mycelial abundance in mixed-species environments, eight new TaqMan((R)) real-time PCR assays were developed for five arbuscular mycorrhizal fungal (AMF, Glomeromycota) taxa. The assays targeted genes encoding 18S rRNA or actin, and were tested on DNA from cloned gene fragments, from spores, mycelia, and from root-free soil, and on reverse-transcribed rRNA templates from entire mycelia and from colonized roots. The assays showed high specificity, sensitivity, and reproducibility, enabling reliable quantitation over broad ranges of template molecules. From cultured mycelia, DNA and RNA measures both correlated with spore number rather than extraradical hyphal length, and epifluorescence microscopy identified pronounced heterogeneity in vitality and nuclear distribution in hyphae. Root colonization was also spatially heterogeneous, as shown by a mixing experiment with root fragments of different length. Therefore, although real-time PCR can reproducibly and accurately quantify AMF nucleic acids, these are poorly correlated with visual measures because of spatial heterogeneity.

  2. Development of Upgraded Magnetic Instrumentation for CERN Real-Time Reference Field Measurement Systems

    CERN Document Server

    Buzio, M; Galbraith, P; Golluccio, G; Giloteaux, D; Gilardoni, S; Petrone, C; Walckiers, L

    2010-01-01

    The control of five of the accelerators in the CERN injector chain (PS, PS Booster, SPS, LEIR and AD) is based upon real-time measurements in a reference magnet. These so-called “B-train” systems include a field marker to signal the achievement of a given field value, complemented by one or more pick-up coils to integrate flux changes. Recently, some concerns were raised about long-term reliability and performance improvements, in terms of both resolution and operational flexibility, for these systems. This paper reports the status of three related R&D activities, namely: the development of a novel dynamic NMR field marker for the PS; a campaign aimed at the detailed measurement of the magnetic state of a PS main magnet; and the design of a standardized electronic signal acquisition and conditioning system.

  3. Real-time measurement of sub-PPM concentrations of airborne chemicals in semiconductor manufacturing.

    Science.gov (United States)

    Corn, M; Cohen, R

    1993-01-01

    Real-time mass spectroscopy (ICAMS) can provide hourly or daily estimates of employee exposure. Field calibration of the unit indicated essentially linear response from 0.01 (Cellosolve Acetate) and 0.03 ppm (Diglyme) to 1 ppm in semiconductor cleanrooms. The instrument can be programmed for 4 minute readings on a single compound, or for rotation among several chemicals, each requiring 4 minute dwell times for analysis. In contrast to full shift personal sampling methods to measure exposure, ICAMS offers insights into the occurrence of peak exposures. In addition, in the occupational environment ICAMS results can be integrated to estimate full-shift within a zone exposures. Thus, the ICAMS extends measurement sensitivities below those currently available and offers a viable alternative to personal sampling in the semiconductor industry.

  4. Performance modeling and measurement of real-time multiprocessors with time-shared buses

    Science.gov (United States)

    Woodbury, Michael H.; Shin, Kang G.

    1988-01-01

    A closed queueing network model is constructed to address workload effects on computer performance for a highly reliable unibus multiprocessor used in real-time control. The queueing model consists of multiserver nodes and a nonpreemptive priority queue. Use of this model requires partitioning the workload into task classes. The time average steady-state solution of the queueing model directly produces useful results that are necessary in performance evaluation. The model is experimentally justified with the Fault-Tolerant Multiprocessor (FTMP) located at the NASA AIRLAB. Extensive experiments are performed on FTMP with a synthetic workload generator (SWG) to directly measure performance parameters, such as processor idle time, system bus contention, and task processing times. These measurements determine values for parameters in the queueing model. Experimental and analytic results are then compared.

  5. Quasi-real-time photon pulse duration measurement by analysis of FEL radiation spectra

    Energy Technology Data Exchange (ETDEWEB)

    Engel, Robin, E-mail: robin.engel@uni-oldenburg.de [Deutsches Elektronen-Synchrotron, Notkestrasse 85, D-22603 Hamburg (Germany); Institut für Physik, Carl von Ossietzky Universität Oldenburg, D-26111 Oldenburg (Germany); Institut für Laser und Optik, Hochschule Emden/Leer, University of Applied Sciences, Constantiaplatz 4, D-26723 Emden (Germany); Düsterer, Stefan; Brenner, Günter [Deutsches Elektronen-Synchrotron, Notkestrasse 85, D-22603 Hamburg (Germany); Teubner, Ulrich [Deutsches Elektronen-Synchrotron, Notkestrasse 85, D-22603 Hamburg (Germany); Institut für Physik, Carl von Ossietzky Universität Oldenburg, D-26111 Oldenburg (Germany); Institut für Laser und Optik, Hochschule Emden/Leer, University of Applied Sciences, Constantiaplatz 4, D-26723 Emden (Germany)

    2016-01-01

    Considering the second-order spectral correlation function of SASE-FEL radiation allows a real-time observation of the photon pulse duration during spectra acquisition. For photon diagnostics at free-electron lasers (FELs), the determination of the photon pulse duration is an important challenge and a complex task. This is especially true for SASE FELs with strongly fluctuating pulse parameters. However, most techniques require an extensive experimental setup, data acquisition and evaluation time, limiting the usability in all-day operation. In contrast, the presented work uses an existing approach based on the analysis of statistical properties of measured SASE FEL spectra and implements it as a software tool, integrated in FLASH’s data acquisition system. This allows the calculation of the average pulse durations from a set of measured spectral distributions with only seconds of delay, whenever high-resolution spectra are recorded.

  6. MEASUREMENT ACCURACY OF NON-CONTACT DISCHARGE MEASUREMENT METHOD USING RIVER MONITORING MOVIE AND DEVELOPMENT OF QUASI REAL TIME MEASUREMENT SYSTEM

    Science.gov (United States)

    Fujita, Ichiro; Hara, Hiroki; Yorozuya, Atsuhiro

    Monitoring of river is significant both in flow management and river usage. Although river monitoring systems have been installed in many rivers in Japan, most of the existing system do not record images and utilize them for quantitative measurement of the flow. Already proposed methods for quantitative measurement include image analysis techniques such as large scale particle image velocimetry (LSPIV) or space-time image velocimetry (STIV) proposed by the authors. However, there still resides skepticism about the accuracy of the methods. Therefore, we conducted concurrent measurements with ADCP to verify the performance of the image analysis techniques. At the same time, we established a quasi-real time system of discharge measurement by introducing a novel image collection system with high image compressibility.

  7. Real-Time Quantitative Analysis of Valproic Acid in Exhaled Breath by Low Temperature Plasma Ionization Mass Spectrometry

    Science.gov (United States)

    Gong, Xiaoxia; Shi, Songyue; Gamez, Gerardo

    2017-04-01

    Real-time analysis of exhaled human breath is a rapidly growing field in analytical science and has great potential for rapid and noninvasive clinical diagnosis and drug monitoring. In the present study, an LTP-MS method was developed for real-time, in-vivo and quantitative analysis of γ-valprolactone, a metabolite of valproic acid (VPA), in exhaled breath without any sample pretreatment. In particular, the effect of working conditions and geometry of the LTP source on the ions of interest, protonated molecular ion at m/z 143 and ammonium adduct ion at m/z 160, were systematically characterized. Tandem mass spectrometry (MS/MS) with collision-induced dissociation (CID) was carried out in order to identify γ-valprolactone molecular ions ( m/z 143), and the key fragment ion ( m/z 97) was used for quantitation. In addition, the fragmentation of ammonium adduct ions to protonated molecular ions was performed in-source to improve the signal-to-noise ratio. At optimum conditions, signal reproducibility with an RSD of 8% was achieved. The concentration of γ-valprolactone in exhaled breath was determined for the first time to be 4.83 (±0.32) ng/L by using standard addition method. Also, a calibration curve was obtained with a linear range from 0.7 to 22.5 ng/L, and the limit of detection was 0.18 ng/L for γ-valprolactone in standard gas samples. Our results show that LTP-MS is a powerful analytical platform with high sensitivity for quantitative analysis of volatile organic compounds in human breath, and can have potential applications in pharmacokinetics or for patient monitoring and treatment.

  8. Visualisation of Radioactivity in Real-Time on a Tablet Measured by a Hybrid Pixel Detector

    CERN Document Server

    AUTHOR|(SzGeCERN)749233; Bantel, Michael; Grünhaupt, Ulrich

    This work explores a method to visualise and interact with radioactivity over time and space by means of augmented reality on a screen. A prototype, iPadPix, was built to demonstrate use as an intuitive new tool for educative and training purposes. Measured by a hybrid pixel detector, Timepix, traces of radioactive decays are displayed in real- time on a mobile device. Its detection principle and properties are detailed as well as the calibration of the sensor. An embedded board is used to process and forward the sensor data to a tablet over a wireless network connection. Software was developed to processes and overlay signatures of ionising radiation and particles on a live camera feed. It is described here and published as open source.

  9. Real time chromametry measurement for food quality detection using mobile device

    Science.gov (United States)

    Witjaksono, Gunawan; Mohamad Hussin, Nur Haziqah Farah Binti; Abdelkreem Saeed Rabih, Almur; Alfa, Sagir

    2017-09-01

    Freshness of the food is the main factor in determining the quality and safety of the consumed food and hence consumers satisfaction. Current technologies for food quality determination depend on colour changing labels to indicate the freshness level, which is subjective to human eyes. The goal of this paper is to design and develop chromatic algorithm based on RGB colour reading and correlation with pH values for real time determination of freshness level of shrimp. The results show that the developed algorithm is able to measure, analyse and display the freshness level of food directly on the screen of a mobile app technology. The mobile app is developed on Android platform and is tested in the shrimp freshness range by stating whether it is “fresh, good or spoiled”.

  10. Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction.

    Science.gov (United States)

    Abdeldaim, Guma M K; Strålin, Kristoffer; Kirsebom, Leif A; Olcén, Per; Blomberg, Jonas; Herrmann, Björn

    2009-08-01

    A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 10(4) DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

  11. The quantitative real-time PCR applications in the monitoring of marine harmful algal bloom (HAB) species.

    Science.gov (United States)

    Penna, Antonella; Antonella, Penna; Galluzzi, Luca; Luca, Galluzzi

    2013-10-01

    In the last decade, various molecular methods (e.g., fluorescent hybridization assay, sandwich hybridization assay, automatized biosensor detection, real-time PCR assay) have been developed and implemented for accurate and specific identification and estimation of marine toxic microalgal species. This review focuses on the recent quantitative real-time PCR (qrt-PCR) technology developed for the control and monitoring of the most important taxonomic phytoplankton groups producing biotoxins with relevant negative impact on human health, the marine environment, and related economic activities. The high specificity and sensitivity of the qrt-PCR methods determined by the adequate choice of the genomic target gene, nucleic acid purification protocol, quantification through the standard curve, and type of chemical detection method make them highly efficient and therefore applicable to harmful algal bloom phenomena. Recent development of qrt-PCR-based assays using the target gene of toxins, such as saxitoxin compounds, has allowed more precise quantification of toxigenic species (i.e., Alexandrium catenella) abundance. These studies focus only on toxin-producing species in the marine environment. Therefore, qrt-PCR technology seems to offer the advantages of understanding the ecology of harmful algal bloom species and facilitating the management of their outbreaks.

  12. Comparison of real-time relative workload measurements in rail signallers

    NARCIS (Netherlands)

    van Broekhoven, Rob; Siegel, A.W.; Schraagen, Johannes Martinus Cornelis; Noordzij, Matthijs Leendert; Milius, Birgit; Naumann, Anja

    2016-01-01

    This exploratory field study investigated the weak resilience signals of workload in a rail traffic control room. The goals of this research are to see whether real-time system information of a rail control post can be used to predict workload of a rail signaller in real-time (Siegel & Schraagen,

  13. Comparative Evaluation of Four Real-Time PCR Methods for the Quantitative Detection of Epstein-Barr Virus from Whole Blood Specimens.

    Science.gov (United States)

    Buelow, Daelynn; Sun, Yilun; Tang, Li; Gu, Zhengming; Pounds, Stanley; Hayden, Randall

    2016-07-01

    Monitoring of Epstein-Barr virus (EBV) load in immunocompromised patients has become integral to their care. An increasing number of reagents are available for quantitative detection of EBV; however, there are little published comparative data. Four real-time PCR systems (one using laboratory-developed reagents and three using analyte-specific reagents) were compared with one another for detection of EBV from whole blood. Whole blood specimens seeded with EBV were used to determine quantitative linearity, analytical measurement range, lower limit of detection, and CV for each assay. Retrospective testing of 198 clinical samples was performed in parallel with all methods; results were compared to determine relative quantitative and qualitative performance. All assays showed similar performance. No significant difference was found in limit of detection (3.12-3.49 log10 copies/mL; P = 0.37). A strong qualitative correlation was seen with all assays that used clinical samples (positive detection rates of 89.5%-95.8%). Quantitative correlation of clinical samples across assays was also seen in pairwise regression analysis, with R(2) ranging from 0.83 to 0.95. Normalizing clinical sample results to IU/mL did not alter the quantitative correlation between assays. Quantitative EBV detection by real-time PCR can be performed over a wide linear dynamic range, using three different commercially available reagents and laboratory-developed methods. EBV was detected with comparable sensitivity and quantitative correlation for all assays. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  14. Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR

    Directory of Open Access Journals (Sweden)

    Amin Forootan

    2017-06-01

    Full Text Available Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we present methods to determine the limit of detection (LoD and the limit of quantification (LoQ as applicable to qPCR. These are based on standard statistical methods as recommended by regulatory bodies adapted to qPCR and complemented with a novel approach to estimate the precision of LoD.

  15. Real-time molecular profiling of photochemically induced rat thrombosis in vivo through quantitative Raman analysis of blood

    Science.gov (United States)

    Lin, M. M.; Shen, A. G.; Yao, H. L.; Zhang, Z. Z.; Hu, J. M.

    2014-11-01

    A device of an animal thrombosis model in vivo coupled with a Raman system for near-surface blood vessels is proposed in this letter. The dual-function set up is capable of simultaneously establishing a photochemically induced artificial thrombus model and collecting in vivo Raman data of both arterial and venous blood, and it provides the first observation of rat thrombosis under the physiological conditions from the beginning to the final form. The real-time and quantitative molecular profiling of flowing blood and the spectra of blood cells in the process of thrombosis provides an insight into the occurring mechanism of thrombosis and a promising method for the in vivo screening of new antithrombotic and thrombolytic drugs.

  16. Identification of three internal feeders from Korla fragrant pears by quantitative real-time polymerase chain reaction.

    Science.gov (United States)

    Wang, Fengjun; Feng, Junli; Ye, Sudan; Li, Xin; Huang, Hannian; Hua, Peng

    2017-02-21

    Cydia pomonella, Euzophera pyriella Yang, and Grapholitha molesta are destructive pest species of Korla fragrant pears in Xinjiang. They are also quarantine pests of concern in a number of countries. Identification of these small pest larvae by morphological characters is difficult, and misidentifications will influence appropriate quarantine decisions. Here, a 520 bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was first amplified and sequenced from each species, and a diagnostic region was observed. Subsequently, the species-specific primer and probe sets of quantitative real-time PCR (qPCR) were designed, which amplified a 114-116 bp fragment of COI genes. This method was validated by amplification DNA extracted from single, multiple, and mixed pest samples. Results indicated that this method allows rapid discrimination and reliable identification of larvae, pupae, and adult specimens of all three species, which could help the international export trading of Korla fragrant pears and related products.

  17. Quantitative and real-time effects of carbon quantum dots on single living HeLa cell membrane permeability.

    Science.gov (United States)

    Kong, Weiqian; Liu, Juan; Liu, Ruihua; Li, Hao; Liu, Yang; Huang, Hui; Li, Kunyang; Liu, Jian; Lee, Shuit-Tong; Kang, Zhenhui

    2014-05-21

    The interaction between carbon quantum dots (CQDs) and a single living cell was explored in real time. Here, we provide the quantitative data on the permeability of the HeLa cell membrane in the presence of CQDs with different surface functional groups (CQDs terminated with -OH/-COOH (CQD-OH), -PEG (CQD-PEG), and -NH2 (CQD-NH2)). Although these CQDs have very low toxicity towards HeLa cells, they still increase the cell membrane permeability by 8%, 13%, and 19% for CQD-PEG, CQD-OH, and CQD-NH2, respectively, and this kind of permeability was irreversible. These observations are valuable for promoting the bio-applications of carbon nanostructures in living systems.

  18. Best Practice for Rainfall Measurement, Torrential Flood Monitoring and Real Time Alerting System in Serbia

    Science.gov (United States)

    Stefanovic, Milutin; Milojevic, Mileta; Zlatanovic, Nikola

    2014-05-01

    Serbia occupies 88.000 km2 and its confined zone menaced with torrent flood occupies 50.000km2. Floods on large rivers and torrents are the most frequent natural disasters in Serbia. This is the result of a geographic position and relief of Serbia. Therefore, defense from these natural disasters has been institutionalized since the 19th century. Through its specialized bodies and public companies, the State organized defense from floods on large rivers and protection of international and other main roads. The Topčiderska River is one of a number of rivers in Serbia that is a threat to both urban and rural environments. In this text, general characteristics of this river will be illustrated, as well as the historical natural hazards that have occurred in the part of Belgrade near Topčiderska River. Belgrade is the capital of Serbia, its political, administrative and financial center, which means that there are significant financial capacities and human resources for investments in all sectors, and specially in the water resources sector. Along the Topčiderska catchment there are many industrial, traffic and residential structures that are in danger of floods and flood protection is more difficult with rapid high flows. The goal is to use monitoring on the Topčiderska River basin to set up a modern system for monitoring in real time and forecast of torrential floods. This paper represents a system of remote detection and monitoring of torrential floods and rain measurements in real time on Topciderka river and ready for a quick response.

  19. Influence of PCR reagents on DNA polymerase extension rates measured on real-time PCR instruments.

    Science.gov (United States)

    Montgomery, Jesse L; Wittwer, Carl T

    2014-02-01

    Radioactive DNA polymerase activity methods are cumbersome and do not provide initial extension rates. A simple extension rate assay would enable study of basic assumptions about PCR and define the limits of rapid PCR. A continuous assay that monitors DNA polymerase extension using noncovalent DNA dyes on common real-time PCR instruments was developed. Extension rates were measured in nucleotides per second per molecule of polymerase. To initiate the reaction, a nucleotide analog was heat activated at 95 °C for 5 min, the temperature decreased to 75 °C, and fluorescence monitored until substrate exhaustion in 30-90 min. The assay was linear with time for over 40% of the reaction and for polymerase concentrations over a 100-fold range (1-100 pmol/L). Extension rates decreased continuously with increasing monovalent cation concentrations (lithium, sodium, potassium, cesium, and ammonium). Melting-temperature depressors had variable effects. DMSO increased rates up to 33%, whereas glycerol had little effect. Betaine, formamide, and 1,2-propanediol decreased rates with increasing concentrations. Four common noncovalent DNA dyes inhibited polymerase extension. Heat-activated nucleotide analogs were 92% activated after 5 min, and hot start DNA polymerases were 73%-90% activated after 20 min. Simple DNA extension rate assays can be performed on real-time PCR instruments. Activity is decreased by monovalent cations, DNA dyes, and most melting temperature depressors. Rational inclusion of PCR components on the basis of their effects on polymerase extension is likely to be useful in PCR, particularly rapid-cycle or fast PCR.

  20. Diagnostic accuracy of quantitative real-time PCR assay versus clinical and Gram stain identification of bacterial vaginosis.

    Science.gov (United States)

    Menard, J-P; Mazouni, C; Fenollar, F; Raoult, D; Boubli, L; Bretelle, F

    2010-12-01

    The purpose of this investigation was to determine the diagnostic accuracy of quantitative real-time polymerase chain reaction (PCR) assay in diagnosing bacterial vaginosis versus the standard methods, the Amsel criteria and the Nugent score. The Amsel criteria, the Nugent score, and results from the molecular tool were obtained independently from vaginal samples of 163 pregnant women who reported abnormal vaginal symptoms before 20 weeks gestation. To determine the performance of the molecular tool, we calculated the kappa value, sensitivity, specificity, and positive and negative predictive values. Either or both of the Amsel criteria (≥3 criteria) and the Nugent score (score ≥7) indicated that 25 women (15%) had bacterial vaginosis, and the remaining 138 women did not. DNA levels of Gardnerella vaginalis or Atopobium vaginae exceeded 10(9) copies/mL or 10(8) copies/mL, respectively, in 34 (21%) of the 163 samples. Complete agreement between both reference methods and high concentrations of G. vaginalis and A. vaginae was found in 94.5% of women (154/163 samples, kappa value = 0.81, 95% confidence interval 0.70-0.81). The nine samples with discordant results were categorized as intermediate flora by the Nugent score. The molecular tool predicted bacterial vaginosis with a sensitivity of 100%, a specificity of 93%, a positive predictive value of 73%, and a negative predictive value of 100%. The quantitative real-time PCR assay shows excellent agreement with the results of both reference methods for the diagnosis of bacterial vaginosis.

  1. Successful Validation of RNA Purification and Quantitative Real-Time PCR Analysis of Gene Expression on the International Space Station

    Science.gov (United States)

    Tran, L.; Parra, Macarena P.; Jung, J.; Boone, T.; Schonfeld, Julie; Almeida, Eduardo

    2017-01-01

    The NASA Ames WetLab-2 system was developed to offer new on-orbit gene expression analysis capabilities to ISS researchers and can be used to conduct on-orbit RNA isolation and quantitative real time PCR (RT-qPCR) analysis of gene expression from a wide range of biological samples ranging from microbes to mammalian tissues. On orbit validation included three quantitative PCR (qPCR) runs using an E. coli genomic DNA template pre-loaded at three different concentrations. The flight Ct values for the DNA standards showed no statistically significant differences relative to ground controls although there was increased noise in Ct curves, likely due to microgravity-related bubble retention in the optical windows. RNA was successfully purified from both E. coli and mouse liver samples and successfully generated singleplex, duplex and triplex data although with higher standard deviations than ground controls, also likely due to bubbles. Using volunteer science activities, a potential bubble reduction strategy was tested and resulted in smooth amplification curves and tighter Cts between replicates. The WetLab-2 validation experiment demonstrates a novel molecular biology workbench on ISS which allows scientists to purify and stabilize RNA, and to conduct RT-qPCR analyses on-orbit with rapid results. This novel ability is an important step towards utilizing ISS as a National Laboratory facility with the capability to conduct and adjust science experiments in real time without sample return, and opens new possibilities for rapid medical diagnostics and biological environmental monitoring on ISS.

  2. Quantitative Detection of Methanotrophs in Soil by Novel pmoA-Targeted Real-Time PCR Assays

    Science.gov (United States)

    Kolb, Steffen; Knief, Claudia; Stubner, Stephan; Conrad, Ralf

    2003-01-01

    Methane oxidation in soils is mostly accomplished by methanotrophic bacteria. Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome. Comparison of 16S ribosomal DNA and pmoA (α subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the α and γ subclasses of Proteobacteria: the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene. Detection limits were between 101 and 102 target molecules per reaction for all assays. Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus, Methylomicrobium album, and Methylosinus trichosporium recovered almost all the added bacteria. Only the Methylosinus-specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs. Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 ± 1.4) × 106 pmoA molecules g−1 for all methanotrophs. The Methylosinus group was predominant (2.7 × 106 ± 1.1 × 106 target molecules g−1). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 × 106 ± 0.9 × 106 target molecules g of soil−1). On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 × 104 target molecules g of soil−1. Our results showed that pmoA-targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil. This approach will thus be useful for quantitative analysis of the community structure of

  3. Development of a Rapid Beam Emittance Measurement System using a Real-Time Beam Profile Monitor

    Science.gov (United States)

    Kamakura, Keita; Hatanaka, Kichiji; Fukuda, Mitsuhiro; Yorita, Tetsuhiko; Ueda, Hiroshi; Saito, Takane; Morinobu, Shunpei; Nagayama, Keiichi; Tamura, Hitoshi; Yasuda, Yuusuke

    2016-06-01

    We have developed a rapid beam emittance measurement system for the injection beam of the K140 azimuthally varying field (AVF) cyclotron at Research Center for Nuclear Physics (RCNP). So far, a conventional emittance monitor has been used in a section of a medium energy beam transport (MEBT) system to evaluate the quality of the injected beam to the K400 ring cyclotron. Two kinds of emittance monitors were supplemented in the low energy beam line for evaluation of ion beams from ion sources. One of them is a conventional type consisting of two sets of position-variable slits and a three-wire profile monitor (TPM), similar to the one installed in the MEBT system of the AVF cyclotron. It takes about 30 min to get emittances in both the horizontal and vertical planes. For quick emittance measurements, we have developed a new system equipped with a set of fast moving slits with a fixed gap and a real-time beam profile monitor (BPM83) with a rotating helical wire. With this system the measurement time was considerably reduced to 70 s for both the horizontal and vertical emittances. Moreover the data analysis and graphical processing were completely automated. The overall measurement and analysis time was successfully minimized within 75 s. This rapid emittance measurement system has contributed to improve the beam quality by optimizing parameters of ion sources and the beam transport system.

  4. Real-Time Measurement of Width and Height of Weld Beads in GMAW Processes

    Directory of Open Access Journals (Sweden)

    Jesús Emilio Pinto-Lopera

    2016-09-01

    Full Text Available Associated to the weld quality, the weld bead geometry is one of the most important parameters in welding processes. It is a significant requirement in a welding project, especially in automatic welding systems where a specific width, height, or penetration of weld bead is needed. This paper presents a novel technique for real-time measuring of the width and height of weld beads in gas metal arc welding (GMAW using a single high-speed camera and a long-pass optical filter in a passive vision system. The measuring method is based on digital image processing techniques and the image calibration process is based on projective transformations. The measurement process takes less than 3 milliseconds per image, which allows a transfer rate of more than 300 frames per second. The proposed methodology can be used in any metal transfer mode of a gas metal arc welding process and does not have occlusion problems. The responses of the measurement system, presented here, are in a good agreement with off-line data collected by a common laser-based 3D scanner. Each measurement is compare using a statistical Welch’s t-test of the null hypothesis, which, in any case, does not exceed the threshold of significance level α = 0.01, validating the results and the performance of the proposed vision system.

  5. Real-Time Measurement of Width and Height of Weld Beads in GMAW Processes

    Science.gov (United States)

    Pinto-Lopera, Jesús Emilio; S. T. Motta, José Mauricio; Absi Alfaro, Sadek Crisostomo

    2016-01-01

    Associated to the weld quality, the weld bead geometry is one of the most important parameters in welding processes. It is a significant requirement in a welding project, especially in automatic welding systems where a specific width, height, or penetration of weld bead is needed. This paper presents a novel technique for real-time measuring of the width and height of weld beads in gas metal arc welding (GMAW) using a single high-speed camera and a long-pass optical filter in a passive vision system. The measuring method is based on digital image processing techniques and the image calibration process is based on projective transformations. The measurement process takes less than 3 milliseconds per image, which allows a transfer rate of more than 300 frames per second. The proposed methodology can be used in any metal transfer mode of a gas metal arc welding process and does not have occlusion problems. The responses of the measurement system, presented here, are in a good agreement with off-line data collected by a common laser-based 3D scanner. Each measurement is compare using a statistical Welch’s t-test of the null hypothesis, which, in any case, does not exceed the threshold of significance level α = 0.01, validating the results and the performance of the proposed vision system. PMID:27649198

  6. Quantitative detection method for Roundup Ready soybean in food using duplex real-time PCR MGB chemistry.

    Science.gov (United States)

    Samson, Maria Cristina; Gullì, Mariolina; Marmiroli, Nelson

    2010-07-01

    Methodologies that enable the detection of genetically modified organisms (GMOs) (authorized and non-authorized) in food and feed strongly influence the potential for adequate updating and implementation of legislation together with labeling requirements. Quantitative polymerase chain reaction (qPCR) systems were designed to boost the sensitivity and specificity on the identification of GMOs in highly degraded DNA samples; however, such testing will become economically difficult to cope with due to increasing numbers of approved genetically modified (GM) lines. Multiplexing approaches are therefore in development to provide cost-efficient solution. Construct-specific primers and probe were developed for quantitative analysis of Roundup Ready soybean (RRS) event glyphosate-tolerant soybean (GTS) 40-3-2. The lectin gene (Le1) was used as a reference gene, and its specificity was verified. RRS- and Le1-specific quantitative real-time PCR (qRTPCR) were optimized in a duplex platform that has been validated with respect to limit of detection (LOD) and limit of quantification (LOQ), as well as accuracy. The analysis of model processed food samples showed that the degradation of DNA has no adverse or little effects on the performance of quantification assay. In this study, a duplex qRTPCR using TaqMan minor groove binder-non-fluorescent quencher (MGB-NFQ) chemistry was developed for specific detection and quantification of RRS event GTS 40-3-2 that can be used for practical monitoring in processed food products.

  7. Real-Time Quantitative Analysis of H2, He, O2, and Ar by Quadrupole Ion Trap Mass Spectrometry

    Science.gov (United States)

    Ottens, Andrew K.; Harrison, W. W.; Griffin, Timothy P.; Helms, William R.; Voska, N. (Technical Monitor)

    2002-01-01

    The use of a quadrupole ion trap mass spectrometer for quantitative analysis of hydrogen and helium as well as other permanent gases is demonstrated. The customized instrument utilizes the mass selective instability mode of mass analysis as with commercial instruments; however, this instrument operates at a greater RF trapping frequency and without a buffer gas. With these differences, a useable mass range from 2 to over 50 Da is achieved, as required by NASA for monitoring the Space Shuttle during a launch countdown. The performance of the ion trap is evaluated using part-per-million concentrations of hydrogen, helium, oxygen and argon mixed into a nitrogen gas stream. Relative accuracy and precision when quantitating the four analytes were better than the NASA-required minimum of 10% error and 5% deviation, respectively. Limits of detection were below the NASA requirement of 25-ppm hydrogen and 100-ppm helium; those for oxygen and argon were slightly higher than the requirement. The instrument provided adequate performance at fast data recording rates, demonstrating the utility of an ion trap mass spectrometer as a real-time quantitative monitoring device for permanent gas analysis.

  8. Comparison of droplet digital PCR and quantitative real-time PCR in mcrA-based methanogen community analysis

    Directory of Open Access Journals (Sweden)

    Tae Gwan Kim

    2014-12-01

    Full Text Available Two different quantitative PCR platforms, droplet digital PCR (dd-PCR and quantitative real-time PCR (qPCR, were compared in a mcrA-based methanogen community assay that quantifies ten methanogen sub-groups. Both technologies exhibited similar PCR efficiencies over at least four orders of magnitude and the same lower limits of detection (8 copies μL-DNA extract−1. The mcrA-based methanogen communities in three full-scale anaerobic digesters were examined using the two technologies. dd-PCR detected seven groups from the digesters, while qPCR did five groups, indicating that dd-PCR is more sensitive for DNA quantification. Linear regression showed quantitative agreements between both of the technologies (R2 = 0.59–0.98 in the five groups that were concurrently detected. Principal component analysis from the two datasets consistently indicated a substantial difference in the community composition among the digesters and revealed similar levels of differentiation among the communities. The combined results suggest that dd-PCR is more promising for examining methanogenic archaeal communities in biotechnological processes.

  9. Use of quantitative real-time PCR for direct detection of serratia marcescens in marine and other aquatic environments.

    Science.gov (United States)

    Joyner, Jessica; Wanless, David; Sinigalliano, Christopher D; Lipp, Erin K

    2014-03-01

    Serratia marcescens is the etiological agent of acroporid serratiosis, a distinct form of white pox disease in the threatened coral Acropora palmata. The pathogen is commonly found in untreated human waste in the Florida Keys, which may contaminate both nearshore and offshore waters. Currently there is no direct method for detection of this bacterium in the aquatic or reef environment, and culture-based techniques may underestimate its abundance in marine waters. A quantitative real-time PCR assay was developed to detect S. marcescens directly from environmental samples, including marine water, coral mucus, sponge tissue, and wastewater. The assay targeted the luxS gene and was able to distinguish S. marcescens from other Serratia species with a reliable quantitative limit of detection of 10 cell equivalents (CE) per reaction. The method could routinely discern the presence of S. marcescens for as few as 3 CE per reaction, but it could not be reliably quantified at this level. The assay detected environmental S. marcescens in complex sewage influent samples at up to 761 CE ml(-1) and in septic system-impacted residential canals in the Florida Keys at up to 4.1 CE ml(-1). This detection assay provided rapid quantitative abilities and good sensitivity and specificity, which should offer an important tool for monitoring this ubiquitous pathogen that can potentially impact both human health and coral health.

  10. Real-time microscopic 3D shape measurement based on optimized pulse-width-modulation binary fringe projection

    Science.gov (United States)

    Hu, Yan; Chen, Qian; Feng, Shijie; Tao, Tianyang; Li, Hui; Zuo, Chao

    2017-07-01

    In recent years, tremendous progress has been made in 3D measurement techniques, contributing to the realization of faster and more accurate 3D measurement. As a representative of these techniques, fringe projection profilometry (FPP) has become a commonly used method for real-time 3D measurement, such as real-time quality control and online inspection. To date, most related research has been concerned with macroscopic 3D measurement, but microscopic 3D measurement, especially real-time microscopic 3D measurement, is rarely reported. However, microscopic 3D measurement plays an important role in 3D metrology and is indispensable in some applications in measuring micro scale objects like the accurate metrology of MEMS components of the final devices to ensure their proper performance. In this paper, we proposed a method which effectively combines optimized binary structured patterns with a number-theoretical phase unwrapping algorithm to realize real-time microscopic 3D measurement. A slight defocusing of our optimized binary patterns can considerably alleviate the measurement error based on four-step phase-shifting FPP, providing the binary patterns with a comparable performance to ideal sinusoidal patterns. The static measurement accuracy can reach 8 μm, and the experimental results of a vibrating earphone diaphragm reveal that our system can successfully realize real-time 3D measurement of 120 frames per second (FPS) with a measurement range of 8~\\text{mm}× 6~\\text{mm} in lateral and 8 mm in depth.

  11. Combination of optical shape measurement and augmented reality for task support: II. Real-time feedback of shape measurement results

    Science.gov (United States)

    Yamauchi, Makoto; Iwamoto, Kazuyo

    2010-05-01

    Line heating is a skilled task in shipbuilding to shape the outer plates of ship hulls. Real-time information on the deformation of the plates during the task would be helpful to workers performing this process. Therefore, we herein propose an interactive scheme for supporting workers performing line heating; the system provides such information through an optical shape measurement instrument combined with an augmented reality (AR) system. The instrument was designed and fabricated so that the measured data were represented using coordinates based on fiducial markers. Since the markers were simultaneously used in the AR system for the purpose of positioning, the data could then be displayed to the workers through a head-mounted display as a virtual image overlaid on the plates. Feedback of the shape measurement results was thus performed in real time using the proposed system.

  12. Application of the Nordtest method for "real-time" uncertainty estimation of on-line field measurement.

    Science.gov (United States)

    Näykki, Teemu; Virtanen, Atte; Kaukonen, Lari; Magnusson, Bertil; Väisänen, Tero; Leito, Ivo

    2015-10-01

    Field sensor measurements are becoming more common for environmental monitoring. Solutions for enhancing reliability, i.e. knowledge of the measurement uncertainty of field measurements, are urgently needed. Real-time estimations of measurement uncertainty for field measurement have not previously been published, and in this paper, a novel approach to the automated turbidity measuring system with an application for "real-time" uncertainty estimation is outlined based on the Nordtest handbook's measurement uncertainty estimation principles. The term real-time is written in quotation marks, since the calculation of the uncertainty is carried out using a set of past measurement results. There are two main requirements for the estimation of real-time measurement uncertainty of online field measurement described in this paper: (1) setting up an automated measuring system that can be (preferably remotely) controlled which measures the samples (water to be investigated as well as synthetic control samples) the way the user has programmed it and stores the results in a database, (2) setting up automated data processing (software) where the measurement uncertainty is calculated from the data produced by the automated measuring system. When control samples with a known value or concentration are measured regularly, any instrumental drift can be detected. An additional benefit is that small drift can be taken into account (in real-time) as a bias value in the measurement uncertainty calculation, and if the drift is high, the measurement results of the control samples can be used for real-time recalibration of the measuring device. The procedure described in this paper is not restricted to turbidity measurements, but it will enable measurement uncertainty estimation for any kind of automated measuring system that performs sequential measurements of routine samples and control samples/reference materials in a similar way as described in this paper.

  13. Wearable Biomedical Measurement Systems for Assessment of Mental Stress of Combatants in Real Time

    Directory of Open Access Journals (Sweden)

    Fernando Seoane

    2014-04-01

    Full Text Available The Spanish Ministry of Defense, through its Future Combatant program, has sought to develop technology aids with the aim of extending combatants’ operational capabilities. Within this framework the ATREC project funded by the “Coincidente” program aims at analyzing diverse biometrics to assess by real time monitoring the stress levels of combatants. This project combines multidisciplinary disciplines and fields, including wearable instrumentation, textile technology, signal processing, pattern recognition and psychological analysis of the obtained information. In this work the ATREC project is described, including the different execution phases, the wearable biomedical measurement systems, the experimental setup, the biomedical signal analysis and speech processing performed. The preliminary results obtained from the data analysis collected during the first phase of the project are presented, indicating the good classification performance exhibited when using features obtained from electrocardiographic recordings and electrical bioimpedance measurements from the thorax. These results suggest that cardiac and respiration activity offer better biomarkers for assessment of stress than speech, galvanic skin response or skin temperature when recorded with wearable biomedical measurement systems.

  14. Real-time energy measurement of high repetition rate ultrashort laser pulses using pulse integration and FPGA processing

    Science.gov (United States)

    Tang, Qi-jie; Yang, Dong-xu; Wang, Jian; Feng, Yi; Zhang, Hong-fei; Chen, Teng-yun

    2016-11-01

    Real-time energy measurement using pulse integration method for high repetition rate ultrashort laser pulses based on FPGA (Field-Programmable Gate Array) and high-speed pipeline ADC (Analog-to-Digital Convertor) is introduced in this paper. There are two parts contained in this method: pulse integration and real-time data processing. The pulse integration circuit will convert the pulse to the step type signals which are linear to the laser pulse energy. Through the real-time data processing part, the amplitude of the step signals will be obtained by ADC sampling and conducting calculation in real time in FPGA. The test result shows that the method with good linearity (4.770%) and without pulse measurement missing is suitable for ultrashort laser pulses with high repetition rate up to 100 MHz.

  15. In-Situ Real Time Measurements of Molten Glass Properties, Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Robert De Saro; Joe Craparo

    2007-12-16

    Energy Research Company (ERCo) of Staten Island, NY has developed a sensor capable of measuring in situ and in real time, both the elemental composition and the temperature of molten glass. A prototype sensor has been designed, constructed and tested in ERCo's laboratory. The sensor was used to collect atomic emission spectra from molten fiberglass via Laser Induced Breakdown Spectroscopy (LIBS). From these spectra, we were able to readily identify all elements of interest (B, Si, Ca, Fe, Mg, Na, Sr, Al). The high signal-to-background signals achieved suggest that data from the sensor can be used to determine elemental concentrations, either through calibration curves or using ERCo's calibrationless method. ERCo's technology fits in well with DOE's Glass Industry Technology Roadmap which emphasizes the need for accurate process and feedstock sensors. Listed first under technological barriers to increased production efficiency is the 'Inability to accurately measure and control the production process'. A large-scale glass melting furnace, developed by SenCer Inc. of Penn Yan, NY was installed in ERCo's laboratory to ensure that a large enough quantity of glass could be melted and held at temperature in the presence of the water-cooled laser sensor without solidifying the glass.

  16. The friction measuring tool: Real-time estimates of sandface pressure during fracture treatments

    Energy Technology Data Exchange (ETDEWEB)

    Graham, R.L.; Amick, P.C.; Shaw, J.S. [Reuben L. Graham, Inc., Charleston, WV (United States); Hill, D.G.

    1994-12-31

    This paper describes a Friction Measuring Tool (FMT) and how it is used to estimate pressure loss from friction in the wellbore while a foam fracture treatment is being pumped. Combined with standard surface equipment, such as densitometers, flowmeters, and pressure transducers, this tool (located on the surface) offers real-time estimates of bottomhole treating pressures during the fracture treatment. Field examples are presented comparing calculated bottomhole treating pressures, based on the FMT, to actual pressures measured with downhole electronic pressure gauges (installed in the casing just below the treated zone) during stimulation treatments. In addition to the provided field examples which verify application of the FMT, the theory and mathematical background underlying the use of the FMT are described. Construction of the tool, actual use in the field, and trouble-shooting are also presented. This tool is also applicable to foam for coiled tubing operations or stable foam drilling. Development of the FMT is one product of a cooperative research program sponsored by the Gas Research Institute (GRI) where one of the broad research objectives is improved stimulation techniques.

  17. Dosimetric Characteristics of an EMF Delivery System Based on a Real-Time Impedance Measurement Device.

    Science.gov (United States)

    Garcia-Fernandez, Miguel A; Percherancier, Yann; Lagroye, Isabelle; O'Connor, Rodney P; Veyret, Bernard; Arnaud-Cormos, Delia; Leveque, Philippe

    2016-11-01

    In this paper, the dosimetric characterization of an EMF exposure setup compatible with real-time impedance measurements of adherent biological cells is proposed. The EMF are directly delivered to the 16-well format plate used by the commercial xCELLigence apparatus. Experiments and numerical simulations were carried out for the dosimetric analysis. The reflection coefficient was less than -10 dB up to 180 MHz and this exposure system can be matched at higher frequencies up to 900 and 1800 MHz. The specific absorption rate (SAR) distribution within the wells containing the biological medium was calculated by numerical finite-difference time domain simulations and results were verified by temperature measurements at 13.56 MHz. Numerical SAR values were obtained at the microelectrode level where the biological cells were exposed to EMF including 13.56, 900, and 1800 MHz. At 13.56 MHz, the SAR values, within the cell layer and the 270-μL volume of medium, are 1.9e3 and 3.5 W/kg/incident mW, respectively.

  18. Real-Time Operation Of A Multipurpose Multi-Reservoir System Using A Distributed Hydrological Model And Quantitative Precipitation Forecast

    Science.gov (United States)

    Saavedra Valeriano, O. C.; Koike, T.; Yang, K.; Yang, D.

    2007-12-01

    Taking advantage of a distributed hydrological model's capabilities such as capturing spatial heterogeneity, this study couples a physically based hydrological model with embedded dam network operation to a heuristic model for real-time operation. The input rainfall is a meso-scale quantitative precipitation forecast at 0.125 degrees resolution issued every 6 hours. It was analyzed 3 different series and the complete 18 hours lead-time. The system attempts to 1) reduce flood peaks down stream and 2) replenish water level at reservoirs after flood event. The proposed scheme takes advantage of the heuristic algorithm in order to evaluate different release combination sets automatically based on stochastic seeding considering the dam constraints and objective function. Latter is defined to minimize the absolute difference between the forecasted flood volume at protecting point and the total released volume from reservoirs. To estimate the flood volume a desirable discharge is to be set at protecting point. The desirable discharge is defined as the average of observed values exceeding the mean annual discharge; however, this can be modified according to flood warning levels and water resources management. The optimization variables are the release-inflow ratios. In addition, it was introduced the standard deviation of the error forecast as a weight in the objective function. The developed system was applied to upper Tone River in Japan using up to three multipurpose reservoirs. The efficiency of the system's response was evident reducing the flood peaks and volume at protecting point comparing the optimized releases against observed data. This approach has shown feasibility to be used by dam operators as a real-time reference tool for more efficient water resources management.

  19. [Comparison of two quantitative methods of endobronchial ultrasound real-time elastography for evaluating intrathoracic lymph nodes].

    Science.gov (United States)

    Mao, X W; Yang, J Y; Zheng, X X; Wang, L; Zhu, L; Li, Y; Xiong, H K; Sun, J Y

    2017-06-12

    Objective: To compare the clinical value of two quantitative methods in analyzing endobronchial ultrasound real-time elastography (EBUS-RTE) images for evaluating intrathoracic lymph nodes. Methods: From January 2014 to April 2014, EBUS-RTE examination was performed in patients who received EBUS-TBNA examination in Shanghai Chest Hospital. Each intrathoracic lymph node had a selected EBUS-RTE image. Stiff area ratio and mean hue value of region of interest (ROI) in each image were calculated respectively. The final diagnosis of lymph node was based on the pathologic/microbiologic results of EBUS-TBNA, pathologic/microbiologic results of other examinations and clinical following-up. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy were evaluated for distinguishing malignant and benign lesions. Results: Fifty-six patients and 68 lymph nodes were enrolled in this study, of which 35 lymph nodes were malignant and 33 lymph nodes were benign. The stiff area ratio and mean hue value of benign and malignant lesions were 0.32±0.29, 0.62±0.20 and 109.99±28.13, 141.62±17.52, respectively, and statistical differences were found in both of those two methods ( t =-5.14, P methods can be used for analyzing EBUS-RTE images quantitatively, having the value of differentiating benign and malignant intrathoracic lymph nodes, and the stiff area ratio is better than the mean hue value between the two methods.

  20. A quantitative real time polymerase chain reaction approach for estimating processed animal proteins in feed: preliminary data

    Directory of Open Access Journals (Sweden)

    Maria Cesarina Abete

    2013-04-01

    Full Text Available Lifting of the ban on the use of processed animal proteins (PAPs from non-ruminants in non-ruminant feed is in the wind, avoiding intraspecies recycling. Discrimination of species will be performed through polymerase chain reaction (PCR, which is at a moment a merely qualitative method. Nevertheless, quantification of PAPs in feed is needed. The aim of this study was to approach the quantitative determination of PAPs in feed through Real Time (RT-PCR technique; three different protocols picked up from the literature were tested. Three different kind of matrices were examined: pure animal meals (bovine, chicken and pork; one feed sample certified by the European reference laboratory on animal proteins (EURL AP in feed spiked with 0.1% bovine meal; and genomic DNAs from bovine, chicken and pork muscles. The limit of detection (LOD of the three protocols was set up. All the results obtained from the three protocols considered failed in the quantification process, most likely due to the uncertain copy numbers of the analytical targets chosen. This preliminary study will allow us to address further investigations, with the purpose of developing a RT-PCR quantitative method.

  1. Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations.

    Directory of Open Access Journals (Sweden)

    Jennifer C Ewald

    Full Text Available Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory molecule in the control of glycolysis and lipid metabolism. Mass spectrometric and NMR based metabolomics allow measuring citrate concentrations, but only with limited spatial and temporal resolution. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Förster resonance energy transfer (FRET. We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concentration range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation.

  2. Identification of three novel OA1 gene mutations identified in three families misdiagnosed with congenital nystagmus and carrier status determination by real-time quantitative PCR assay

    Directory of Open Access Journals (Sweden)

    Hamel Christian

    2003-01-01

    Full Text Available Abstract Background X-linked ocular albinism type 1 (OA1 is caused by mutations in OA1 gene, which encodes a membrane glycoprotein localised to melanosomes. OA1 mainly affects pigment production in the eye, resulting in optic changes associated with albinism including hypopigmentation of the retina, nystagmus, strabismus, foveal hypoplasia, abnormal crossing of the optic fibers and reduced visual acuity. Affected Caucasian males usually appear to have normal skin and hair pigment. Results We identified three previously undescribed mutations consisting of two intragenic deletions (one encompassing exon 6, the other encompassing exons 7–8, and a point mutation (310delG in exon 2. We report the development of a new method for diagnosis of heterozygous deletions in OA1 gene based on measurement of gene copy number using real-time quantitative PCR from genomic DNA. Conclusion The identification of OA1 mutations in families earlier reported as families with hereditary nystagmus indicate that ocular albinism type 1 is probably underdiagnosed. Our method of real-time quantitative PCR of OA1 exons with DMD exon as external standard performed on the LightCycler™ allows quick and accurate carrier-status assessment for at-risk females.

  3. Realization of a new sensor for a real-time local measurement of tissue optical coefficients

    Science.gov (United States)

    Ettori, Dominique; Tinet, Eric; Nguyen Tien, Tuan; Revel, Franck; Tualle, Jean-Michel; Avrillier, Sigrid

    2004-09-01

    We develop a new sensor for the local in vivo measurement of optical coefficients near the surface of a tissue. To be less sensitive to the heterogeneous surface of the sample, we decided to perform space and time-resolved measurements. The sensor is a bundle of fibres. The excitation light is generated by a mode-locked Ti-Sa laser at 800nm and filtered by a 1.5nm bandwidth dielectric filter in order to limit group velocity dispersion in the monomode excitation fibre. The reflectance light is collected by gradient index fibres at 250μm and 1.3 mm from the source. The detection is performed with a Hamamatsu M5675 synchroscan streak camera. The whole system allows a time resolution of about 5ps. We made comparisons between time and space resolved Monte-Carlo numerical simulations and in vitro experimental data obtained with unskimmed UHT milk which is a known reference medium. The system does not rely on the absolute value of the reflected light intensity nor depend on the intensity ratio between different fibres since the distance between the medium and the fibres as well as the fibres tip cleanness cannot be guaranteed in vivo. As a consequence we use global characteristic of the time resolved curves such as the FWHM and their evolution with the distance from the source. The good agreement between the simulations and the experimental data lets us envisage to use numerically pre-computed tables for a real time determination of the local scattering mean free path and the anisotropy factor . We soon will be able to perform measurements with biological tissues, in vitro in a first time and in vivo in a second time.

  4. A bio-inspired real-time capable artificial lateral line system for freestream flow measurements.

    Science.gov (United States)

    Abels, C; Qualtieri, A; De Vittorio, M; Megill, W M; Rizzi, F

    2016-06-03

    To enhance today's artificial flow sensing capabilities in aerial and underwater robotics, future robots could be equipped with a large number of miniaturized sensors distributed over the surface to provide high resolution measurement of the surrounding fluid flow. In this work we show a linear array of closely separated bio-inspired micro-electro-mechanical flow sensors whose sensing mechanism is based on a piezoresistive strain-gauge along a stress-driven cantilever beam, mimicking the biological superficial neuromasts found in the lateral line organ of fishes. Aiming to improve state-of-the-art flow sensing capability in autonomously flying and swimming robots, our artificial lateral line system was designed and developed to feature multi-parameter freestream flow measurements which provide information about (1) local flow velocities as measured by the signal amplitudes from the individual cantilevers as well as (2) propagation velocity, (3) linear forward/backward direction along the cantilever beam orientation and (4) periodicity of pulses or pulse trains determined by cross-correlating sensor signals. A real-time capable cross-correlation procedure was developed which makes it possible to extract freestream flow direction and velocity information from flow fluctuations. The computed flow velocities deviate from a commercial system by 0.09 m s(-1) at 0.5 m s(-1) and 0.15 m s(-1) at 1.0 m s(-1) flow velocity for a sampling rate of 240 Hz and a sensor distance of 38 mm. Although experiments were performed in air, the presented flow sensing system can be applied to underwater vehicles as well, once the sensors are embedded in a waterproof micro-electro-mechanical systems package.

  5. Measuring real-time streamflow using emerging technologies: Radar, hydroacoustics, and the probability concept

    Science.gov (United States)

    Fulton, J.; Ostrowski, J.

    2008-01-01

    Forecasting streamflow during extreme hydrologic events such as floods can be problematic. This is particularly true when flow is unsteady, and river forecasts rely on models that require uniform-flow rating curves to route water from one forecast point to another. As a result, alternative methods for measuring streamflow are needed to properly route flood waves and account for inertial and pressure forces in natural channels dominated by nonuniform-flow conditions such as mild water surface slopes, backwater, tributary inflows, and reservoir operations. The objective of the demonstration was to use emerging technologies to measure instantaneous streamflow in open channels at two existing US Geological Survey streamflow-gaging stations in Pennsylvania. Surface-water and instream-point velocities were measured using hand-held radar and hydroacoustics. Streamflow was computed using the probability concept, which requires velocity data from a single vertical containing the maximum instream velocity. The percent difference in streamflow at the Susquehanna River at Bloomsburg, PA ranged from 0% to 8% with an average difference of 4% and standard deviation of 8.81 m3/s. The percent difference in streamflow at Chartiers Creek at Carnegie, PA ranged from 0% to 11% with an average difference of 5% and standard deviation of 0.28 m3/s. New generation equipment is being tested and developed to advance the use of radar-derived surface-water velocity and instantaneous streamflow to facilitate the collection and transmission of real-time streamflow that can be used to parameterize hydraulic routing models.

  6. CLEAR PM: Teaching, Outreach, and Research Through Real-Time Particulate Measurements

    Science.gov (United States)

    DeCarlo, P. F.

    2013-12-01

    An understanding of particulate matter (also called aerosols) can be made through measurement. This measurement does not change in value if it is made in a teaching, research, or outreach environment. A grant from the Camille and Henry Dreyfus Foundation provided funding to construct an instrument suite composed of 1-4 second measurements that are displayed in real-time through a software interface. This display module is called CLEAR PM (Chemistry Lessons Enabling Aerosol Realizations through Particulate Measurement), and was conceived to apply across outreach activities, teaching activities, and research activities. The construction and software design of CLEAR PM was done as part of a special topics course for chemistry and engineering graduate students at Drexel University. Measurement principles of the different (research grade) instruments were taught as part of the course, with emphasis put on the fundamental measurements and their limitations, and an introduction to data acquisition software was also integral to the teaching component. As a final project of the course graduate students were required to create a 'teaching' module that illustrates a chemistry or physics concept and utilizes the measurements of CLEAR PM. These modules ranged from gas-phase ozone chemistry creating secondary organic aerosols, to the wavelength dependent absorption profiles of wood smoke versus propane soot. The teaching modules developed by the graduate students have been used in outreach activities sponsored by The Franklin Institute and the Clean Air Council in Philadelphia, where underrepresented groups often make up a large fraction of the audience. CLEAR PM is designed to give students and citizens a hands-on opportunity to see how we measure and understand the world around us. As mentioned previously, the instruments that are part of CLEAR PM are research grade instruments, and are actively being used in research projects in the DeCarlo lab at Drexel to study particulate

  7. Virtual lab based real-time data acquisition, measurement and monitoring platform for solar photovoltaic module

    Directory of Open Access Journals (Sweden)

    Amit Kumar Rohit

    2017-12-01

    Full Text Available The work presents real-time data acquisition and monitoring of solar photovoltaic modules using LabVIEW. A graphical program has been developed to obtain efficiency and fill factor of solar PV module. A front panel is designed, displaying all the acquired data such as; voltage, current, solar radiation, ambient temperature, humidity, Current vs. Voltage and Power vs. Voltage graphs which make it very useful to understand the performance behavior of the solar photovoltaic module in real time. Data acquisition and monitoring for solar panels of different ratings are carried out. This tool is an effective platform for experimental study in the laboratory of different solar photovoltaic modules with access to real-time data. Keywords: Photovoltaic, Instruments, Labview software, Characteristics, Sensors

  8. Real-time emission factor measurements of isocyanic acid from light duty gasoline vehicles.

    Science.gov (United States)

    Brady, James M; Crisp, Timia A; Collier, Sonya; Kuwayama, Toshihiro; Forestieri, Sara D; Perraud, Véronique; Zhang, Qi; Kleeman, Michael J; Cappa, Christopher D; Bertram, Timothy H

    2014-10-07

    Exposure to gas-phase isocyanic acid (HNCO) has been previously shown to be associated with the development of atherosclerosis, cataracts and rheumatoid arthritis. As such, accurate emission inventories for HNCO are critical for modeling the spatial and temporal distribution of HNCO on a regional and global scale. To date, HNCO emission rates from light duty gasoline vehicles, operated under driving conditions, have not been determined. Here, we present the first measurements of real-time emission factors of isocyanic acid from a fleet of eight light duty gasoline-powered vehicles (LDGVs) tested on a chassis dynamometer using the Unified Driving Cycle (UC) at the California Air Resources Board (CARB) Haagen-Smit test facility, all of which were equipped with three-way catalytic converters. HNCO emissions were observed from all vehicles, in contrast to the idealized laboratory measurements. We report the tested fleet averaged HNCO emission factors, which depend strongly on the phase of the drive cycle; ranging from 0.46 ± 0.13 mg kg fuel(-1) during engine start to 1.70 ± 1.77 mg kg fuel(-1) during hard acceleration after the engine and catalytic converter were warm. The tested eight-car fleet average fuel based HNCO emission factor was 0.91 ± 0.58 mg kg fuel(-1), within the range previously estimated for light duty diesel-powered vehicles (0.21-3.96 mg kg fuel(-1)). Our results suggest that HNCO emissions from LDGVs represent a significant emission source in urban areas that should be accounted for in global and regional models.

  9. Real-time indoor and outdoor measurements of black carbon at primary schools

    Science.gov (United States)

    Reche, C.; Rivas, I.; Pandolfi, M.; Viana, M.; Bouso, L.; Àlvarez-Pedrerol, M.; Alastuey, A.; Sunyer, J.; Querol, X.

    2015-11-01

    Epidemiological and toxicological studies have demonstrated the association between Black Carbon in indoor and outdoor air and the occurrence of health risks. Data on air quality in schools is of special interest, as children are more vulnerable to health hazards. In this context, indoor and outdoor measurements of real-time Equivalent Black Carbon (EBC) were collected at 39 primary schools located in Barcelona (Spain), with classrooms naturally ventilated under warm weather conditions. A main contribution of road traffic emissions to indoor and outdoor EBC levels was evidenced through different approaches. Simultaneous measurements of EBC levels at schools under different traffic conditions revealed concentrations by 30-35% higher at schools exposed to higher vehicles intensities. Moreover, a significant correlation was obtained between average outdoor EBC levels at different districts of the city and the percentage of surface area in each district used for the road network (R2 = 0.61). Higher indoor than outdoor levels were recorded at some instances when the indoor sampling location was relatively closer to road traffic, even under low outdoor temperatures. Indeed, the average indoor/outdoor EBC ratios for each school correlate moderately between campaigns in spite of significant differences in temperature between sampling periods. These two facts highlight the strong dependency of the EBC levels on the distance to traffic. The peaks of exposure inside the classrooms seemed to be determined by outdoor concentrations, as shown by the parallelism between indoor and outdoor mean EBC daily cycles and the similar contribution of traffic rush hours to indoor and outdoor daily mean levels. The airtightness of the classroom was suggested as the responsible for the indoor/outdoor ratios of EBC higher than 1 recorded at nights.

  10. Quantification of measles, mumps and rubella viruses using real-time quantitative TaqMan-based RT-PCR assay.

    Science.gov (United States)

    Ammour, Y; Faizuloev, E; Borisova, T; Nikonova, A; Dmitriev, G; Lobodanov, S; Zverev, V

    2013-01-01

    In this study, a rapid quantitative method using TaqMan-based real-time reverse transcription-polymerase chain reaction (qPCR-RT) has been developed for estimating the titers of measles, mumps and rubella (MMR) viruses in infected cell culture supernatants. The qPCR-RT assay was demonstrated to be a specific, sensitive, efficient and reproducible method. For MMR viral samples obtained during MMR viral propagations in Vero cells at a different multiplicity of infection, titers determined by the qPCR-RT assay have been compared with estimates of infectious virus obtained by a traditional commonly used method for MMR viruses - 50% cell culture infective dose (CCID(50)) assay, in paired samples. Pearson analysis evidenced a significant correlation between both methods for a certain period after viral inoculation. Furthermore, the established qPCR-RT assay was faster and less-laborious. The developed method could be used as an alternative method or a supplementary tool for the routine titer estimation during MMR vaccine production. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Accurate, fast and cost-effective diagnostic test for monosomy 1p36 using real-time quantitative PCR.

    Science.gov (United States)

    Cunha, Pricila da Silva; Pena, Heloisa B; D'Angelo, Carla Sustek; Koiffmann, Celia P; Rosenfeld, Jill A; Shaffer, Lisa G; Stofanko, Martin; Gonçalves-Dornelas, Higgor; Pena, Sérgio Danilo Junho

    2014-01-01

    Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5-0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescent in situ hybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR of PRKCZ and SKI is a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.

  12. Accurate, Fast and Cost-Effective Diagnostic Test for Monosomy 1p36 Using Real-Time Quantitative PCR

    Directory of Open Access Journals (Sweden)

    Pricila da Silva Cunha

    2014-01-01

    Full Text Available Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5–0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH, which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescent in situ hybridization (FISH, and/or multiplex ligation-dependent probe amplification (MLPA all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR of PRKCZ and SKI is a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.

  13. Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve.

    Science.gov (United States)

    Rueda-Martínez, Carmen; Fernández, M Carmen; Soto-Navarrete, María Teresa; Jiménez-Navarro, Manuel; Durán, Ana Carmen; Fernández, Borja

    2016-01-01

    Bicuspid aortic valve (BAV) is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40%) incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR) assays. A total of 51 adult (180-240 days old) and 56 old (300-440 days old) animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30), or to the affected strain of hamsters with TAV (n = 45) or BAV (n = 32). The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta.

  14. Reference gene selection for real-time quantitative PCR analysis of the mouse uterus in the peri-implantation period.

    Directory of Open Access Journals (Sweden)

    Pengfei Lin

    Full Text Available The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNorm(PLUS, NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.

  15. Identification and validation of reference genes for quantitative real-time PCR in Drosophila suzukii (Diptera: Drosophilidae.

    Directory of Open Access Journals (Sweden)

    Yifan Zhai

    Full Text Available To accurately evaluate gene expression levels and obtain more accurate quantitative real-time RT-PCR (qRT-PCR data, normalization relative to reliable reference gene(s is required. Drosophila suzukii, is an invasive fruit pest native to East Asia, and recently invaded Europe and North America, the stability of its reference genes have not been previously investigated. In this study, ten candidate reference genes (RPL18, RPS3, AK, EF-1β, TBP, NADH, HSP22, GAPDH, Actin, α-Tubulin, were evaluated for their suitability as normalization genes under different biotic (developmental stage, tissue and population, and abiotic (photoperiod, temperature conditions. The three statistical approaches (geNorm, NormFinder and BestKeeper and one web-based comprehensive tool (RefFinder were used to normalize analysis of the ten candidate reference genes identified α-Tubulin, TBP and AK as the most stable candidates, while HSP22 and Actin showed the lowest expression stability. We used three most stable genes (α-Tubulin, TBP and AK and one unstably expressed gene to analyze the expression of P-glycoprotein in abamectin-resistant and sensitive strains, and the results were similar to reference genes α-Tubulin, TBP and AK, which show good stability, while the result of HSP22 has a certain bias. The three validated reference genes can be widely used for quantification of target gene expression with qRT-PCR technology in D.suzukii.

  16. Evaluation of reference genes for gene expression studies in radish (Raphanus sativus L.) using quantitative real-time PCR.

    Science.gov (United States)

    Xu, Yuanyuan; Zhu, Xianwen; Gong, Yiqin; Xu, Liang; Wang, Yan; Liu, Liwang

    2012-08-03

    Real-time quantitative reverse transcription PCR (RT-qPCR) is a rapid and reliable method for gene expression studies. Normalization based on reference genes can increase the reliability of this technique; however, recent studies have shown that almost no single reference gene is universal for all possible experimental conditions. In this study, eight frequently used reference genes were investigated, including Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Actin2/7 (ACT), Tubulin alpha-5 (TUA), Tubulin beta-1 (TUB), 18S ribosomal RNA (18SrRNA), RNA polymerase-II transcription factor (RPII), Elongation factor 1-b (EF-1b) and Translation elongation factor 2 (TEF2). Expression stability of candidate reference genes was examined across 27 radish samples, representing a range of tissue types, cultivars, photoperiodic and vernalization treatments, and developmental stages. The eight genes in these sample pools displayed a wide range of Ct values and were variably expressed. Two statistical software packages, geNorm and NormFinder showed that TEF2, RPII and ACT appeared to be relatively stable and therefore the most suitable for use as reference genes. These results facilitate selection of desirable reference genes for accurate gene expression studies in radish. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. Universal real-time PCR assay for quantitation and size evaluation of residual cell DNA in human viral vaccines.

    Science.gov (United States)

    André, Murielle; Reghin, Sylviane; Boussard, Estelle; Lempereur, Laurent; Maisonneuve, Stéphane

    2016-05-01

    Residual host cellular DNA (rcDNA) is one of the principal risk associated with continuous cell lines derived medicines such as viral vaccines. To assess rcDNA degradation, we suggest two quantitative real-time PCR assays designed to separately quantify target sequences shorter and longer than the 200 bp risk limit, the relative abundance of both targets reflecting the extent of rcDNA fragmentation. The conserved multicopy ribosomal 18S RNA gene was targeted to detect host cell templates from most mammalian cell substrates commonly used in the manufacture of human viral vaccines. The detection range of the method was assessed on purified DNA templates from different animal origins. The standard calibrator origin and structural conformation were shown crucial to achieve accurate quantification. Artificial mixtures of PCR products shorter and longer than 200 bp were used as a model to check the ability of the assay to estimate the fragment size distribution. The method was successfully applied to a panel of Vero cell derived vaccines and could be used as a universal method for determination of both content and size distribution of rcDNA in vaccines. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  18. A multiplex quantitative real-time polymerase chain reaction panel for detecting neurologic pathogens in dogs with meningoencephalitis.

    Science.gov (United States)

    Han, Jae-Ik; Chang, Dong-Woo; Na, Ki-Jeong

    2015-01-01

    Meningoencephalitis (ME) is a common inflammatory disorder of the central nervous system in dogs. Clinically, ME has both infectious and non-infectious causes. In the present study, a multiplex quantitative real-time polymerase chain reaction (mqPCR) panel was optimized for the detection of eight canine neurologic pathogens (Blastomyces dermatitidis, Cryptococcus spp., Neospora caninum, Borrelia burgdorferi, Bartonella spp., Toxoplasma gondii, Ehrlichia canis, and canine distemper virus [CDV]). The mqPCR panel was subsequently applied to 53 cerebrospinal fluid (CSF) samples collected from dogs with ME. The analytic sensitivity (i.e., limit of detection, expressed as molecules per 1 mL of recombinant vector) was 3.8 for CDV, 3.7 for Ehrlichia canis, 3.7 for Bartonella spp., 3.8 for Borrelia burgdorferi, 3.7 for Blastomyces dermatitidis, 3.7 for Cryptococcus spp., 38 for Neospora caninum, and 3.7 for Toxoplasma gondii. Among the tested CSF samples, seven (15%) were positive for the following pathogens in decreasing order of frequency: Cryptococcus spp. (3/7), Blastomyces dermatitidis (2/7), and Borrelia burgdorferi (2/7). In summary, use of an mqPCR panel with high analytic sensitivity as an initial screen for infectious agents in dogs with ME could facilitate the selection of early treatment strategies and improve outcomes.

  19. Application of quantitative real-time PCR for enumeration of total bacterial, archaeal, and yeast populations in kimchi.

    Science.gov (United States)

    Park, Eun-Jin; Chang, Ho-Won; Kim, Kyoung-Ho; Nam, Young-Do; Roh, Seong Woon; Bae, Jin-Woo

    2009-12-01

    Kimchi is a Korean traditional fermented food made of brined vegetables, with a variety of spices. Various microorganisms are associated with the kimchi fermentation process. This study was undertaken in order to apply quantitative real-time PCR targeting the 16S and 26S rRNA genes for the investigation of dynamics of bacterial, archaeal, and yeast communities during fermentation of various types of kimchi. Although the total bacterial and archaeal rRNA gene copy numbers increased during kimchi fermentation, the number of yeasts was not significantly altered. In 1 ng of bulk DNA, the mean number of rRNA gene copies for all strains of bacteria was 5.45 x 10(6) which was 360 and 50 times greater than those for archaea and yeast, respectively. The total gene copy number for each group of microorganisms differed among the different types of kimchi, although the relative ratios among them were similar. The common dominance of bacteria in the whole microbial communities of various types of kimchi suggests that bacteria play a principal role in the kimchi fermentation process.

  20. Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean

    Directory of Open Access Journals (Sweden)

    Renata Stolf-Moreira

    2011-01-01

    Full Text Available The objective of this work was to validate, by quantitative PCR in real time (RT-qPCR, genes to be used as reference in studies of gene expression in soybean in drought-stressed trials. Four genes commonly used in soybean were evaluated: Gmβ-actin, GmGAPDH, GmLectin and GmRNAr18S. Total RNA was extracted from six samples: three from roots in a hydroponic system with different drought intensities (0, 25, 50, 75 and 100 minutes of water stress, and three from leaves of plants grown in sand with different soil moistures (15, 5 and 2.5% gravimetric humidity. The raw cycle threshold (Ct data were analyzed, and the efficiency of each primer was calculated for an overall analysis of the Ct range among the different samples. The GeNorm application was used to evaluate the best reference gene, according to its stability. The GmGAPDH was the least stable gene, with the highest mean values of expression stability (M, and the most stable genes, with the lowest M values, were the Gmβ-actin and GmRNAr18S, when both root and leaves samples were tested. These genes can be used in RT-qPCR as reference gene for expression analysis.

  1. Real-time quantitative PCR detection of WT1 and M-BCR-ABL expressions in chronic myeloid leukemia.

    Science.gov (United States)

    Szántó, Annamária; Pap, Zsuzsánna; Dénes, Lóránd; Benedek Lázár, Erzsébet; Horváth, Adrienne; Tunyogi, Alíz Beáta; Baróti, Beáta Ágota; Pávai, Zoltán

    2015-01-01

    The Philadelphia chromosome and the resulting BCR-ABL fusion gene represent the hallmark event in chronic myeloid leukemia (CML) and their discoveries radically changed the management of these patients. Currently Wilms tumor 1 gene (WT1) is intensively investigated as high WT1 expression levels have been demonstrated in case of multiple solid tumors and malignant hematological syndromes (acute myeloid and lymphoid leukemia, myelodysplastic syndromes and chronic myeloid leukemia). The aim of our study was to investigate the WT1 expression in CML patients and its possible contribution to disease evolution. In the Laboratory of Molecular Biology, University of Medicine and Pharmacy of Tirgu Mures, Romania, we regularly determined the M-BCR-ABL and WT1 expression levels by RQ-PCR (real-time quantitative polymerase chain reaction) testing in case of 19 CML patients: six patients monitorized from the diagnosis and 13 patients first tested during therapy. Eight CML (four advanced stage and four CP) patients showed high WT1 expression level, and in case of 11 patients the WT1 expression levels were undetectable or lower than 0.02%. The only significant difference between the high and low WT1 expression groups was represented by the clinical stage. In the majority of pretreated patients (10 out of 13 patients), the WT1 expression levels were low or undetectable. High WT1 expression in CML patients is detected especially in the advanced stages of the disease. Efficient Imatinib therapy may contribute to low WT1 levels in CP patients.

  2. Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product.

    Science.gov (United States)

    Fazelahi, Mansoureh; Kia, Vahid; Kaghazian, Hooman; Paryan, Mahdi

    2017-11-26

    Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli's and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (10 1 to 10 7 copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.

  3. DNA and RNA Extraction and Quantitative Real-Time PCR-Based Assays for Biogas Biocenoses in an Interlaboratory Comparison

    Science.gov (United States)

    Lebuhn, Michael; Derenkó, Jaqueline; Rademacher, Antje; Helbig, Susanne; Munk, Bernhard; Pechtl, Alexander; Stolze, Yvonne; Prowe, Steffen; Schwarz, Wolfgang H.; Schlüter, Andreas; Liebl, Wolfgang; Klocke, Michael

    2016-01-01

    Five institutional partners participated in an interlaboratory comparison of nucleic acid extraction, RNA preservation and quantitative Real-Time PCR (qPCR) based assays for biogas biocenoses derived from different grass silage digesting laboratory and pilot scale fermenters. A kit format DNA extraction system based on physical and chemical lysis with excellent extraction efficiency yielded highly reproducible results among the partners and clearly outperformed a traditional CTAB/chloroform/isoamylalcohol based method. Analytical purpose, sample texture, consistency and upstream pretreatment steps determine the modifications that should be applied to achieve maximum efficiency in the trade-off between extract purity and nucleic acid recovery rate. RNA extraction was much more variable, and the destination of the extract determines the method to be used. RNA stabilization with quaternary ammonium salts was an as satisfactory approach as flash freezing in liquid N2. Due to co-eluted impurities, spectrophotometry proved to be of limited value for nucleic acid qualification and quantification in extracts obtained with the kit, and picoGreen® based quantification was more trustworthy. Absorbance at 230 nm can be extremely high in the presence of certain chaotropic guanidine salts, but guanidinium isothiocyanate does not affect (q)PCR. Absolute quantification by qPCR requires application of a reliable internal standard for which correct PCR efficiency and Y-intercept values are important and must be reported. PMID:28952569

  4. Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils.

    Science.gov (United States)

    Ledderose, Carola; Heyn, Jens; Limbeck, Elisabeth; Kreth, Simone

    2011-10-20

    The choice of reliable reference genes is a prerequisite for valid results when analyzing gene expression with real-time quantitative PCR (qPCR). This method is frequently applied to study gene expression patterns in immune cells, yet a thorough validation of potential reference genes is still lacking for most leukocyte subtypes and most models of their in vitro stimulation. In the current study, we evaluated the expression stability of common reference genes in two widely used cell culture models-anti-CD3/CD28 activated T cells and lipopolysaccharide stimulated neutrophils-as well as in unselected untreated leukocytes. The mRNA expression of 17 (T cells), 7 (neutrophils) or 8 (unselected leukocytes) potential reference genes was quantified by reverse transcription qPCR, and a ranking of the preselected candidate genes according to their expression stability was calculated using the programs NormFinder, geNorm and BestKeeper. IPO8, RPL13A, TBP and SDHA were identified as suitable reference genes in T cells. TBP, ACTB and SDHA were stably expressed in neutrophils. TBP and SDHA were also the most stable genes in untreated total blood leukocytes. The critical impact of reference gene selection on the estimated target gene expression is demonstrated for IL-2 and FIH expression in T cells. The study provides a shortlist of suitable reference genes for normalization of gene expression data in unstimulated and stimulated T cells, unstimulated and stimulated neutrophils and in unselected leukocytes.

  5. Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

    Directory of Open Access Journals (Sweden)

    Ledderose Carola

    2011-10-01

    Full Text Available Abstract Background The choice of reliable reference genes is a prerequisite for valid results when analyzing gene expression with real-time quantitative PCR (qPCR. This method is frequently applied to study gene expression patterns in immune cells, yet a thorough validation of potential reference genes is still lacking for most leukocyte subtypes and most models of their in vitro stimulation. In the current study, we evaluated the expression stability of common reference genes in two widely used cell culture models-anti-CD3/CD28 activated T cells and lipopolysaccharide stimulated neutrophils-as well as in unselected untreated leukocytes. Results The mRNA expression of 17 (T cells, 7 (neutrophils or 8 (unselected leukocytes potential reference genes was quantified by reverse transcription qPCR, and a ranking of the preselected candidate genes according to their expression stability was calculated using the programs NormFinder, geNorm and BestKeeper. IPO8, RPL13A, TBP and SDHA were identified as suitable reference genes in T cells. TBP, ACTB and SDHA were stably expressed in neutrophils. TBP and SDHA were also the most stable genes in untreated total blood leukocytes. The critical impact of reference gene selection on the estimated target gene expression is demonstrated for IL-2 and FIH expression in T cells. Conclusions The study provides a shortlist of suitable reference genes for normalization of gene expression data in unstimulated and stimulated T cells, unstimulated and stimulated neutrophils and in unselected leukocytes.

  6. Quantitation of MYC gene expression in sporadic breast tumors with a real-time reverse transcription-PCR assay.

    Science.gov (United States)

    Bièche, I; Laurendeau, I; Tozlu, S; Olivi, M; Vidaud, D; Lidereau, R; Vidaud, M

    1999-06-15

    MYC gene overexpression was identified recently as a downstream step at the end of the Wnt/APC/beta-catenin pathway dysregulation observed in colorectal cancer (T-C. He et al., Science (Washington DC), 281: 1509-1512, 1998). It thus appears that an excess of c-myc protein is a primary cause of numerous cancers. In breast cancer, MYC has been studied mostly at the DNA level because of the poor quality of available antibodies against the protein product. The renewed interest in MYC calls for a sensitive and accurate method for analyzing MYC overexpression in breast tumors. We have developed a real-time quantitative reverse transcription-PCR assay based on TaqMan fluorescence methodology to quantify the MYC mRNA copy number. We validated the method on a large series of breast tumors. MYC gene overexpression was observed in 29 of 134 (22%) breast tumor RNAs, ranging from 3.2 to 19 times the level in normal breast tissues. These data imply that dysregulated MYC gene expression is potentially involved in the pathogenesis of breast cancer, especially by favoring local cell proliferation. We also found that MYC gene overexpression was rarely due to an increased MYC gene copy number in breast cancer. This new, simple, rapid, and semiautomated method will be useful for screening cancer patients for MYC overexpression and will prove a powerful tool in large, randomized, prospective, cooperative group trials and in the MYC-based therapy project.

  7. Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR.

    Science.gov (United States)

    Yin, Zhiyuan; Ke, Xiwang; Huang, Dingxuan; Gao, Xiaoning; Voegele, Ralf T; Kang, Zhensheng; Huang, Lili

    2013-09-01

    Valsa mali var. mali (Vmm), is the predominant species of apple valsa canker in China. Modern analysis of genes involved in virulence or pathogenicity usually implicate gene expression analysis most often performed using real-time quantitative polymerase chain reaction (RT-qPCR). However, for relative gene expression analysis pertinent reference genes have to be validated before using them as internal reference. This has not been reported for Vmm, so far. Therefore, eight commonly used housekeeping genes (ACT, CYP, EF1-α, G6PDH, GAPDH, L13, TUB, and UBQ) were cloned and evaluated for their expression stability by geNorm and NormFinder. Overall, all of the candidate reference genes were found to be suitable for gene expression analysis. After analysis of 10 samples from different strains and abiotic stress treatments, G6PDH appeared to be the most suitable reference gene, whereas GAPDH was the least suitable. Moreover, taking G6PDH combined with L13 or CYP as reference genes, improved the reliability of RT-qPCR significantly. The influence of the reference system on expression data was demonstrated by analyzing Vmmpg-1 encoding an endo-polygalacturonase gene. Pectinases are considered key pathogenicity factors for this fungus. In order to better understand the role of pectinases in pathogenicity of Vmm, RT-qPCR was used for expression analysis. Our results may provide a guideline for future studies on gene expression of V. mali var. mali by using RT-qPCR.

  8. Development of real-time quantitative polymerase chain reaction assays to track treatment response in retinoid resistant acute promyelocytic leukemia

    Directory of Open Access Journals (Sweden)

    Jelena V Jovanovic

    2011-10-01

    Full Text Available Molecular detection of minimal residual disease (MRD has become established to assess remission status and guide therapy in patients with PML-RARA+ acute promyelocytic leukemia (APL. However, there are few data on tracking disease response in patients with rarer retinoid resistant subtypes of APL, characterized by PLZF-RARA and STAT5b-RARA. Despite their relative rarity (<1% of APL we identified 6 cases (PLZF-RARA, n=5; STAT5b-RARA, n=1, established the respective breakpoint junction regions and designed real-time quantitative polymerase chain reaction (RQ-PCR assays to detect leukemic transcripts. The relative level of fusion gene expression in diagnostic samples was comparable to that observed in t(15;17-associated APL, affording assay sensitivities of ~1 in 104-105. Serial samples were available from 2 PLZF-RARA APL patients. One showed persistent PCR positivity, predicting subsequent relapse, and remains in CR2, ~11 years post-autograft. The other, achieved molecular remission (CRm with combination chemotherapy, remaining in CR1 at 6 years. The STAT5b-RARA patient failed to achieve CRm following frontline combination chemotherapy and ultimately proceeded to allogeneic transplant on the basis of a steadily rising fusion transcript level. These data highlight the potential of RQ-PCR detection of MRD to facilitate development of more individualized approaches to the management of rarer molecularly-defined subsets of acute leukemia.

  9. Assessing Reception and Individual Responses to Media Messages: Relevance, Computer Techniques, Quality and Analysis of Real-Time Response Measurements

    Directory of Open Access Journals (Sweden)

    Jürgen MAIER

    2013-07-01

    Full Text Available Media messages have an important impact on political attitudes and behavior. Hence, the question arises - how is media content received at an individual level? This question is difficult to investigate with the social science research methods currently available. Measuring an individual’s real-time reaction to auditory or visual media content using computers, i.e. real-time response (RTR measurement, can be a fruitful approach to address the question. This article describes various RTR techniques, discusses the reliability and validity of RTR measurements and outlines strategies on how to analyze RTR data.

  10. Real-Time Quadrature Measurement of a Single-Photon Wave Packet with Continuous Temporal-Mode Matching.

    Science.gov (United States)

    Ogawa, Hisashi; Ohdan, Hideaki; Miyata, Kazunori; Taguchi, Masahiro; Makino, Kenzo; Yonezawa, Hidehiro; Yoshikawa, Jun-Ichi; Furusawa, Akira

    2016-06-10

    Real-time controls based on quantum measurements are powerful tools for various quantum protocols. However, their experimental realization has been limited by mode mismatch between the temporal mode of quadrature measurement and that heralded by photon detection. Here, we demonstrate real-time quadrature measurement of a single-photon wave packet induced by photon detection by utilizing continuous temporal-mode matching between homodyne detection and an exponentially rising temporal mode. Single photons in exponentially rising modes are also expected to be useful resources for interactions with other quantum systems.

  11. Quantitative analysis of the improvement in high zoom maritime tracking due to real-time image enhancement

    Science.gov (United States)

    Bachoo, Asheer K.; de Villiers, Jason P.; Nicolls, Fred; le Roux, Francois P. J.

    2011-05-01

    This work aims to evaluate the improvement in the performance of tracking small maritime targets due to real-time enhancement of the video streams from high zoom cameras on pan-tilt pedestal. Due to atmospheric conditions these images can frequently have poor contrast, or exposure of the target if it is far and thus small in the camera's field of view. A 300mm focal length lens and machine vision camera were mounted on a pan-tilt unit and used to observe the False Bay near Simon's Town, South Africa. A ground truth data-set was created by performing a least squares geo-alignment of the camera system and placing a differential global position system receiver on a target boat, thus allowing the boat's position in the camera's field of view to be determined. Common tracking techniques including level-sets, Kalman filters and particle filters were implemented to run on the central processing unit of the tracking computer. Image enhancement techniques including multi-scale tone mapping, interpolated local histogram equalisation and several sharpening techniques were implemented on the graphics processing unit. This allowed the 1.3 mega-pixel 20 frames per second video stream to be processed in real-time. A quantified measurement of each tracking algorithm's robustness in the presence of sea-glint, low contrast visibility and sea clutter - such as white caps is performed on the raw recorded video data. These results are then compared to those obtained using data enhanced with the algorithms described.

  12. Real-time measurement of outdoor worker's exposure to solar ultraviolet radiation in Pretoria, South Africa

    Directory of Open Access Journals (Sweden)

    Mmathapelo Makgabutlane

    2015-05-01

    Full Text Available The city of Pretoria in South Africa receives considerable solar ultraviolet radiation (UVR because of its low latitude (22–35°S and relatively clear skies. Certain meteorological factors affect the amount of solar UVR that reaches the ground; the most dominant factors being stratospheric ozone, cloud cover and solar zenith angle. It is known that overexposure to solar UVR may lead to the development of adverse health conditions, the most significant being skin cancer. Outdoor workers spend a significant amount of time outside and are thus susceptible to this risk. In this case study, we estimated, for the first time, the real-time solar UVR exposure of an outdoor worker in Pretoria. Measurements were made on 27 and 28 May 2013 using a handheld ultraviolet index (UVI meter calibrated against a science-grade biometer at the South African Weather Service in Pretoria. Personal exposure estimation was used to discern the pattern in diurnal and annual sunburn risk for the outdoor worker. Ambient UVR levels ranged from 0 UVI to 4.66 UVI and the outdoor worker’s potential exposure estimates regularly exceeded 80% of these levels depending on the time of day. The risk of sunburn was evident; however, actual incidents would depend on individual skin photosensitivity and melanin content, as well as sun protection used. Further research is needed to determine the personal exposure estimations of outdoor workers in other provinces in which solar UVR levels may be equally high, or higher than those in Pretoria.

  13. A Comprehensive Statistically-Based Method to Interpret Real-Time Flowing Measurements

    Energy Technology Data Exchange (ETDEWEB)

    Pinan Dawkrajai; Keita Yoshioka; Analis A. Romero; Ding Zhu; A.D. Hill; Larry W. Lake

    2005-10-01

    This project is motivated by the increasing use of distributed temperature sensors for real-time monitoring of complex wells (horizontal, multilateral and multi-branching wells) to infer the profiles of oil, gas, and water entry. Measured information can be used to interpret flow profiles along the wellbore including junction and build section. In this second project year, we have completed a forward model to predict temperature and pressure profiles in complex wells. As a comprehensive temperature model, we have developed an analytical reservoir flow model which takes into account Joule-Thomson effects in the near well vicinity and multiphase non-isothermal producing wellbore model, and couples those models accounting mass and heat transfer between them. For further inferences such as water coning or gas evaporation, we will need a numerical non-isothermal reservoir simulator, and unlike existing (thermal recovery, geothermal) simulators, it should capture subtle temperature change occurring in a normal production. We will show the results from the analytical coupled model (analytical reservoir solution coupled with numerical multi-segment well model) to infer the anomalous temperature or pressure profiles under various conditions, and the preliminary results from the numerical coupled reservoir model which solves full matrix including wellbore grids. We applied Ramey's model to the build section and used an enthalpy balance to infer the temperature profile at the junction. The multilateral wellbore temperature model was applied to a wide range of cases varying fluid thermal properties, absolute values of temperature and pressure, geothermal gradients, flow rates from each lateral, and the trajectories of each build section.

  14. An Aptamer-Based Nanobiosensor for Real-Time Measurements of ATP Dynamics

    DEFF Research Database (Denmark)

    Özalp, Cengiz; Nielsen, Lise Junker; Olsen, Lars Folke

    2010-01-01

    A nanosensor based on a new DNA aptamer can show changes in intracellular concentration of ATP of 0.5 to 8 mM and thereby the kinetics of ATP-consuming reactions in real-time. The biosensor was protected against nuclease attack by being buried in a polyacrylamide nanoparticle. This strategy can...

  15. WetLab-2: Tools for Conducting On-Orbit Quantitative Real-Time Gene Expression Analysis on ISS

    Science.gov (United States)

    Parra, Macarena; Almeida, Eduardo; Boone, Travis; Jung, Jimmy; Schonfeld, Julie

    2014-01-01

    The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR system, the Cepheid SmartCycler and will fly it in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid ramp times and the ability to detect up to four separate fluorescent channels at one time enabling multiplex assays that can be used for normalization and to study multiple genes of interest in each module. The team is currently working with Cepheid to enable the downlink of data from the ISS to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project has adapted commercial technology to design a module that can lyse cells and extract RNA of sufficient quality and quantity for use in qRT-PCR reactions while using a housekeeping gene to normalize RNA concentration and integrity. The WetLab-2 system is capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. The ability to conduct qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples. The system can be used to validate terrestrial analyses of samples returned from ISS by providing on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experiment parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Researchers will also be able to sample multigenerational changes in organisms. Finally, the system can be

  16. Whole-genome analysis of pseudorabies virus gene expression by real-time quantitative RT-PCR assay

    Directory of Open Access Journals (Sweden)

    Petrovszki Pál

    2009-10-01

    Full Text Available Abstract Background Pseudorabies virus (PRV, a neurotropic herpesvirus of pigs, serves as an excellent model system with which to investigate the herpesvirus life cycle both in cultured cells and in vivo. Real-time RT-PCR is a very sensitive, accurate and reproducible technique that can be used to detect very small amounts of RNA molecules, and it can therefore be applied for analysis of the expression of herpesvirus genes from the very early period of infection. Results In this study, we have developed and applied a quantitative reverse transcriptase-based real-time PCR technique in order to profile transcription from the whole genome of PRV after lytic infection in porcine kidney cells. We calculated the relative expression ratios in a novel way, which allowed us to compare different PRV genes with respect to their expression dynamics, and to divide the PRV genes into distinct kinetic classes. This is the first publication on the whole-genome analysis of the gene expression of an alpha-herpesvirus by qRT2-PCR. We additionally established the kinetic properties of uncharacterized PRV genes and revised or confirmed data on PRV genes earlier examined by traditional methods such as Northern blot analysis. Our investigations revealed that genes with the same expression properties form clusters on the PRV genome: nested overlapping genes belong in the same kinetic class, while most convergent genes belong in different kinetic classes. Further, we detected inverse relationships as concerns the expressions of EP0 and IE180 mRNAs and their antisense partners. Conclusion Most (if not all PRV genes begin to be expressed from the onset of viral expression. No sharp boundary was found between the groups of early and late genes classified on the basis of their requirement for viral DNA synthesis. The expressions of the PRV genes were analyzed, categorized and compared by qRT2-PCR assay, with the average of the minimum cycle threshold used as a control for

  17. Unmanned Airborne System Deployment at Turrialba Volcano for Real Time Eruptive Cloud Measurements

    Science.gov (United States)

    Diaz, J. A.; Pieri, D. C.; Fladeland, M. M.; Bland, G.; Corrales, E.; Alan, A., Jr.; Alegria, O.; Kolyer, R.

    2015-12-01

    The development of small unmanned aerial systems (sUAS) with a variety of instrument packages enables in situ and proximal remote sensing measurements of volcanic plumes, even when the active conditions of the volcano do not allow volcanologists and emergency response personnel to get too close to the erupting crater. This has been demonstrated this year by flying a sUAS through the heavy ash driven erupting volcanic cloud of Turrialba Volcano, while conducting real time in situ measurement of gases over the crater summit. The event also achieved the collection of newly released ash samples from the erupting volcano. The interception of the Turrialba ash cloud occurred during the CARTA 2015 field campaign carried out as part of an ongoing program for remote sensing satellite calibration and validation purposes, using active volcanic plumes. These deployments are timed to support overflights of the Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) onboard the NASA Terra satellite on a bimonthly basis using airborne platforms such as tethered balloons, free-flying fixed wing small UAVs at altitudes up to 12.5Kft ASL within about a 5km radius of the summit crater. The onboard instrument includes the MiniGas payload which consists of an array of single electrochemical and infrared gas detectors (SO2, H2S CO2), temperature, pressure, relative humidity and GPS sensors, all connected to an Arduino-based board, with data collected at 1Hz. Data are both stored onboard and sent by telemetry to the ground operator within a 3 km range. The UAV can also carry visible and infrared cameras as well as other payloads, such as a UAV-MS payload that is currently under development for mass spectrometer-based in situ measurements. The presentation describes the ongoing UAV- based in situ remote sensing validation program at Turrialba Volcano, the results of a fly-through the eruptive cloud, as well as future plans to continue these efforts. Work presented here was

  18. Successful Validation of Sample Processing and Quantitative Real-Time PCR Capabilities on the International Space Station

    Science.gov (United States)

    Parra, Macarena; Jung, Jimmy; Almeida, Eduardo; Boone, Travis; Schonfeld, Julie; Tran, Luan

    2016-01-01

    ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. The WetLab-2 Project is supported by the Research Integration Office in the ISS Program.

  19. Time-resolved measurements with intense ultrashort laser pulses: a 'molecular movie' in real time

    Science.gov (United States)

    Rudenko, A.; Ergler, Th; Feuerstein, B.; Zrost, K.; Schröter, C. D.; Moshammer, R.; Ullrich, J.

    2007-11-01

    We report on the high-resolution multidimensional real-time mapping of H2+ and D2+ nuclear wave packets performed employing time-resolved three-dimensional Coulomb explosion imaging with intense laser pulses. Exploiting a combination of a "reaction microscope" spectrometer and a pump-probe setup with two intense 6-7 fs laser pulses, we simultaneously visualize both vibrational and rotational motion of the molecule, and obtain a sequence of snapshots of the squared ro-vibrational wave function with time-step resolution of ~ 0.3 fs, allowing us to reconstruct a real-time movie of the ultrafast molecular motion. We observe fast dephasing, or 'collapse' of the vibrational wave packet and its subsequent revival, as well as signatures of rotational excitation. For D2+ we resolve also the fractional revivals resulting from the interference between the counter-propagating parts of the wave packet.

  20. XpertTrack: Precision Autonomous Measuring Device Developed for Real Time Shipments Tracker

    Directory of Open Access Journals (Sweden)

    Liviu Viman

    2016-03-01

    Full Text Available This paper proposes a software and hardware solution for real time condition monitoring applications. The proposed device, called XpertTrack, exchanges data through the GPRS protocol over a GSM network and monitories temperature and vibrations of critical merchandise during commercial shipments anywhere on the globe. Another feature of this real time tracker is to provide GPS and GSM positioning with a precision of 10 m or less. In order to interpret the condition of the merchandise, the data acquisition, analysis and visualization are done with 0.1 °C accuracy for the temperature sensor, and 10 levels of shock sensitivity for the acceleration sensor. In addition to this, the architecture allows increasing the number and the types of sensors, so that companies can use this flexible solution to monitor a large percentage of their fleet.

  1. IMU-based Real-time Pose Measurement system for Anterior Pelvic Plane in Total Hip Replacement Surgeries.

    Science.gov (United States)

    Zhe Cao; Shaojie Su; Hao Tang; Yixin Zhou; Zhihua Wang; Hong Chen

    2017-07-01

    With the aging of population, the number of Total Hip Replacement Surgeries (THR) increased year by year. In THR, inaccurate position of the implanted prosthesis may lead to the failure of the operation. In order to reduce the failure rate and acquire the real-time pose of Anterior Pelvic Plane (APP), we propose a measurement system in this paper. The measurement system includes two parts: Initial Pose Measurement Instrument (IPMI) and Real-time Pose Measurement Instrument (RPMI). IPMI is used to acquire the initial pose of the APP, and RPMI is used to estimate the real-time pose of the APP. Both are composed of an Inertial Measurement Unit (IMU) and magnetometer sensors. To estimate the attitude of the measurement system, the Extended Kalman Filter (EKF) is adopted in this paper. The real-time pose of the APP could be acquired together with the algorithm designed in the paper. The experiment results show that the Root Mean Square Error (RMSE) is within 1.6 degrees, which meets the requirement of THR operations.

  2. Quantitative real-time RT-PCR in sentinel lymph nodes from melanoma patients. Detection of melanocytic mRNA predicts disease-free survival

    DEFF Research Database (Denmark)

    Riber-Hansen, Rikke; Abrahamsen, Helene Nortvig; Sorensen, Boe Sandahl

    2008-01-01

    Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) for specific melanoma markers is more sensitive than histology for detecting cells of melanocytic origin in sentinel lymph nodes (SLNs) in cutaneous melanoma. The clinical significance of a positive qRT-PCR analysis...... is unclear. We performed qRT-PCR for the presence of MART-1 and tyrosinase in SLNs from 93 melanoma patients, and then followed these patients clinically (median follow-up time 43.5 months). We found a significant correlation between disease progression and presence of MART-1 mRNA in SLNs (p=0.......02), but no correlation with the amount of MART-1 mRNA as measured by qRT-PCR. No correlation between histology and recurrence was detected, recurrence rates being low in both histology-negative (12%) and -positive (15%) patients. We found a significant difference in disease recurrence between patients positive by both...

  3. [A comparative study between real time monitor KH-3000 and conventional Durham sampler measuring airborne pollen].

    Science.gov (United States)

    Yoda, Shigetoshi; Enomoto, Tadao; Shibano, Akira; Ikeda, Hiroki; Yajin, Shinji; Dake, Yoshihiro; Harada, Tamotsu

    2005-08-01

    Real time monitoring of airborne pollen has gradually increased because monitoring is laborsaving and provides better real-time information. A problem arose, however, due to differences between the KH3000 (Yamato Co. Ltd) monitor and the conventional Durham sampler pointed out in results of airborne pollen monitoring in Wakayama in 2004. We compared the two monitors for airborne pollen in Wakayama in 2004, which less dispersed than usual. The peak monitored by the KH-3000 monitor was not consistent with the prime period of Japanese cedar and cypress pollen dispersion, especially in February and April, although they correlated highly in March. The inconsistency in February is thought to be caused by snow, and that in April by falsely monitoring beech-tree airborne pollen-which is similar in size-in addition to Japanese cedar and cypress pollen. This report points out the need to take these conditions (snow and other plants pollen) into account when a real time monitor is used for collecting pollen information.

  4. Detection and quantification of viable Bacillus cereus group species in milk by propidium monoazide quantitative real-time PCR.

    Science.gov (United States)

    Cattani, Fernanda; Barth, Valdir C; Nasário, Jéssica S R; Ferreira, Carlos A S; Oliveira, Sílvia D

    2016-04-01

    The Bacillus cereus group includes important spore-forming bacteria that present spoilage capability and may cause foodborne diseases. These microorganisms are traditionally evaluated in food using culturing methods, which can be laborious and time-consuming, and may also fail to detect bacteria in a viable but nonculturable state. The purpose of this study was to develop a quantitative real-time PCR (qPCR) combined with a propidium monoazide (PMA) treatment to analyze the contamination of UHT milk by B. cereus group species viable cells. Thirty micrograms per milliliter of PMA was shown to be the most effective concentration for reducing the PCR amplification of extracellular DNA and DNA from dead cells. The quantification limit of the PMA-qPCR assay was 7.5 × 10(2) cfu/mL of milk. One hundred thirty-five UHT milk samples were analyzed to evaluate the association of PMA to qPCR to selectively detect viable cells. The PMA-qPCR was able to detect B. cereus group species in 44 samples (32.6%), whereas qPCR without PMA detected 78 positive samples (57.8%). Therefore, the PMA probably inhibited the amplification of DNA from cells that were killed during UHT processing, which avoided an overestimation of bacterial cells when using qPCR and, thus, did not overvalue potential health risks. A culture-based method was also used to detect and quantify B. cereus sensu stricto in the same samples and showed positive results in 15 (11.1%) samples. The culture method and PMA-qPCR allowed the detection of B. cereus sensu stricto in quantities compatible with the infective dose required to cause foodborne disease in 3 samples, indicating that, depending on the storage conditions, even after UHT treatment, infective doses may be reached in ready-to-consume products. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. Threshold microsclerotial inoculum for cotton verticillium wilt determined through wet-sieving and real-time quantitative PCR.

    Science.gov (United States)

    Wei, Feng; Fan, Rong; Dong, Haitao; Shang, Wenjing; Xu, Xiangming; Zhu, Heqin; Yang, Jiarong; Hu, Xiaoping

    2015-02-01

    Quantification of Verticillium dahliae microsclerotia is an important component of wilt management on a range of crops. Estimation of microsclerotia by dry or wet sieving and plating of soil samples on semiselective medium is a commonly used technique but this method is resource-intensive. We developed a new molecular quantification method based on Synergy Brands (SYBR) Green real-time quantitative polymerase chain reaction of wet-sieving samples (wet-sieving qPCR). This method can detect V. dahliae microsclerotia as low as 0.5 CFU g(-1) of soil. There was a high correlation (r=0.98) between the estimates of conventional plating analysis and the new wet-sieving qPCR method for 40 soil samples. To estimate the inoculum threshold for cotton wilt, >400 soil samples were taken from the rhizosphere of individual plants with or without visual wilt symptoms in experimental and commercial cotton fields at the boll-forming stage. Wilt inoculum was estimated using the wet-sieving qPCR method and related to wilt development. The estimated inoculum threshold varied with cultivar, ranging from 4.0 and 7.0 CFU g(-1) of soil for susceptible and resistant cultivars, respectively. In addition, there was an overall relationship of wilt incidence with inoculum density across 31 commercial fields where a single composite soil sample was taken at each field, with an estimated inoculum threshold of 11 CFU g(-1) of soil. These results suggest that wilt risk can be predicted from the estimated soil inoculum density using the new wet-sieving qPCR method. We recommend the use of 4.0 and 7.0 CFU g(-1) as an inoculum threshold on susceptible and resistant cultivars, respectively, in practical risk prediction schemes.

  6. Quantitative real-time PCR method with internal amplification control to quantify cyclopiazonic acid producing molds in foods.

    Science.gov (United States)

    Rodríguez, Alicia; Werning, María L; Rodríguez, Mar; Bermúdez, Elena; Córdoba, Juan J

    2012-12-01

    A quantitative TaqMan real-time PCR (qPCR) method that includes an internal amplification control (IAC) to quantify cyclopiazonic acid (CPA)-producing molds in foods has been developed. A specific primer pair (dmaTF/dmaTR) and a TaqMan probe (dmaTp) were designed on the basis of dmaT gene which encodes the enzyme dimethylallyl tryptophan synthase involved in the biosynthesis of CPA. The IAC consisted of a 105 bp chimeric DNA fragment containing a region of the hly gene of Listeria monocytogenes. Thirty-two mold reference strains representing CPA producers and non-producers of different mold species were used in this study. All strains were tested for CPA production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the designed qPCR method was demonstrated by the high linear relationship of the standard curves relating to the dmaT gene copy numbers and the Ct values obtained from the different CPA producers tested. The ability of the qPCR protocol to quantify CPA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1-4 log cfu/g in the different food matrices. The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g. This qPCR protocol including an IAC showed good efficiency to quantify CPA-producing molds in naturally contaminated foods avoiding false negative results. This method could be used to monitor the CPA producers in the HACCP programs to prevent the risk of CPA formation throughout the food chain. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Selection and validation of appropriate reference genes for quantitative real-time PCR analysis in Salvia hispanica.

    Directory of Open Access Journals (Sweden)

    Rahul Gopalam

    Full Text Available Quantitative real-time polymerase chain reaction (qRT-PCR has become the most popular choice for gene expression studies. For accurate expression analysis, it is pertinent to select a stable reference gene to normalize the data. It is now known that the expression of internal reference genes varies considerably during developmental stages and under different experimental conditions. For Salvia hispanica, an economically important oilseed crop, there are no reports of stable reference genes till date. In this study, we chose 13 candidate reference genes viz. Actin11 (ACT, Elongation factor 1-alpha (EF1-α, Eukaryotic translation initiation factor 3E (ETIF3E, alpha tubulin (α-TUB, beta tubulin (β-TUB, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Cyclophilin (CYP, Clathrin adaptor complex (CAC, Serine/threonine-protein phosphatase 2A (PP2A, FtsH protease (FtsH, 18S ribosomal RNA (18S rRNA, S-adenosyl methionine decarboxylase (SAMDC and Rubisco activase (RCA and the expression levels of these genes were assessed in a diverse set of tissue samples representing vegetative stages, reproductive stages and various abiotic stress treatments. Two of the widely used softwares, geNorm and Normfinder were used to evaluate the expression stabilities of these 13 candidate reference genes under different conditions. Results showed that GAPDH and CYP expression remain stable throughout in the different abiotic stress treatments, CAC and PP2A expression were relatively stable under reproductive stages and α-TUB, PP2A and ETIF3E were found to be stably expressed in vegetative stages. Further, the expression levels of Diacylglycerol acyltransferase (DGAT1, a key enzyme in triacylglycerol synthesis was analyzed to confirm the validity of reference genes identified in the study. This is the first systematic study of selection of reference genes in S. hispanica, and will benefit future expression studies in this crop.

  8. Systematic evaluation of three microRNA profiling platforms: microarray, beads array, and quantitative real-time PCR array.

    Directory of Open Access Journals (Sweden)

    Bin Wang

    Full Text Available BACKGROUND: A number of gene-profiling methodologies have been applied to microRNA research. The diversity of the platforms and analytical methods makes the comparison and integration of cross-platform microRNA profiling data challenging. In this study, we systematically analyze three representative microRNA profiling platforms: Locked Nucleic Acid (LNA microarray, beads array, and TaqMan quantitative real-time PCR Low Density Array (TLDA. METHODOLOGY/PRINCIPAL FINDINGS: The microRNA profiles of 40 human osteosarcoma xenograft samples were generated by LNA array, beads array, and TLDA. Results show that each of the three platforms perform similarly regarding intra-platform reproducibility or reproducibility of data within one platform while LNA array and TLDA had the best inter-platform reproducibility or reproducibility of data across platforms. The endogenous controls/probes contained in each platform have been observed for their stability under different treatments/environments; those included in TLDA have the best performance with minimal coefficients of variation. Importantly, we identify that the proper selection of normalization methods is critical for improving the inter-platform reproducibility, which is evidenced by the application of two non-linear normalization methods (loess and quantile that substantially elevated the sensitivity and specificity of the statistical data assessment. CONCLUSIONS: Each platform is relatively stable in terms of its own microRNA profiling intra-reproducibility; however, the inter-platform reproducibility among different platforms is low. More microRNA specific normalization methods are in demand for cross-platform microRNA microarray data integration and comparison, which will improve the reproducibility and consistency between platforms.

  9. Culture-independent identification and quantification of Gallibacterium anatis (G. anatis) by real-time quantitative PCR.

    Science.gov (United States)

    Wang, Chong; Robles, Francisco; Ramirez, Saul; Riber, Anja Brinch; Bojesen, Anders Miki

    2016-10-01

    Gallibacterium is a genus within the family Pasteurellaceae characterized by a high level of phenotypic and genetic diversity. No diagnostic method has yet been described, which allows species-specific identification of Gallibacterium anatis. The aim of this study was to develop a real-time quantitative PCR (qPCR) method allowing species-specific identification and quantification of G. anatis. A G. anatis specific DNA sequence was identified in the gyrase subunit B gene (gyrB) and used to design a TaqMan probe and corresponding primers. The specificity of the assay was tested on 52 bacterial strains. Twenty-two of the strains represented all of the presently available 13 phenotypic variants of G. anatis originating from different geographical locations. Nine strains represented each of the additional six Gallibacterium species and 21 strains represented other poultry associated bacterial species of the families Pasteurellaceae, Enterobacteriaceae and Flavobacteriaceae. Regarding specificity none of non-G. anatis strains tested positive with the proposed assay. To test and compare the qPCR method's ability to detect G. anatis from field samples, the sensitivity was compared to a previously published conventional PCR method and culture-based identification, respectively. The detection rates were 97%, 78% and 34% for the current qPCR, the conventional PCR and the culture-based identification method, respectively. The qPCR assay was able to detect the gene gyrB in serial dilutions of 10(8) colony forming units (CFU)/ml to as low as 10(0) CFU/ml copies. The proposed qPCR method is thus highly specific, sensitive and reproducible. In conclusion, we have developed a qPCR method that allows species-specific identification of G. anatis.

  10. Identification and validation of reference genes for quantitative real-time PCR normalization and its applications in lycium.

    Directory of Open Access Journals (Sweden)

    Shaohua Zeng

    Full Text Available Lycium barbarum and L. ruthenicum are extensively used as traditional Chinese medicinal plants. Next generation sequencing technology provides a powerful tool for analyzing transcriptomic profiles of gene expression in non-model species. Such gene expression can then be confirmed with quantitative real-time polymerase chain reaction (qRT-PCR. Therefore, use of systematically identified suitable reference genes is a prerequisite for obtaining reliable gene expression data. Here, we calculated the expression stability of 18 candidate reference genes across samples from different tissues and grown under salt stress using geNorm and NormFinder procedures. The geNorm-determined rank of reference genes was similar to those defined by NormFinder with some differences. Both procedures confirmed that the single most stable reference gene was ACNTIN1 for L. barbarum fruits, H2B1 for L. barbarum roots, and EF1α for L. ruthenicum fruits. PGK3, H2B2, and PGK3 were identified as the best stable reference genes for salt-treated L. ruthenicum leaves, roots, and stems, respectively. H2B1 and GAPDH1+PGK1 for L. ruthenicum and SAMDC2+H2B1 for L. barbarum were the best single and/or combined reference genes across all samples. Finally, expression of salt-responsive gene NAC, fruit ripening candidate gene LrPG, and anthocyanin genes were investigated to confirm the validity of the selected reference genes. Suitable reference genes identified in this study provide a foundation for accurately assessing gene expression and further better understanding of novel gene function to elucidate molecular mechanisms behind particular biological/physiological processes in Lycium.

  11. Identification and validation of reference genes for quantitative real-time PCR normalization and its applications in lycium.

    Science.gov (United States)

    Zeng, Shaohua; Liu, Yongliang; Wu, Min; Liu, Xiaomin; Shen, Xiaofei; Liu, Chunzhao; Wang, Ying

    2014-01-01

    Lycium barbarum and L. ruthenicum are extensively used as traditional Chinese medicinal plants. Next generation sequencing technology provides a powerful tool for analyzing transcriptomic profiles of gene expression in non-model species. Such gene expression can then be confirmed with quantitative real-time polymerase chain reaction (qRT-PCR). Therefore, use of systematically identified suitable reference genes is a prerequisite for obtaining reliable gene expression data. Here, we calculated the expression stability of 18 candidate reference genes across samples from different tissues and grown under salt stress using geNorm and NormFinder procedures. The geNorm-determined rank of reference genes was similar to those defined by NormFinder with some differences. Both procedures confirmed that the single most stable reference gene was ACNTIN1 for L. barbarum fruits, H2B1 for L. barbarum roots, and EF1α for L. ruthenicum fruits. PGK3, H2B2, and PGK3 were identified as the best stable reference genes for salt-treated L. ruthenicum leaves, roots, and stems, respectively. H2B1 and GAPDH1+PGK1 for L. ruthenicum and SAMDC2+H2B1 for L. barbarum were the best single and/or combined reference genes across all samples. Finally, expression of salt-responsive gene NAC, fruit ripening candidate gene LrPG, and anthocyanin genes were investigated to confirm the validity of the selected reference genes. Suitable reference genes identified in this study provide a foundation for accurately assessing gene expression and further better understanding of novel gene function to elucidate molecular mechanisms behind particular biological/physiological processes in Lycium.

  12. Fibroblast growth factor receptor 1 amplification in non-small cell lung cancer by quantitative real-time PCR.

    Directory of Open Access Journals (Sweden)

    Shirish M Gadgeel

    Full Text Available Amplification of the fibroblast growth factor receptor 1 (FGFR1 gene has been described in tumors of non-small-cell lung cancer (NSCLC patients. Prior reports showed conflicting rates of amplification frequency and clinical relevance.We developed a reliable real-time quantitative PCR assay to assess the frequency of FGFR1 amplification and assessed the optimal cutoff level of amplification for clinical application.In a training cohort of 203 NSCLCs, we established that a 3.5-fold amplification optimally divided patients into groups with different survival rates with a clear threshold level. Those with FGFR1 amplification levels above 3.5-fold had an inferior survival. These data were confirmed in a validation cohort of 142 NSCLC. After adjusting for age, sex, performance status, stage, and histology, patients with FGFR1 amplification levels above 3.5 fold had a hazard ratio of 2.91 (95% CI- 1.14, 7.41; pvalue-0.025 for death in the validation cohort. The rates of FGFR1 amplification using the cutoff level of 3.5 were 5.1% in squamous cell and 4.1% in adenocarcinomas. There was a non-significant trend towards higher amplifications rates in heavy smokers (> 15 pack-years of cigarette consumption as compared to light smokers.Our data suggest that a 3.5-fold amplification of FGFR1 is of clinical importance in NSCLC. Our cutpoint analysis showed a clear threshold effect for the impact of FGFR1 amplification on patients' survival, which can be used as an initial guide for patient selection in trials assessing efficacy of novel FGFR inhibitors.

  13. Absolute quantification of microRNAs in green tea (Camellia sinensis) by stem-loop quantitative real-time PCR.

    Science.gov (United States)

    Hou, Ying-Hui; Jeyaraj, Anburaj; Zhang, Xiao; Wei, Chao-Ling

    2017-07-01

    There are some studies to show that food-derived plant microRNAs (miRNAs) may be detected in mammals. The research evidence has provoked a considerable debate whether plant-derived miRNAs exert the same regulatory functions as endogenous animal miRNAs. To test the hypothesis, methods of highly sensitive absolute quantification miRNAs have been developed. However, absolute miRNA quantification of green tea has not yet been reported. This study is the first to build an absolute quantification method to detect miRNAs level in green tea using stem-loop quantitative real-time PCR (qRT-PCR). Two miRNAs, csn-miR164 (a conserved miRNA) and csn-miRn329 (a tea-specific miRNA), were selected as examples for the detection and absolute quantification of miRNAs in green tea samples using stem-loop qRT-PCR. The content of csn-miR164 was significantly higher in the Yuexi Cuilan (YX) samples than in the Shucheng Orchid (SC) samples. The content of csn-miRn329 was found to be high at the start of processing in leaf tissues in both the withering and soaking experiments, after which it gradually decreased with time. To the best of our knowledge, this is the first report to absolutely quantify the miRNAs present in green tea. This method will help to further investigate the possibility that tea-derived miRNAs may play an important role on defending against various diseases in humans. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  14. Ensuring good quality RNA for quantitative real-time PCR isolated from renal proximal tubular cells using laser capture microdissection.

    Science.gov (United States)

    Yee, Jie Yin; Limenta, Lie Michael George; Rogers, Keith; Rogers, Susan Mary; Tay, Vanessa S Y; Lee, Edmund J D

    2014-01-27

    In order to provide gene expression profiles of different cell types, the primary step is to isolate the specific cells of interest via laser capture microdissection (LCM), followed by extraction of good quality total RNA sufficient for quantitative real-time polymerase chain reaction (qPCR) analysis. This LCM-qPCR strategy has allowed numerous gene expression studies on specific cell populations, providing valuable insights into specific cellular changes in diseases. However, such strategy imposed challenges as cells of interests are often available in limited quantities and quality of RNA may be compromised during long periods of time spent on collection of cells and extraction of total RNA; therefore, it is crucial that protocols for sample preparation should be optimised according to different cell populations. We made several modifications to existing protocols to improve the total RNA yield and integrity for downstream qPCR analyses. A modified condensed hematoxylin and eosin (H&E) staining protocol was developed for the identification of rat renal proximal tubular cells (PTCs). It was then determined that a minimal of eight thousands renal PTCs were required to meet the minimal total RNA yield required for downstream qPCR. RNA integrity was assessed using at every progressive step of sample preparation. Therefore, we decided that the shortened H&E staining, together with microdissection should be performed consecutively within twenty minutes for good quality for gene expression analysis. These modified protocols were later applied on six individual rat samples. A panel of twenty rat renal drug transporters and five housekeeping genes showed Ct values below thirty-five, confirming the expression levels of these drug transporters can be detected. We had successfully optimized the protocols to achieve sufficient good quality total RNA from microdissected rat renal PTCs for gene expression profiling via qPCR. This protocol may be suitable for researchers who are

  15. Identification and testing of reference genes for Sesame gene expression analysis by quantitative real-time PCR.

    Science.gov (United States)

    Wei, Libin; Miao, Hongmei; Zhao, Ruihong; Han, Xiuhua; Zhang, Tide; Zhang, Haiyang

    2013-03-01

    Sesame (Sesamum indicum L.) is an ancient and important oilseed crop. However, few sesame reference genes have been selected for quantitative real-time PCR until now. Screening and validating reference genes is a requisite for gene expression normalization in sesame functional genomics research. In this study, ten candidate reference genes, i.e., SiACT, SiUBQ6, SiTUB, Si18S rRNA, SiEF1α, SiCYP, SiHistone, SiDNAJ, SiAPT and SiGAPDH, were chosen and examined systematically in 32 sesame samples. Three qRT-PCR analysis methods, i.e., geNorm, NormFinder and BestKeeper, were evaluated systematically. Results indicated that all ten candidate reference genes could be used as reference genes in sesame. SiUBQ6 and SiAPT were the optimal reference genes for sesame plant development; SiTUB was suitable for sesame vegetative tissue development, SiDNAJ for pathogen treatment, SiHistone for abiotic stress, SiUBQ6 for bud development and SiACT for seed germination. As for hormone treatment and seed development, SiHistone, SiCYP, SiDNAJ or SiUBQ6, as well as SiACT, SiDNAJ, SiTUB or SiAPT, could be used as reference gene, respectively. To illustrate the suitability of these reference genes, we analyzed the expression variation of three functional sesame genes of SiSS, SiLEA and SiGH in different organs using the optimal qRT-PCR system for the first time. The stability levels of optimal and worst reference genes screened for seed development, anther sterility and plant development were validated in the qRT-PCR normalization. Our results provided a reference gene application guideline for sesame gene expression characterization using qRT-PCR system.

  16. Real time magnetic field and flux measurements for tokamak control using a multi-core PCI Express system

    Energy Technology Data Exchange (ETDEWEB)

    Giannone, L. [Max-Planck-Institut fuer Plasmaphysik, IPP-EURATOM Association, 85748 Garching (Germany)], E-mail: Louis.Giannone@ipp.mpg.de; Schneider, W.; McCarthy, P.J.; Sips, A.C.C.; Treutterer, W.; Behler, K.; Eich, T.; Fuchs, J.C.; Hicks, N.; Kallenbach, A.; Maraschek, M.; Mlynek, A.; Neu, G.; Pautasso, G.; Raupp, G.; Reich, M.; Schuhbeck, K.H.; Stober, J.; Volpe, F.; Zehetbauer, T. [Max-Planck-Institut fuer Plasmaphysik, IPP-EURATOM Association, 85748 Garching (Germany)] (and others)

    2009-06-15

    The existing real time system for the position and shape control in ASDEX Upgrade has been extended to calculate magnetic flux surfaces in real time using a multi-core PCI Express system running LabVIEW RT. The availability of reflective memory for LabVIEW RT will allow this system to be connected to the control system and other diagnostics in a multi-platform real time network. The measured response of each magnetic probe to the individual poloidal field coil currents in the absence of plasma current is compared to the calculated value. Prior to a tokamak discharge this comparison can be used to check for failure of the magnetic probe, flux loop or integrator.

  17. Real-time In Situ Electron Spin Resonance Measurements on Fungal Spores of Penicillium digitatum during Exposure of Oxygen Plasmas

    CERN Document Server

    Ishikawa, Kenji; Tanaka, Hiromasa; Tamiya, Kazuhiro; Hashizume, Hiroshi; Ohta, Takayuki; Ito, Masafumi; Iseki, Sachiko; Takeda, Keigo; Kondo, Hiroki; Sekine, Makoto; Hori, Masaru

    2012-01-01

    We report the kinetic analysis of free radicals on fungal spores of Penicillium digitatum interacted with atomic oxygen generated plasma electric discharge using real time in situ electron spin resonance (ESR) measurements. We have obtained information that the ESR signal from the spores was observed and preliminarily assignable to semiquinone radical with a g-value of around 2.004 and a line width of approximately 5G. The decay of the signal is possibly linked to the inactivation of the fungal spore. The real-time in situ ESR has proven to be a useful method to elucidate plasma-induced surface reactions on biological specimens.

  18. Real-time micro-vibration multi-spot synchronous measurement within a region based on heterodyne interference

    Science.gov (United States)

    Lan, Ma; Xiao, Wen; Chen, Zonghui; Hao, Hongliang; Pan, Feng

    2018-01-01

    Real-time micro-vibration measurement is widely used in engineering applications. It is very difficult for traditional optical detection methods to achieve real-time need in a relatively high frequency and multi-spot synchronous measurement of a region at the same time,especially at the nanoscale. Based on the method of heterodyne interference, an experimental system of real-time measurement of micro - vibration is constructed to satisfy the demand in engineering applications. The vibration response signal is measured by combing optical heterodyne interferometry and a high-speed CMOS-DVR image acquisition system. Then, by extracting and processing multiple pixels at the same time, four digital demodulation technique are implemented to simultaneously acquire the vibrating velocity of the target from the recorded sequences of images. Different kinds of demodulation algorithms are analyzed and the results show that these four demodulation algorithms are suitable for different interference signals. Both autocorrelation algorithm and cross-correlation algorithm meet the needs of real-time measurements. The autocorrelation algorithm demodulates the frequency more accurately, while the cross-correlation algorithm is more accurate in solving the amplitude.

  19. A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods.

    Science.gov (United States)

    Nixon, Gavin J; Svenstrup, Helle F; Donald, Carol E; Carder, Caroline; Stephenson, Judith M; Morris-Jones, Stephen; Huggett, Jim F; Foy, Carole A

    2014-12-01

    Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These 'isothermal' methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

  20. A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods

    Directory of Open Access Journals (Sweden)

    Gavin J. Nixon

    2014-12-01

    Full Text Available Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR. There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These ‘isothermal’ methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT, akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

  1. Real-time measurement of outdoor worker’s exposure to solar ultraviolet radiation in Pretoria, South Africa

    CSIR Research Space (South Africa)

    Makgabutlane, M

    2015-05-01

    Full Text Available significant amount of time outside and are thus susceptible to this risk. In this case study, we estimated, for the first time, the real-time solar UVR exposure of an outdoor worker in Pretoria. Measurements were made on 27 and 28 May 2013 using a handheld...

  2. Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR.

    Directory of Open Access Journals (Sweden)

    Deshui Liu

    Full Text Available Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant-pathogen interactions. Quantitative real-time PCR (qPCR is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1α, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41 were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X. Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N

  3. Evaluation of some software measuring displacements using GPS in real-time

    Science.gov (United States)

    Langbein, John

    2006-01-01

    For the past decade, the USGS has been monitoring deformation at various locations in the western United States using continuous GPS. The main focus of these measurements are estimates of displacement averaged over one day. Essentially, these consist of recording at 30 seconds intervals the carrier-frequency phase-data (equivalent to travel-time) between a GPS receiver and the GPS satellite network. In turn, these observations, which are converted to pseudo—ranges, are processed using one of the “research grade” programs (GIPSY, Zumberge et al., or GAMIT, wwwgpsg.mit.edu/~simon/gtgk) to estimate the position of the GPS receiver averaged over 24 hours. However, it is possible and desirable to estimate the position of the receiver (actually the antenna) more frequently and to do this within a few seconds of the time actual measurement (known as real-time). A recent example, the 2004 Magnitude 6, Parkfield, California earthquake, demonstrated that having GPS estimates of position more frequently than simply a daily average is required if one requires discrimination between co-seismic and post-seismic deformation (Langbein et al., 2006). The high-rate estimates of position obtained at Parkfield show that post-seismic deformation started less than one-hour after the mainshock and that this deformation was roughly the same magnitude as the co-seismic deformation. The high-rate solutions for Parkfield were done by others including Yehuda Bock at UCSD and Kristine Larson at U. of Colorado, but not the USGS. The Parkfield experience points out the need for an in-house capability by the USGS to be able to accurately measure co-seismic displacements and other rapid, deformation signals using GPS. This applies to both the Earthquake and Volcano Hazard programs. Although at many locations where we monitor deformation, we have strainmeters and tiltmeters in addition to GPS which, in principle, are far more sensitive to rapid deformation over periods of less than a day

  4. Quantitative magnetic resonance temperature mapping for real-time monitoring of radiofrequency ablation of the liver: an ex vivo study

    Energy Technology Data Exchange (ETDEWEB)

    Seror, Olivier [ERT CNRS/Universite Victor Segalen Bordeaux 2, Laboratoire d' Imagerie Moleculaire et Fonctionnelle, Bordeaux (France); Assistance Publique-Hopitaux de Paris/CHU Paris XIII, Service de Radiologie Hopital Jean Verdier, Bondy (France); Universite Paris 13, UPRES EA 3409, UFR SMBH, Bobigny (France); Lepetit-Coiffe, Matthieu; Quesson, Bruno; Moonen, Chrit T.W [ERT CNRS/Universite Victor Segalen Bordeaux 2, Laboratoire d' Imagerie Moleculaire et Fonctionnelle, Bordeaux (France); Trillaud, Herve [ERT CNRS/Universite Victor Segalen Bordeaux 2, Laboratoire d' Imagerie Moleculaire et Fonctionnelle, Bordeaux (France); Hopital Saint-Andre, CHU Bordeaux, Service de Radiologie, Bordeaux (France)

    2006-10-15

    We evaluated the feasibility and accuracy of real-time magnetic resonance (MR) thermometry for monitoring radiofrequency (RF) ablation in the liver. Continuous MR temperature mapping was used to monitor bipolar RF ablations performed in ex vivo livers with and without flow using two parallel electrodes. Macroscopic inspection of ablation zones was compared with thermal dose maps (TDm) and T1-weighted inversion recovery turbo spin echo (IR-TSE) images for their size and shape and the influence of flow. Pearson's correlation (r), Bland and Altman tests and kappa ({chi}K) tests were performed. The mean differences in ablation zone size between macroscopic and TDm and IR-TSE measurements were +4 mm and -2 mm, respectively. TDm was well correlated with macroscopy (r=0.77 versus r=0.44 for IR-TSE). TDm was found to be more precise for shape recognition ({chi}K=0.73 versus {chi}K=0.55 for IR-TSE) and for detection of an intact ring of liver due to the cooling effect of flow which was impossible with IR-TSE. Simultaneous monitoring of RF ablation by MR thermometry is feasible and reliable for predicting the shape of ablation zones and the impact of the heat-sink effect of flow. Further studies are needed to confirm these results in vivo. (orig.)

  5. Design of a real time quantitative PCR assay to assess global mRNA amplification of small size specimens for microarray hybridisation

    Science.gov (United States)

    Choesmel, V; Foucault, F; Thiery, J P; Blin, N

    2004-01-01

    Background: Low RNA yields from clinical samples are a limiting step for microarray technology. Aims: To design an accurate real time quantitative polymerase chain reaction (PCR) assay to assess the crucial step of global mRNA amplification performed before microarray hybridisation, using less than 1 µg total RNA. Methods: Three RNA extraction procedures were compared for small size samples. Total RNA was amplified from universal RNA or the BC-H1 breast cancer micrometastatic cell line using three different protocols. Real time quantitative PCR technology was used for accurate measurement of urokinase plasminogen activator receptor and cytokeratin 8 RNA amplification rates and ratios, using primer sets binding at various distances from the 3′ end of transcripts. A 50 mer oligomeric array targeting 87 genes potentially involved in breast cancer metastatic progression was built and hybridised with amplified RNA. Results: Eighteen nanograms of total RNA could be purified from 1000 BC-H1 micrometastatic cells. Amplification rates of 25 000 to 100 000 were achieved with as little as 10 ng of starting material. However, results were highly variable, depending on the amount of starting material, gene characteristics, sample quality, and protocols used. Oligomeric array hybridisation with 20 µg reference RNA resulted in specific and reproducible signals for 83% of the genes, whereas mRNA amplification from less than 400 ng of starting material resulted in selective detection of signals from highly expressed genes. Conclusions: Improvements in the design of global mRNA amplification procedures and oligomeric arrays are needed to extract informative gene expression data from clinical samples containing limited cell numbers. PMID:15563668

  6. A Human Fecal Contamination Score for Ranking Recreational Sites using the HF183/BacR287 Quantitative Real-Time PCR Method

    Science.gov (United States)

    Human fecal pollution of recreational waters remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality research and manag...

  7. Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification ▿

    Science.gov (United States)

    Yankson, Kweku K.; Steck, Todd R.

    2009-01-01

    We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil. PMID:19633108

  8. MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments

    NARCIS (Netherlands)

    Bustin, S.A.; Beaulieu, J.F.; Huggett, J.; Jaggi, R.; Kibenge, F.S.; Olsvik, P.A.; Penning, L.C.|info:eu-repo/dai/nl/110369181; Toegel, S.

    2010-01-01

    MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments Stephen A Bustin1 , Jean-François Beaulieu2 , Jim Huggett3 , Rolf Jaggi4 , Frederick SB Kibenge5 , Pål A Olsvik6 , Louis C Penning7 and Stefan Toegel8 1 Centre for

  9. Detection of micrometastatic breast cancer by means of real time quantitative RT-PCR and immunostaining in perioperative blood samples and sentinel nodes

    NARCIS (Netherlands)

    Schroder, CP; Ruiters, MHJ; de Jong, S; Tiebosch, ATMG; Wesseling, J; de Vries, J; Hoekstra, HJ; de Leij, LFMH; de Vries, EGE; Veenstra, R.

    2003-01-01

    The aim of our study was to detect micrometastatic breast cancer by epithelial glycoprotein-2 (EGP-2) and cytokeratin 19 (CK19), using immunostaining and real time quantitative reverse transcriptase-polymerise chain reaction (qRT-PCR). Fifty-eight breast cancer patients, 52 primary tumors, 75

  10. The applicability of TaqMan-based quantitative real-time PCR assays for detecting and enumeratIng Cryptosporidium spp. oocysts in the environment

    Science.gov (United States)

    Molecular detection methods such as PCR have been extensively used to type Cryptosporidium oocysts detected in the environment. More recently, studies have developed quantitative real-time PCR assays for detection and quantification of microbial contaminants in water as well as ...

  11. Assessment of offsite, real-time dose measurement systems for emergency situations

    Energy Technology Data Exchange (ETDEWEB)

    Maeck, W.J.; Hoffman, L.G.; Staples, B.A.; Keller, J.H.

    1982-04-01

    An evaluation is made of the effectiveness of fixed, real-time monitoring systems around nuclear power stations in determining the magnitude of unmonitored releases. The effects of meteorological conditions on the accuracy with which the magnitude of unmonitored releases is determined and the uncertainties inherent in defining these meteorological conditions are discussed. The number and placement of fixed field detectors in a system is discussed, and the data processing equipment required to convert field detector output data into release rate information is described. Cost data relative to the purchase and installation of specific systems are given, as well as the characteristics and information return for a system purchased at an arbitrary cost.

  12. An FPGA Architecture for Extracting Real-Time Zernike Coefficients from Measured Phase Gradients

    Science.gov (United States)

    Moser, Steven; Lee, Peter; Podoleanu, Adrian

    2015-04-01

    Zernike modes are commonly used in adaptive optics systems to represent optical wavefronts. However, real-time calculation of Zernike modes is time consuming due to two factors: the large factorial components in the radial polynomials used to define them and the large inverse matrix calculation needed for the linear fit. This paper presents an efficient parallel method for calculating Zernike coefficients from phase gradients produced by a Shack-Hartman sensor and its real-time implementation using an FPGA by pre-calculation and storage of subsections of the large inverse matrix. The architecture exploits symmetries within the Zernike modes to achieve a significant reduction in memory requirements and a speed-up of 2.9 when compared to published results utilising a 2D-FFT method for a grid size of 8×8. Analysis of processor element internal word length requirements show that 24-bit precision in precalculated values of the Zernike mode partial derivatives ensures less than 0.5% error per Zernike coefficient and an overall error of DSP blocks independent of the Shack-Hartmann grid size. Block RAM usage is <16% for Shack-Hartmann grid sizes up to 32×32.

  13. Real-time fiber Bragg grating measurement system using temperature-controlled Fourier domain mode locking laser

    Science.gov (United States)

    Yamaguchi, Tatsuya; Shinoda, Yukitaka

    2017-06-01

    We constructed a temperature-controlled Fourier domain mode locking (TC-FDML) laser capable of high-speed wavelength sweeping and developed a real-time fiber Bragg grating (FBG) measurement system. The TC-FDML laser can perform high-speed wavelength sweeping at a sweep frequency of 50.7 kHz with a scan range of ˜60 nm in the 1.55-μm band. This system uses a data acquisition system mounting an analog/digital converter and field programmable gate array that enables real-time FBG measurement at a sampling frequency of 250 MHz. Using bidirectional wavelength sweeping by the TC-FDML laser, the system has a measurement time resolution of 9.9 μs. We show that the developed system can measure high-speed vibrations of several kHz and perform simultaneous and continuous measurements of multiple FBGs for a period of one hour.

  14. Charm and J/psi cross section measurements at 13 TeV with real-time calibration

    CERN Multimedia

    CERN. Geneva

    2015-01-01

    The LHC's sqrt(s) = 13 TeV proton-proton collisions open a new regime in which the predictions of QCD may be precisely tested via production measurements. LHCb undertook an Early 2015 Measurements campaign to coordinate the operational and analysis activities that are required for rapid completion of such production measurements. The Early Measurements campaign is now bearing fruit with the recent publication of J/psi cross-sections and the imminent publication of charm hadron cross-sections. These are the first results to rely on LHCb's new real-time calibration system, in which the sub-detectors are promptly calibrated and the full event reconstruction of the software High Level Trigger has analysis-quality precision. This seminar will discuss the LHCb real-time calibration system and our recent charm and J/psi production measurements at sqrt(s) = 13 TeV.

  15. Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment

    Directory of Open Access Journals (Sweden)

    Li Qingdi

    2012-06-01

    Full Text Available Abstract Background The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata. Results The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25 remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1α, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4 were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-ΔΔCT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-ΔΔCT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13 as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such

  16. Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

    Science.gov (United States)

    Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

    2014-01-01

    Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate

  17. Screening suitable reference genes for normalization in reverse transcription quantitative real-time PCR analysis in melon.

    Science.gov (United States)

    Kong, Qiusheng; Yuan, Jingxian; Niu, Penghui; Xie, Junjun; Jiang, Wei; Huang, Yuan; Bie, Zhilong

    2014-01-01

    Melon (Cucumis melo. L) is not only an economically important cucurbitaceous crop but also an attractive model for studying many biological characteristics. Screening appropriate reference genes is essential to reverse transcription quantitative real-time PCR (RT-qPCR), which is key to many studies involving gene expression analysis. In this study, 14 candidate reference genes were selected, and the variations in their expression in roots and leaves of plants subjected to biotic stress, abiotic stress, and plant growth regulator treatment were assessed by RT-qPCR. The stability of the expression of the selected genes was determined and ranked using geNorm and NormFinder. geNorm identified the two most stable genes for each set of conditions: CmADP and CmUBIep across all samples, CmUBIep and CmRPL in roots, CmRAN and CmACT in leaves, CmADP and CmRPL under abiotic stress conditions, CmTUA and CmACT under biotic stress conditions, and CmRAN and CmACT under plant growth regulator treatments. NormFinder determined CmRPL to be the best reference gene in roots and under biotic stress conditions and CmADP under the other experimental conditions. CmUBC2 and CmPP2A were not found to be suitable under many experimental conditions. The catalase family genes CmCAT1, CmCAT2, and CmCAT3 were identified in melon genome and used as target genes to validate the reliability of identified reference genes. The catalase family genes showed the most upregulation 3 days after inoculation with Fusarium wilt in roots, after which they were downregulated. Their levels of expression were significantly overestimated when the unsuitable reference gene was used for normalization. These results not only provide guidelines for the selection of reference genes for gene expression analyses in melons but may also provide valuable information for studying the functions of catalase family genes in stress responses.

  18. Quantitative real-time PCR and fluorescence in situ hybridization approaches for enumerating Brevundimonas diminuta in drinking water.

    Science.gov (United States)

    Donofrio, Robert S; Bestervelt, Lorelle L; Saha, Ratul; Bagley, Susan T

    2010-09-01

    Brevundimonas diminuta is a small Gram-negative bacterium used for validation of membranes and filters used in the pharmaceutical and drinking water treatment industries. Current assays are time consuming, nonselective, and may be subject to interference by competing indigenous microorganisms. The focus of this study is to develop rapid and specific enumeration methodologies for B. diminuta. Quantitative real-time polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) assays were developed based on the gyrB (1,166 bp) and rpoD (829 bp) gene sequences of B. diminuta ATCC 19146. Species-specific primers and probes were designed, and a 100-200 bp segment of each gene was targeted in the qPCR studies. For both the qPCR and FISH assays, an internal 25 bp sequence was selected for use as a TaqMan probe (labeled with 6-FAM and a Black Hole Quencher). Probe specificity studies, conducted against Gram-negative and Gram-positive reference strains as well as environmental strains, revealed high specificity of the primer/probe pairs to B. diminuta. Sensitivities of the qPCR reactions using purified genomic DNA from B. diminuta were determined to be 0.89 pg for rpoD and 8.9 pg for gyrB. The feasibility of using whole-cell B. diminuta suspensions directly with the rpoD qPCR protocol was also evaluated. The greatest sensitivity observed for B. diminuta was 1 x 10(3) colony forming units (CFU) per mL when tryptic soy broth was used as the growth medium. When compared with direct microscopic enumeration using a 5' 6-FAM FISH probe, traditional plating methods showed significant underestimation of B. diminuta concentration (P = 0.01) when this organism was cultivated in saline lactose broth. The results of this investigation demonstrate that qPCR and FISH are effective methods for rapid (drinking water filtration systems.

  19. O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis.

    Science.gov (United States)

    Mekonnen, Solomon A; Beissner, Marcus; Saar, Malkin; Ali, Solomon; Zeynudin, Ahmed; Tesfaye, Kassahun; Adbaru, Mulatu G; Battke, Florian; Poppert, Sven; Hoelscher, Michael; Löscher, Thomas; Bretzel, Gisela; Herbinger, Karl-Heinz

    2017-10-02

    Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis.

  20. Screening suitable reference genes for normalization in reverse transcription quantitative real-time PCR analysis in melon.

    Directory of Open Access Journals (Sweden)

    Qiusheng Kong

    Full Text Available Melon (Cucumis melo. L is not only an economically important cucurbitaceous crop but also an attractive model for studying many biological characteristics. Screening appropriate reference genes is essential to reverse transcription quantitative real-time PCR (RT-qPCR, which is key to many studies involving gene expression analysis. In this study, 14 candidate reference genes were selected, and the variations in their expression in roots and leaves of plants subjected to biotic stress, abiotic stress, and plant growth regulator treatment were assessed by RT-qPCR. The stability of the expression of the selected genes was determined and ranked using geNorm and NormFinder. geNorm identified the two most stable genes for each set of conditions: CmADP and CmUBIep across all samples, CmUBIep and CmRPL in roots, CmRAN and CmACT in leaves, CmADP and CmRPL under abiotic stress conditions, CmTUA and CmACT under biotic stress conditions, and CmRAN and CmACT under plant growth regulator treatments. NormFinder determined CmRPL to be the best reference gene in roots and under biotic stress conditions and CmADP under the other experimental conditions. CmUBC2 and CmPP2A were not found to be suitable under many experimental conditions. The catalase family genes CmCAT1, CmCAT2, and CmCAT3 were identified in melon genome and used as target genes to validate the reliability of identified reference genes. The catalase family genes showed the most upregulation 3 days after inoculation with Fusarium wilt in roots, after which they were downregulated. Their levels of expression were significantly overestimated when the unsuitable reference gene was used for normalization. These results not only provide guidelines for the selection of reference genes for gene expression analyses in melons but may also provide valuable information for studying the functions of catalase family genes in stress responses.

  1. Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Lycoris aurea

    Science.gov (United States)

    Ma, Rui; Xu, Sheng; Zhao, Yucheng; Xia, Bing; Wang, Ren

    2016-01-01

    Lycoris aurea (L' Hér.) Herb, a perennial grass species, produces a unique variety of pharmacologically active Amaryllidaceae alkaloids. However, the key enzymes and their expression pattern involved in the biosynthesis of Amaryllidaceae alkaloids (especially for galanthamine) are far from being fully understood. Quantitative real-time polymerase chain reaction (qRT-PCR), a commonly used method for quantifying gene expression, requires stable reference genes to normalize its data. In this study, to choose the appropriate reference genes under different experimental conditions, 14 genes including YLS8 (mitosis protein YLS8), CYP2 (Cyclophilin 2), CYP 1 (Cyclophilin 1), TIP41 (TIP41-like protein), EXP2 (Expressed protein 2), PTBP1 (Polypyrimidine tract-binding protein 1), EXP1 (Expressed protein 1), PP2A (Serine/threonine-protein phosphatase 2A), β-TUB (β-tubulin), α-TUB (α-tubulin), EF1-α (Elongation factor 1-α), UBC (Ubiquitin-conjugating enzyme), ACT (Actin) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were selected from the transcriptome datasets of L. aurea. And then, expressions of these genes were assessed by qRT-PCR in various tissues and the roots under different treatments. The expression stability of the 14 candidates was analyzed by three commonly used software programs (geNorm, NormFinder, and BestKeeper), and their results were further integrated into a comprehensive ranking based on the geometric mean. The results show the relatively stable genes for each subset as follows: (1) EXP1 and TIP41 for all samples; (2) UBC and EXP1 for NaCl stress; (3) PTBP1 and EXP1 for heat stress, polyethylene glycol (PEG) stress and ABA treatment; (4) UBC and CYP2 for cold stress; (5) PTBP1 and PP2A for sodium nitroprusside (SNP) treatment; (6) CYP1 and TIP41 for methyl jasmonate (MeJA) treatment; and (7) EXP1 and TIP41 for various tissues. The reliability of these results was further enhanced through comparison between part qRT-PCR result and RNA

  2. Comparison of lung cancer cell lines representing four histopathological subtypes with gene expression profiling using quantitative real-time PCR

    Directory of Open Access Journals (Sweden)

    Kawaguchi Makoto

    2010-01-01

    Full Text Available Abstract Background Lung cancers are the most common type of human malignancy and are intractable. Lung cancers are generally classified into four histopathological subtypes: adenocarcinoma (AD, squamous cell carcinoma (SQ, large cell carcinoma (LC, and small cell carcinoma (SC. Molecular biological characterization of these subtypes has been performed mainly using DNA microarrays. In this study, we compared the gene expression profiles of these four subtypes using twelve human lung cancer cell lines and the more reliable quantitative real-time PCR (qPCR. Results We selected 100 genes from public DNA microarray data and examined them by DNA microarray analysis in eight test cell lines (A549, ABC-1, EBC-1, LK-2, LU65, LU99, STC 1, RERF-LC-MA and a normal control lung cell line (MRC-9. From this, we extracted 19 candidate genes. We quantified the expression of the 19 genes and a housekeeping gene, GAPDH, with qPCR, using the same eight cell lines plus four additional validation lung cancer cell lines (RERF-LC-MS, LC-1/sq, 86-2, and MS-1-L. Finally, we characterized the four subtypes of lung cancer cell lines using principal component analysis (PCA of gene expression profiling for 12 of the 19 genes (AMY2A, CDH1, FOXG1, IGSF3, ISL1, MALL, PLAU, RAB25, S100P, SLCO4A1, STMN1, and TGM2. The combined PCA and gene pathway analyses suggested that these genes were related to cell adhesion, growth, and invasion. S100P in AD cells and CDH1 in AD and SQ cells were identified as candidate markers of these lung cancer subtypes based on their upregulation and the results of PCA analysis. Immunohistochemistry for S100P and RAB25 was closely correlated to gene expression. Conclusions These results show that the four subtypes, represented by 12 lung cancer cell lines, were well characterized using qPCR and PCA for the 12 genes examined. Certain genes, in particular S100P and CDH1, may be especially important for distinguishing the different subtypes. Our results

  3. Real-time measurement of kidney tubule fluid nitric oxide concentrations in early diabetes: disparate changes in different rodent models.

    Science.gov (United States)

    Levine, David Z; Iacovitti, Michelle

    2006-08-01

    There are several reports indicating that nitric oxide (NO) plays a role in the kidney hyperfiltration seen in the early stages of diabetes mellitus (DM). Whole kidney GFR and single nephron GFR (SNGFR) have been reported to decrease after nitric oxide synthase (NOS) inhibition. To date, no direct, in vivo, quantitative NO measurements have been made within the kidney in any models of early diabetes. To assess the possible association of changes in tubular fluid nitric oxide concentrations (TF [NO]) with early diabetes, a specially modified NO electrode with a tip diameter of about 7 microm was used to measure NO in single tubules in seven rodent groups. In the Sprague-Dawley (SD) rat model, TF [NO] increased by 50% after streptozotocin (STZ) induced DM1. In the B6129G2/J mouse, control TF [NO] was more than twice the rat control value and fell by 50% after STZ treatment. In three other groups of mice-db/db (B6.Cg-m+/+Lepr(db)/J) Type II diabetic (DM2) mouse, db/m (its heterozygote), and the corresponding wild type (WT)-TF [NO] was also much higher than in the rat, and unlike the B6129G2/J STZ diabetic mouse, did not change after the onset of diabetes. Blood glucose concentrations were similar in the three diabetic groups. Accordingly, in different rodent models of diabetes, in vivo TF [NO], measured in real time, varies significantly in control animals and directionally in different models of DM1 and DM2.

  4. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    Energy Technology Data Exchange (ETDEWEB)

    Huang, S-H; Tsai, M-H; Lin, C-W [Department of Biotechnology, College of Health Science, Asia University, Wufeng, Taichung, Taiwan (China); Yang, T-C; Chuang, P-H [Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan (China); Tsai, I-S; Lu, H-C [Nanotechnology Research Center, Feng Chia University, Taichung, Taiwan (China); Wan Lei; Lin, Y-J [Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung, Taiwan (China); Lai, C-H [Department of Microbiology and Immunology, China Medical University, Taichung, Taiwan (China)], E-mail: cwlin@mail.cmu.edu.tw

    2008-10-08

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  5. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    Science.gov (United States)

    Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

    2008-10-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  6. Culture-independent identification and quantification of Gallibacterium anatis (G. anatis) by real-time quantitative PCR

    DEFF Research Database (Denmark)

    Wang, Chong; Robles, Francisco; Ramirez, Saul

    2016-01-01

    Gallibacterium is a genus within the family Pasteurellaceae characterized by a high level of phenotypic and genetic diversity. No diagnostic method has yet been described, which allows species-specific identification of Gallibacterium anatis. The aim of this study was to develop a real-time quant...

  7. Development of a quantitative real-time detection assay for hepatitis B virus DNA and comparison with two commercial assays

    NARCIS (Netherlands)

    Pas, S D; Fries, E; De Man, R A; Osterhaus, A D; Niesters, H G

    A highly reproducible and sensitive real-time detection assay based on TaqMan technology was developed for the detection of hepatitis B virus (HBV) DNA and compared with two commercially available assays. The assay was validated with the Viral Quality Control panel, which also includes EUROHEP HBV

  8. Development of a quantitative real-time detection assay for hepatitis B virus DNA and comparison with two commercial assays

    NARCIS (Netherlands)

    S.D. Pas (Suzan); E. Fries; H.G.M. Niesters (Bert); R.A. de Man (Robert); A.D.M.E. Osterhaus (Albert)

    2000-01-01

    textabstractA highly reproducible and sensitive real-time detection assay based on TaqMan technology was developed for the detection of hepatitis B virus (HBV) DNA and compared with two commercially available assays. The assay was validated with the Viral Quality Control panel,

  9. Real time- and control software for the new orbit measurement system for the CERN SPS

    CERN Document Server

    De Vries, J C; Boccard, C; Bogey, T; Coussemaeker, D; Dach, M; Gras, J J; Hiller, H; Jackson, S; Rybalchenko, K; Brazier, J C L

    1999-01-01

    The 240 channel SPS Orbit acquisition system is implemented on a PowerPC under the LynxOS operating system, making use of multi threaded real-time capabilities. The acquired data is transferred efficiently by DMA via the PCI bus into the main memory. System configuration aspects were implemented in a Broker architecture, where individual threads communicate with an Oracle database and the acquisition systems. This Broker hides the implementation details of the front-end systems. A versatile configuration client is provided in Java, to provide both local graphical user interfaces and remote WWW access using a dedicated gateway to the SL equipment layer. The timing diagnostics of the acquisition system are provided in a LabView application integrating oscilloscope control and channel multiplex control. This paper describes in detail the technical solutions implemented and reports on the arguments, which have led to particular choices.

  10. Alchemy: A Web 2.0 Real-time Quality Assurance Platform for Human Immunodeficiency Virus, Hepatitis C Virus, and BK Virus Quantitation Assays.

    Science.gov (United States)

    Agosto-Arroyo, Emmanuel; Coshatt, Gina M; Winokur, Thomas S; Harada, Shuko; Park, Seung L

    2017-01-01

    The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT). Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs) contain all the data elements necessary to do accurate quality assurance (QA) reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV) quantitation, hepatitis C virus (HCV) quantitation, and BK virus (BKV) quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45-60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician-supervised resident/fellow programming projects as learning

  11. Alchemy: A web 2.0 real-time quality assurance platform for human immunodeficiency Virus, hepatitis C Virus, and BK Virus quantitation assays

    Directory of Open Access Journals (Sweden)

    Emmanuel Agosto-Arroyo

    2017-01-01

    Full Text Available Background: The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT. Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs contain all the data elements necessary to do accurate quality assurance (QA reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Methods: Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV quantitation, hepatitis C virus (HCV quantitation, and BK virus (BKV quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Results: Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45–60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Conclusions: Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician

  12. Real-time interferometric monitoring and measuring of photopolymerization based stereolithographic additive manufacturing process: sensor model and algorithm

    Science.gov (United States)

    Zhao, X.; Rosen, D. W.

    2017-01-01

    As additive manufacturing is poised for growth and innovations, it faces barriers of lack of in-process metrology and control to advance into wider industry applications. The exposure controlled projection lithography (ECPL) is a layerless mask-projection stereolithographic additive manufacturing process, in which parts are fabricated from photopolymers on a stationary transparent substrate. To improve the process accuracy with closed-loop control for ECPL, this paper develops an interferometric curing monitoring and measuring (ICM&M) method which addresses the sensor modeling and algorithms issues. A physical sensor model for ICM&M is derived based on interference optics utilizing the concept of instantaneous frequency. The associated calibration procedure is outlined for ICM&M measurement accuracy. To solve the sensor model, particularly in real time, an online evolutionary parameter estimation algorithm is developed adopting moving horizon exponentially weighted Fourier curve fitting and numerical integration. As a preliminary validation, simulated real-time measurement by offline analysis of a video of interferograms acquired in the ECPL process is presented. The agreement between the cured height estimated by ICM&M and that measured by microscope indicates that the measurement principle is promising as real-time metrology for global measurement and control of the ECPL process.

  13. Real-time measurements of particulate matter emissions from stationary sources with ELPI and TEOM series 7000

    Energy Technology Data Exchange (ETDEWEB)

    Louer, P.; Chaucherie, X. [Sechaud Environnement, Maizieres-Les-Metz (France)

    2006-07-01

    The Electrical Low Pressure Impactor (ELPI) from DEKATI Ltd. has the capability to provide real-time measurement of fine (PM2.5) and ultrafine (PM 0.1) particle size distribution and concentrations. The ELPI was compared with the TEOM Series 7000 Source Particulate Monitor, from Rupprecht and Pataschnick, to determine its reliability to measure particle mass concentrations from stationary sources. The ELPI measures in real-time particles concentrations and particles sizes from 0.03 to 10 {mu}m in diameter. The operation is based on particle charging in an unipolar diode charger, size classification in an inertial impactor and electrical detection with a multi-channel electrometer. The TEOM Series 7000 provides in real-time high resolution, in situ direct mass measurement of particulate concentrations, using the TEOM technology (Tapered Element Oscillating Microbalance). Measurements of particulate matter (PM) emissions form the ELPI and TEOM Series 7000 were conducted at coke plant located in France. Results indicated a good correlation between the ELPI and TEOM Series 7000 with a tendency for the ELPI to measure higher mass concentration. 14 refs., 3 figs., 2 tabs.

  14. Real-time measurement of ice growth during simulated and natural icing conditions using ultrasonic pulse-echo techniques

    Science.gov (United States)

    Hansman, R. J., Jr.; Kirby, M. S.

    1986-01-01

    Results of tests to measure ice accretion in real-time using ultrasonic pulse-echo techniques are presented. Tests conducted on a 10.2 cm diameter cylinder exposed to simulated icing conditions in the NASA Lewis Icing Research Tunnel and on an 11.4 cm diameter cylinder exposed to natural icing conditions in flight are described. An accuracy of + or - 0.5 mm is achieved for real-time ice thickness measurements. Ice accretion rate is determined by differentiating ice thickness with respect to time. Icing rates measured during simulated and natural icing conditions are compared and related to icing cloud parameters. The ultrasonic signal characteristics are used to detect the presence of surface water on the accreting ice shape and thus to distinguish between dry ice growth and wet growth. The surface roughness of the accreted ice is shown to be related to the width of the echo signal received from the ice surface.

  15. Real-time systems

    OpenAIRE

    Badr, Salah M.; Bruztman, Donald P.; Nelson, Michael L.; Byrnes, Ronald Benton

    1992-01-01

    This paper presents an introduction to the basic issues involved in real-time systems. Both real-time operating sys and real-time programming languages are explored. Concurrent programming and process synchronization and communication are also discussed. The real-time requirements of the Naval Postgraduate School Autonomous Under Vehicle (AUV) are then examined. Autonomous underwater vehicle (AUV), hard real-time system, real-time operating system, real-time programming language, real-time sy...

  16. Development of quantitative gene-specific real-time RT-PCR assays for the detection of measles virus in clinical specimens.

    Science.gov (United States)

    Hummel, Kimberly B; Lowe, Luis; Bellini, William J; Rota, Paul A

    2006-03-01

    Real-time RT-PCR assays targeting sequences in the measles virus (MV) nucleoprotein (N), fusion (F), and hemagglutinin (H) genes were developed for the detection of MV RNA in clinical specimens. Four primer and probe sets each for the N, F, and H genes were evaluated and reaction conditions optimized. Using dilution series of synthetic RNAs, the limits of detection were determined to be approximately 10 copies for each target RNA/reaction. The relationship between C(t) values and RNA concentration was linear within a range of 10-10(6) RNA copies/reaction, and intra- and inter-assay variability was low. The N gene-specific real-time assay detected MV RNA in 100% of clinical samples from confirmed measles cases compared to 41% by standard RT-PCR. The MV H and F gene-specific real-time assays detected MV RNA in 93% and 82% of these specimens, respectively. Real-time assays could detect RNA from strains representing each active genotype of MV and were also highly specific, as no false positives were identified when samples known to contain other respiratory viruses were tested. Real-time RT-PCR assays will be available to support routine measles laboratory surveillance, to facilitate research projects on pathogenesis that require sensitive and quantitative detection of MV RNA, and to aid in the investigation of serious disease sequelae resulting from natural measles infection or vaccination with measles-containing vaccines.

  17. An advanced vision-based system for real-time displacement measurement of high-rise buildings

    Science.gov (United States)

    Lee, Jong-Han; Ho, Hoai-Nam; Shinozuka, Masanobu; Lee, Jong-Jae

    2012-12-01

    This paper introduces an advanced vision-based system for dynamic real-time displacement measurement of high-rise buildings using a partitioning approach. The partitioning method is based on the successive estimation of relative displacements and rotational angles at several floors using a multiple vision-based displacement measurement system. In this study, two significant improvements were made to realize the partitioning method: (1) time synchronization, (2) real-time dynamic measurement. Displacement data and time synchronization information are wirelessly transferred via a network using the TCP/IP protocol. The time synchronization process is periodically conducted by the master system to guarantee the system time at the master and slave systems are synchronized. The slave system is capable of dynamic real-time measurement and it is possible to economically expand measurement points at slave levels using commercial devices. To verify the accuracy and feasibility of the synchronized multi-point vision-based system and partitioning approach, many laboratory tests were carried out on a three-story steel frame model. Furthermore, several tests were conducted on a five-story steel frame tower equipped with a hybrid mass damper to experimentally confirm the effectiveness of the proposed system.

  18. Determination of Cytochrome P450 2D6 (CYP2D6 Gene Copy Number by Real-Time Quantitative PCR

    Directory of Open Access Journals (Sweden)

    Laurent Bodin

    2005-01-01

    Full Text Available Gene dosage by real-time quantitative PCR has proved to be accurate for measuring gene copy number. The aim of this study was to apply this approach to the CYP2D6 gene to allow for rapid identification of poor and ultrarapid metabolizers (0, 1, or more than 2 gene copy number. Using the 2−ΔΔCt calculation method and a duplex reaction, the number of CYP2D6 gene copies was determined. Quantitative PCR was performed on 43 samples previously analyzed by Southern blotting and long PCR including 20 samples with a heterozygous deletion, 11 with normal copy number (2 copies, and 12 samples with duplicated genes. The average ratio ranged from 1.02 to 1.28, 1.85 to 2.21, and 2.55 to 3.30, respectively, for the samples with 1 copy, 2 copies, and 3 copies. This study shows that this method is sensitive enough to detect either a heterozygous gene deletion or duplication.

  19. Trace-Level Volatile Quantitation by Direct Analysis in Real Time Mass Spectrometry following Headspace Extraction: Optimization and Validation in Grapes.

    Science.gov (United States)

    Jastrzembski, Jillian A; Bee, Madeleine Y; Sacks, Gavin L

    2017-10-25

    Ambient ionization mass spectrometric (AI-MS) techniques like direct analysis in real time (DART) offer the potential for rapid quantitative analyses of trace volatiles in food matrices, but performance is generally limited by the lack of preconcentration and extraction steps. The sensitivity and selectivity of AI-MS approaches can be improved through solid-phase microextraction (SPME) with appropriate thin-film geometries, for example, solid-phase mesh-enhanced sorption from headspace (SPMESH). This work improves the SPMESH-DART-MS approach for use in food analyses and validates the approach for trace volatile analysis for two compounds in real samples (grape macerates). SPMESH units prepared with different sorbent coatings were evaluated for their ability to extract a range of odor-active volatiles, with poly(dimethylsiloxane)/divinylbenzene giving the most satisfactory results. In combination with high-resolution mass spectrometry (HRMS), detection limits for SPMESH-DART-MS under 4 ng/L in less than 30 s acquisition times could be achieved for some volatiles [3-isobutyl-2-methoxypyrazine (IBMP) and β-damascenone]. A comparison of SPMESH-DART-MS and SPME-GC-MS quantitation of linalool and IBMP demonstrates excellent agreement between the two methods for real grape samples (r2 ≥ 0.90), although linalool measurements appeared to also include isobaric interference.

  20. Analysis of THCA synthase gene expression in cannabis: a preliminary study by real-time quantitative PCR.

    Science.gov (United States)

    Cascini, Fidelia; Passerotti, Stella; Boschi, Ilaria

    2013-09-10

    In this paper we describe analyses performed by Real-Time Reverse-Transcriptase Polymerase Chain Reaction (real-time RT-PCR) on RNA of 12 samples, carried out for forensic purposes to investigate a correlation between tetrahydrocannabinol (THC) concentration in Cannabis and the tetrahydrocannabinol acid synthase (THCAS) gene expression. Samples were obtained from an experimental cultivation of declared potency Cannabis variety seeds and from seizures. The Rubisco gene and the 26S ribosomal RNA gene were used as internal control genes for their constant expression and stability. As results we found minor gene expression in samples from leaves of young plants. Further, grouping results for cannabis samples with similar characteristics, we have found an increased relative expression in samples with the highest percentage of THC coming from seized sample and adult plants. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  1. Quantitative detection of pork in commercial meat products by TaqMan® real-time PCR assay targeting the mitochondrial D-loop region.

    Science.gov (United States)

    Kim, Miju; Yoo, Insuk; Lee, Shin-Young; Hong, Yeun; Kim, Hae-Yeong

    2016-11-01

    The TaqMan® real-time PCR assay using the mitochondrial D-loop region was developed for the quantitative detection of pork in processed meat products. The newly designed primers and probe specifically amplified pork without any cross-reactivity with non-target animal species. The limit of detection of the real-time PCR assay was 0.1pg of heat-treated pork meat and 0.1% (w/w) pork meat in beef and chicken meat mixtures. The quantitative real-time PCR assay was applied to analyze the pork meat content in 22 commercial processed meat products including jerkies, press hams, sausages, hamburger patties and steaks, grilled short rib patties, and nuggets. The developed real-time PCR method was able to detect pork meat in various types of processed meat products that declared the use of pork meat on their label. All processed meat products that declared no use of pork meat showed a negative result in the assay. The method developed in this study showed sensitivity and specificity in the quantification of pork meat in commercial processed meat products. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. A quantitative real-time PCR assay for the identification and enumeration of Alexandrium cysts in marine sediments

    Science.gov (United States)

    Erdner, D. L.; Percy, L.; Keafer, B.; Lewis, J.; Anderson, D. M.

    2010-02-01

    Harmful algal blooms (HABs) are a global problem that affects both human and ecosystem health. One of the most serious and widespread HAB poisoning syndromes is paralytic shellfish poisoning, commonly caused by Alexandrium spp. dinoflagellates. Like many toxic dinoflagellates, Alexandrium produces resistant resting cysts as part of its life cycle. These cysts play a key role in bloom initiation and decline, as well as dispersal and colonization of new areas. Information on cyst numbers and identity is essential for understanding and predicting blooms, yet comprehensive cyst surveys are extremely time- and labor-intensive. Here we describe the development and validation of a quantitative real-time PCR (qPCR) technique for the enumeration of cysts of A. tamarense of the toxic North American/Group I ribotype. The method uses a cloned fragment of the large subunit ribosomal RNA gene as a standard for cyst quantification, with an experimentally determined conversion factor of 28,402±6152 LSU ribosomal gene copies per cyst. Tests of DNA extraction and PCR efficiency show that mechanical breakage is required for adequate cyst lysis, and that it was necessary to dilute our DNA extracts 50-fold in order to abolish PCR inhibition from compounds co-extracted from the sediment. The resulting assay shows a linear response over 6 orders of magnitude and can reliably quantify ≥10 cysts/cm 3 sediment. For method validation, 129 natural sediment samples were split and analyzed in parallel, using both the qPCR and primulin-staining techniques. Overall, there is a significant correlation ( p<0.001) between the cyst abundances determined by the two methods, although the qPCR counts tend to be lower than the primulin values. This underestimation is less pronounced in those samples collected from the top 1 cm of sediment, and more pronounced in those derived from the next 1-3 cm of the core. These differences may be due to the condition of the cysts in the different layers, as the

  3. Rapid detection of aflatoxin producing fungi in food by real-time quantitative loop-mediated isothermal amplification.

    Science.gov (United States)

    Luo, Jie; Vogel, Rudi F; Niessen, Ludwig

    2014-12-01

    Aflatoxins represent a serious risk for human and animal health. They are mainly produced by Aspergillus flavus and Aspergillus parasiticus but also by Aspergillus nomius. Three species specific turbidimeter based real-time LAMP (loop-mediated isothermal amplification) assays were developed to quantify the three species individually in conidial solutions and to define contamination levels in samples of shelled Brazil nuts, maize, and peanuts. Standard curves relating spore numbers to time to threshold (Tt) values were set up for each of the species. Assays had detection limits of 10, 100 and 100 conidia per reaction of A. flavus, A. parasiticus, and A. nomius, respectively. Analysis of contaminated sample materials revealed that the A. nomius specific real-time LAMP assay detected a minimum of 10 conidia/g in Brazil nuts while assays specific for A. flavus and A. parasiticus had detection limits of 10(2) conidia/g and 10(5) conidia/g, respectively in peanut samples as well as 10(4) conidia/g and 10(4) conidia/g, respectively in samples of maize. The real-time LAMP assays developed here appear to be promising tools for the prediction of potential aflatoxigenic risk at an early stage and in all critical control points of the food and feed production chain. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Quantitative detection of Cucumber vein yellowing virus in susceptible and partially resistant plants using real-time PCR.

    Science.gov (United States)

    Picó, Belén; Sifres, Alicia; Nuez, Fernando

    2005-09-01

    A method for the detection of Cucumber vein yellowing virus (CVYV) that combines reverse transcription with real-time PCR (SYBR((R)) Green chemistry) was developed using specific primers designed from a nucleotide sequence of the RNA polymerase gene (NIb) conserved among all the available CVYV strains. This method provided a linear assay over five to six orders of magnitude and reproducibly detected titres as low as 10(3) molecules of the target CVYV cDNA. Real-time PCR gave reproducible results for the quantification of CVYV in young leaves of susceptible and resistant cucumber landraces after mechanical inoculation. Significant differences in the starting amount of target cDNA were found between the analyzed genotypes, indicating differences in viral accumulation that correlated to their different levels of resistance. Real-time PCR results validated our previous findings using slot-blot hybridization, the dominance of the strong resistance to CVYV displayed by C.sat 10, and provided improved reliability and sensitivity of detection. This method has great potential in resistance breeding for germplasm screening, characterization of resistance mechanisms and genetic studies.

  5. Real-Time Analysis of Tenofovir Release Kinetics Using Quantitative Phosphorus (31P) Nuclear Magnetic Resonance Spectroscopy.

    Science.gov (United States)

    Agrahari, Vivek; Meng, Jianing; Purohit, Sudhaunshu S; Oyler, Nathan A; Youan, Bi-Botti C

    2017-10-01

    The dialysis method is classically used for drug separation before analysis, but does not provide direct and real-time drug quantification and has limitations affecting the dialysis rate. In this study, a phosphorus nuclear magnetic resonance (31P-qNMR) method is developed for the real-time quantification of therapeutic molecules in vitro. The release kinetics of model drug, tenofovir (anti-HIV microbicide), was analyzed in vaginal fluid simulant (VFS), seminal fluid simulant (SFS), and human plasma (HP) from chitosan nanofibers (size ∼100-200 nm) using the NMR (direct) method and compared with dialysis/UV-Vis (indirect) method. The assay was linear in VFS/SFS (0.20-5.0 mM), HP (0.30-5.0 mM of drug concentration range) and specific no drug 31P-qNMR chemical shift [∼15 ppm] interference with formulation/media components. Limit of detection values were 0.075/0.10/0.20 mM, whereas limit of quantification values were 0.20/0.20/0.30 mM in VFS/SFS/HP, respectively. The method was robust, precise (%RSE 31P-qNMR provides more accurate, real-time, and direct drug quantification for effective in vitro-in vivo correlation. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  6. [Quantitative detection of Streptococcus mutans in different people with dental caries by real-time polymerase chain reaction].

    Science.gov (United States)

    Zhao, Dong; Wang, Zhan-yong; Wang, Jian-qiu; Xiao, Bai; Zhou, Yan

    2010-04-01

    To establish a quantity detection method of Streptococcus mutans (Sm) and bacteria and compare the relationship between the number of these bacteria and the prevalence of dental caries in different people. With specific primers for a unique sequence in a 14 kb HaeIII restriction fragment consistently presenting during detecting Sm by chromosomal DNA fingerprints, the total number of Sm and bacteria of 99 saliva samples were detected by real-time polymerase chain reaction (PCR) and statistically analyzed. The primers were specific for Sm and the minimum detectable level by real-time PCR was 0.1 microg/L. The total number of bacteria in the dental caries and people without caries was 51.4 x 10(8) cell copies/L and 221.6 x 10(8) cell copies/L respectively, in which the ratio of Sm to bacteria was 0.0193 and 0.0059 respectively. The differences were significantly different between the people with dental caries and those without caries in the total number of bacteria and the ratio of Sm to bacteria. The primers can be used to detect the Sm by real-time PCR. The ratio of Sm to bacteria was closely associated with the prevalence of dental caries.

  7. The Real-Time Dose Measurement Scintillating Fiber Array for Brachytherapy Procedures

    Science.gov (United States)

    Tynes, Lawrence

    2007-03-01

    Brachytherapy is a treatment modality that uses tiny radioactive sources (few mm in length) by delivering enough doses to kill cancer tumors or plaque build-up. The type of sources used in hospitals include both gamma and beta emitters. Presently, the technique suffers from not having a single detector with the capability of providing accurate dose distribution information within sub-mm accuracy. The current standard is based primarily on well chambers and film dosimetry. The Center for Advanced Medical Instrumentation (CAMI) at Hampton University is developing a Scintillating Fiber Based Beta Detector prototype in collaboration with the National Institute for Standards and Technology (NIST) to address this problem. The device is composed of an array of 1x1 mm^2 scintillating fibers optically coupled to photo-multiplier tubes for photon-to-current conversion. A CAMAC LabView based data acquisition system is used for real time data collection and histogramming, data analysis. A set of data were collected at the nearby Bon Secours DePaul Medical Center using a GammaMed 12i HDR after-loader housing a 6.62 mCi Ir-192 source. Preliminary comparison between our device and film dosimetry will be discussed.

  8. Application of quantitative real-time PCR compared to filtration methods for the enumeration of Escherichia coli in surface waters within Vietnam.

    Science.gov (United States)

    Vital, Pierangeli G; Van Ha, Nguyen Thi; Tuyet, Le Thi Hong; Widmer, Kenneth W

    2017-02-01

    Surface water samples in Vietnam were collected from the Saigon River, rural and suburban canals, and urban runoff canals in Ho Chi Minh City, Vietnam, and were processed to enumerate Escherichia coli. Quantification was done through membrane filtration and quantitative real-time polymerase chain reaction (PCR). Mean log colony-forming unit (CFU)/100 ml E. coli counts in the dry season for river/suburban canals and urban canals were log 2.8 and 3.7, respectively, using a membrane filtration method, while using Taqman quantitative real-time PCR they were log 2.4 and 2.8 for river/suburban canals and urban canals, respectively. For the wet season, data determined by the membrane filtration method in river/suburban canals and urban canals samples had mean counts of log 3.7 and 4.1, respectively. While mean log CFU/100 ml counts in the wet season using quantitative PCR were log 3 and 2, respectively. Additionally, the urban canal samples were significantly lower than those determined by conventional culture methods for the wet season. These results show that while quantitative real-time PCR can be used to determine levels of fecal indicator bacteria in surface waters, there are some limitations to its application and it may be impacted by sources of runoff based on surveyed samples.

  9. Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods

    Directory of Open Access Journals (Sweden)

    Akiko Edagawa

    2015-10-01

    Full Text Available We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR, and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%. Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%. In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8% compared with real-time qPCR alone (46/68, 67.6%. Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1% compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%. Legionella was not detected in the remaining six samples (6/68, 8.8%, irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.

  10. Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods.

    Science.gov (United States)

    Edagawa, Akiko; Kimura, Akio; Kawabuchi-Kurata, Takako; Adachi, Shinichi; Furuhata, Katsunori; Miyamoto, Hiroshi

    2015-10-19

    We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR), and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%). Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%). In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8%) compared with real-time qPCR alone (46/68, 67.6%). Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1%) compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%). Legionella was not detected in the remaining six samples (6/68, 8.8%), irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.

  11. Real-time intraocular pressure measurement during phacoemulsification in dogs ex vivo

    Science.gov (United States)

    KANG, Seonmi; PARK, Sangwan; NOH, Hyunwoo; KWAK, Jiyoon; SEO, Kangmoon

    2015-01-01

    This study was performed to evaluate changes in intraocular pressure (IOP) during standard coaxial phacoemulsification using 4 different bottle heights (BHs) and 2 different incision sizes. Coaxial phacoemulsification was performed with a venturi-based machine in 8 enucleated canine eyes through 3.0 and 3.2 mm clear corneal incisions (CCIs). A pressure transducer inserted in the peripheral cornea monitored the IOP in real-time. The surgery was subdivided into 4 stages: sculpt-segment removal, irrigation/aspiration, capsular polishing and viscoelastic removal. The mean IOP and the difference between the maximum and minimum IOPs were calculated at each stage and compared. The ultrasound time and volume of irrigation fluid used were recorded. The mean IOP increased with an elevation in the BH. The mean IOP in the irrigation/aspiration stage was significantly higher than that in the sculpt-segment removal stage at the same BH. The difference between the maximum and minimum IOP at each stage was greater in the 3.2 mm than the 3.0 mm CCIs, although the mean IOP was lower with the 3.2 mm than the 3.0 mm CCIs. The ultrasound time and irrigation fluid volume were greater with the 3.2 mm than the 3.0 mm CCIs. Therefore, fluidic parameters during each stage could be reassessed and adjusted to reduce complications arising from an elevated IOP. Phacoemulsification with 3.0 mm CCIs at a lower BH might lead to less stress on the eye from IOP fluctuations, ultrasound energy and irrigation fluid. PMID:25716691

  12. Correlation between Herrold egg yolk medium culture and real-time quantitative polymerase chain reaction results for Mycobacterium avium subspecies paratuberculosis in pooled fecal and environmental samples.

    Science.gov (United States)

    Aly, Sharif S; Mangold, Beverly L; Whitlock, Robert H; Sweeney, Raymond W; Anderson, Randall J; Jiang, Jiming; Schukken, Ynte H; Hovingh, Ernest; Wolfgang, David; Van Kessel, Jo Ann S; Karns, Jeffrey S; Lombard, Jason E; Smith, Julia M; Gardner, Ian A

    2010-09-01

    Real-time quantitative polymerase chain reaction (qPCR) testing for Mycobacterium avium subspecies paratuberculosis (MAP) in fecal samples is a rapid alternative to culture on Herrold egg yolk medium (HEYM), the traditional antemortem reference test for MAP. Although the sensitivity and specificity of these 2 tests have been estimated based on dichotomized test results, the correlation between real-time qPCR threshold cycle (Ct) values and colony-forming units (CFU) on HEYM for fresh and thawed samples has not been evaluated. The objectives of the present study were to estimate the correlation and association between Ct and CFU in fresh and thawed pooled fecal and environmental samples. Results of HEYM culture of 1,997 pooled fecal samples from cows in 14 herds, and 802 environmental samples from 109 dairies nationwide were negatively (inversely) correlated with their respective real-time qPCR results. The Spearman's rank correlation between Ct and CFU was good (-0.66) in fresh and thawed pooled fecal samples, and excellent (-0.76) and good (-0.61) in fresh and thawed environmental samples, respectively. The correlation varied from good (-0.53) to excellent (-0.90) depending on the number of samples in a fecal pool. Truncated regression models indicated a significant negative association between Ct and CFU in fecal pools and environmental samples. The use of real-time qPCR instead of HEYM can yield rapid, quantitative estimates of MAP load and allow for incorporation of real-time qPCR results of pooled and environmental samples in testing strategies to identify dairy cow groups with the highest MAP shedding.

  13. NanoString nCounter® Approach in Breast Cancer: A Comparative Analysis with Quantitative Real-Time Polymerase Chain Reaction, In Situ Hybridization, and Immunohistochemistry

    OpenAIRE

    Hyeon, Jiyeon; Cho, Soo Youn; Hong, Min Eui; Kang, So Young; Do, Ingu; Im, Young Hyuck; Cho, Eun Yoon

    2017-01-01

    Purpose Accurate testing for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) is essential for breast cancer treatment. At present, immunohistochemistry (IHC)/florescence in situ hybridization (FISH) are widely accepted as the standard testing methods. To investigate the value of NanoString nCounter®, we performed its comparative analysis with IHC/FISH and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) fo...

  14. Validation of Reference Genes for Gene Expression by Quantitative Real-Time RT-PCR in Stem Segments Spanning Primary to Secondary Growth in Populus tomentosa

    OpenAIRE

    Ying Wang; Yajuan Chen; Liping Ding; Jiewei Zhang; Jianhua Wei; Hongzhi Wang

    2016-01-01

    The vertical segments of Populus stems are an ideal experimental system for analyzing the gene expression patterns involved in primary and secondary growth during wood formation. Suitable internal control genes are indispensable to quantitative real time PCR (qRT-PCR) assays of gene expression. In this study, the expression stability of eight candidate reference genes was evaluated in a series of vertical stem segments of Populus tomentosa. Analysis through software packages geNorm, NormFinde...

  15. Can We Trust Real Time Measurements of Lung Deposited Surface Area Concentrations in Dust from Powder Nanomaterials?

    DEFF Research Database (Denmark)

    Levin, Marcus; Witschger, Olivier; Bau, Sebastien

    2016-01-01

    A comparison between various methods for real-time measurements of lung deposited surface area (LDSA) using spherical particles and powder dust with specific surface area ranging from 0.03 to 112 m2 g-1 was conducted. LDSA concentrations measured directly using Nanoparticle Surface Area Monitor...... present. We conclude that there is currently insufficient reliability and comparability between methods in the measurement of LDSA concentrations. Further development is required to enable use of LDSA for reliable dose metric and regulatory enforcement of exposure....

  16. Can We Trust Real Time Measurements of Lung Deposited Surface Area Concentrations in Dust from Powder Nanomaterials?

    DEFF Research Database (Denmark)

    Levin, Marcus; Witschger, Olivier; Bau, Sebastien

    2016-01-01

    A comparison between various methods for real-time measurements of lung deposited surface area (LDSA) using spherical particles and powder dust with specific surface area ranging from 0.03 to 112 m2 g-1 was conducted. LDSA concentrations measured directly using Nanoparticle Surface Area Monitor (...... present. We conclude that there is currently insufficient reliability and comparability between methods in the measurement of LDSA concentrations. Further development is required to enable use of LDSA for reliable dose metric and regulatory enforcement of exposure....

  17. Real-Time In-Situ Measurements for Earthquake Early Warning and Space-Borne Deformation Measurement Mission Support

    Science.gov (United States)

    Kedar, S.; Bock, Y.; Webb, F.; Clayton, R. W.; Owen, S. E.; Moore, A. W.; Yu, E.; Dong, D.; Fang, P.; Jamason, P.; Squibb, M. B.; Crowell, B. W.

    2010-12-01

    In situ geodetic networks for observing crustal motion have proliferated over the last two decades and are now recognized as indispensable tools in geophysical research, along side more traditional seismic networks. The 2007 National Research Council’s Decadal Survey recognizes that space-borne and in situ observations, such as Interferometric Synthetic Aperture Radar (InSAR) and ground-based continuous GPS (CGPS) are complementary in forecasting, in assessing, and in mitigating natural hazards. However, the information content and timeliness of in situ geodetic observations have not been fully exploited, particularly at higher frequencies than traditional daily CGPS position time series. Nor have scientists taken full advantage of the complementary natures of geodetic and seismic data, as well as those of space-based and in situ observations. To address these deficits we are developing real-time CGPS data products for earthquake early warning and for space-borne deformation measurement mission support. Our primary mission objective is in situ verification and validation for DESDynI, but our work is also applicable to other international missions (Sentinel 1a/1b, SAOCOM, ALOS 2). Our project is developing new capabilities to continuously observe and mitigate earthquake-related hazards (direct seismic damage, tsunamis, landslides, volcanoes) in near real-time with high spatial-temporal resolution, to improve the planning and accuracy of space-borne observations. We also are using GPS estimates of tropospheric zenith delay combined with water vapor data from weather models to generate tropospheric calibration maps for mitigating the largest source of error, atmospheric artifacts, in InSAR interferograms. These functions will be fully integrated into a Geophysical Resource Web Services and interactive GPS Explorer data portal environment being developed as part of an ongoing MEaSUREs project and NASA’s contribution to the EarthScope project. GPS Explorer

  18. Advances in LWD pressure measurements: smart, time optimized pretests and on demand real-time transmission applications

    Energy Technology Data Exchange (ETDEWEB)

    Serafim, Robson; Ferraris, Paolo [Schlumberger, Rio de Janeiro, RJ (Brazil)

    2008-07-01

    The StethoScope Logging While Drilling (LWD) Pressure Measurement, introduced in Brazil in 2005, has been extensively used in deep water environment to provide reservoir pressure and mobility in real-time. In the last three years the StethoScope service was further enhanced to allow better real time monitoring using a larger transmission rate, higher RT data resolution and remote visualization. In order to guarantee stable formation pressures with a limited test duration under a wide range of conditions, Time Optimized Pretests (TOP) were developed. These tests adjust automatically drawdown and buildup parameters as a function of formation characteristics (pressure/mobility) without requiring any input from the operator. On-demand frame (ODF), an advanced telemetry triggered automatically during the pressure tests, allowed to increase equivalent transmission rate and resolution and to include quality indices computed downhole. This paper is focused on the TOP and ODF Field Test results in Brazil, which proved to be useful and reliable options for better real-time decisions together with remote monitoring visualization implemented by the RTMonitor program. (author)

  19. Implementation of a real-time automatic onset time detection for surface electromyography measurement systems using NI myRIO

    Directory of Open Access Journals (Sweden)

    Lersviriyanantakul Chaiwat

    2016-01-01

    Full Text Available For using surface electromyography (sEMG in various applications, the process consists of three parts: an onset time detection for detecting the first point of movement signals, a feature extraction for extracting the signal attribution, and a feature classification for classifying the sEMG signals. The first and the most significant part that influences the accuracy of other parts is the onset time detection, particularly for automatic systems. In this paper, an automatic and simple algorithm for the real-time onset time detection is presented. There are two main processes in the proposed algorithm; a smoothing process for reducing the noise of the measured sEMG signals and an automatic threshold calculation process for determining the onset time. The results from the algorithm analysis demonstrate the performance of the proposed algorithm to detect the sEMG onset time in various smoothing-threshold equations. Our findings reveal that using a simple square integral (SSI as the smoothing-threshold equation with the given sEMG signals gives the best performance for the onset time detection. Additionally, our proposed algorithm is also implemented on a real hardware platform, namely NI myRIO. Using the real-time simulated sEMG data, the experimental results guarantee that the proposed algorithm can properly detect the onset time in the real-time manner.

  20. Quantitative detection of Citrus tristeza virus in citrus and aphids by real-time reverse transcription-PCR (TaqMan).

    Science.gov (United States)

    Saponari, Maria; Manjunath, Keremane; Yokomi, Raymond K

    2008-01-01

    A quantitative and multiplex real-time RT-PCR assay was developed to detect Citrus tristeza virus (CTV) along with plant mRNA, which serves as an internal control to ascertain RNA extraction quality. The real-time technique was validated against 39 CTV strains from around the world as well as with the aphid vector, Aphis gossypii, given a 48 h acquisition access period on a CTV source plant. The assay was effective for quantitation of the viral template in infected plants and in single aphids. CTV detection was compared from different plant tissues and for different RNA isolation methods from aphids. Less than 1 fg was consistently detected when RNA transcripts were diluted in extracts from healthy plants while RNA copies carried by single aphids were estimated to be between 12,000 and 13,000,000. The assay was more sensitive and less time consuming than ELISA or traditional RT-PCR. The real-time RT-PCR assay developed is a valuable new tool for detection and titer quantitation of CTV.

  1. Comparative evaluation of a laboratory developed real-time PCR assay and the RealStar®HHV-6 PCR Kit for quantitative detection of human herpesvirus 6.

    Science.gov (United States)

    Yip, Cyril C Y; Sridhar, Siddharth; Cheng, Andrew K W; Fung, Ami M Y; Cheng, Vincent C C; Chan, Kwok-Hung; Yuen, Kwok-Yung

    2017-08-01

    HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar ® HHV-6 PCR Kit. The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated. The diagnostic performance of the in-house HHV-6 qPCR assay was compared with the RealStar ® HHV-6 PCR Kit, using 72 clinical specimens and 17 proficiency testing samples. Linear regression analysis of the quantitative results showed a dynamic range from 2 to 10 log 10 copies/ml and a coefficient of determination (R 2 ) of 0.999 for the in-house assay. A dilution series demonstrated a limit of detection and a limit of quantification of 1.7 log 10 and 2 log 10 copies/ml, respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.27% to 4.37%. A comparison of 27 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house HHV-6 qPCR assay and the RealStar ® HHV-6 PCR Kit (R 2 =0.926; PPCR Kit. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Real-Time Measurement of Electronic Cigarette Aerosol Size Distribution and Metals Content Analysis.

    Science.gov (United States)

    Mikheev, Vladimir B; Brinkman, Marielle C; Granville, Courtney A; Gordon, Sydney M; Clark, Pamela I

    2016-09-01

    Electronic cigarette (e-cigarette) use is increasing worldwide and is highest among both daily and nondaily smokers. E-cigarettes are perceived as a healthier alternative to combustible tobacco products, but their health risk factors have not yet been established, and one of them is lack of data on aerosol size generated by e-cigarettes. We applied a real-time, high-resolution aerosol differential mobility spectrometer to monitor the evolution of aerosol size and concentration during puff development. Particles generated by e-cigarettes were immediately delivered for analysis with minimal dilution and therefore with minimal sample distortion, which is critically important given the highly dynamic aerosol/vapor mixture inherent to e-cigarette emissions. E-cigarette aerosols normally exhibit a bimodal particle size distribution: nanoparticles (11-25nm count median diameter) and submicron particles (96-175nm count median diameter). Each mode has comparable number concentrations (10(7)-10(8) particles/cm(3)). "Dry puff" tests conducted with no e-cigarette liquid (e-liquid) present in the e-cigarette tank demonstrated that under these conditions only nanoparticles were generated. Analysis of the bulk aerosol collected on the filter showed that e-cigarette emissions contained a variety of metals. E-cigarette aerosol size distribution is different from that of combustible tobacco smoke. E-cigarettes generate high concentrations of nanoparticles and their chemical content requires further investigation. Despite the small mass of nanoparticles, their toxicological impact could be significant. Toxic chemicals that are attached to the small nanoparticles may have greater adverse health effects than when attached to larger submicron particles. The e-cigarette aerosol size distribution is different from that of combustible tobacco smoke and typically exhibits a bimodal behavior with comparable number concentrations of nanoparticles and submicron particles. While vaping the e

  3. Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station.

    Directory of Open Access Journals (Sweden)

    Macarena Parra

    Full Text Available The International Space Station (ISS National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen Escherichia coli and mouse liver tissue. Cycle threshold (Ct values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for

  4. Comparison of Enterococcus quantitative polymerase chain reaction analysis results from fresh and marine waters on two real-time instruments.

    Science.gov (United States)

    Sivaganensan, Mano; Varma, Manju; Haugland, Richard A

    2012-11-01

    The U.S. Environmental Protection Agency will be recommending a quantitative polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality. A practical consideration for implementation of this and other qPCR methods is whether the results are comparable on different PCR instruments. In this study, quantitative estimates of Enterococcus densities from marine and freshwater samples were determined by the qPCR method from cycle threshold (Ct) measurements obtained on Applied Biosystems StepOnePlus and Cepheid SmartCycler instruments. Three variations of a comparative Ct model, differing in their sources of calibration data, were used in the estimations. Both traditional and Bayesian statistical modeling approaches were examined in the instrument comparisons. The traditional analysis of variance (ANOVA) approach indicated no significant differences (p>0.05) between mean density estimates from the instruments in two of the three model variations. The Bayesian approach indicated that the 95% Bayesian credible intervals of density estimates from the instruments overlapped in all models; however, the uncertainty of the estimates varied depending on the model. These results support the interchangeable use of the two instruments in the method and also illustrate the importance of defining the source of calibration data used in the comparative Ct model. Published by Elsevier Inc.

  5. Novel quantitative real-time PCR approach to determine safflower (Carthamus tinctorius) adulteration in saffron (Crocus sativus).

    Science.gov (United States)

    Villa, Caterina; Costa, Joana; Oliveira, M Beatriz P P; Mafra, Isabel

    2017-08-15

    This work intended to develop DNA-based methods to detect and quantify safflower as an adulterant in saffron. Species-specific PCR and real-time PCR with EvaGreen dye targeting the ITS region of Carthamus tinctorius L. (safflower) were successfully proposed. The assays allowed absolute and relative sensitivities of 2pg of safflower DNA (∼1.4 DNA copies) and 0.1% of safflower in saffron (Crocus sativus L.), respectively. A normalised real-time PCR approach was also proposed in the range of 0.1-20% (w/w) of safflower in saffron, which was successfully validated and applied to commercial saffron samples (stigmas, powders and seasonings). From 19 samples, three were positive to safflower, though at levels below the limit of detection, suggesting cross-contamination rather than adulteration. In this work, specific, sensitive and accurate tools were proposed to authenticate saffron. To the best of our knowledge, this is the first successful attempt to quantify safflower by a DNA-based approach. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Real-time quantitative analysis of metabolic flux in live cells using a hyperpolarized micromagnetic resonance spectrometer.

    Science.gov (United States)

    Jeong, Sangmoo; Eskandari, Roozbeh; Park, Sun Mi; Alvarez, Julio; Tee, Sui Seng; Weissleder, Ralph; Kharas, Michael G; Lee, Hakho; Keshari, Kayvan R

    2017-06-01

    Metabolic reprogramming is widely considered a hallmark of cancer, and understanding metabolic dynamics described by the conversion rates or "fluxes" of metabolites can shed light onto biological processes of tumorigenesis and response to therapy. For real-time analysis of metabolic flux in intact cells or organisms, magnetic resonance (MR) spectroscopy and imaging methods have been developed in conjunction with hyperpolarization of nuclear spins. These approaches enable noninvasive monitoring of tumor progression and treatment efficacy and are being tested in multiple clinical trials. However, because of their limited sensitivity, these methods require a larger number of cells, on the order of 10(7), which is impractical for analyzing scant target cells or mass-limited samples. We present a new technology platform, a hyperpolarized micromagnetic resonance spectrometer (HMRS), that achieves real-time, 10(3)-fold more sensitive metabolic analysis on live cells. This platform enables quantification of the metabolic flux in a wide range of cell types, including leukemia stem cells, without significant changes in viability, which allows downstream molecular analyses in tandem. It also enables rapid assessment of metabolic changes by a given drug, which may direct therapeutic choices in patients. We further advanced this platform for high-throughput analysis of hyperpolarized molecules by integrating a three-dimensionally printed microfluidic system. The HMRS platform holds promise as a sensitive method for studying metabolic dynamics in mass-limited samples, including primary cancer cells, providing novel therapeutic targets and an enhanced understanding of cellular metabolism.

  7. Self-referencing optrodes for measuring spatially resolved, real-time metabolic oxygen flux in plant systems.

    Science.gov (United States)

    McLamore, Eric S; Jaroch, David; Chatni, M Rameez; Porterfield, D Marshall

    2010-10-01

    The ability to non-invasively measure metabolic oxygen flux is a very important tool for physiologists interested in a variety of questions ranging from basic metabolism, growth/development, and stress adaptation. Technologies for measuring oxygen concentration near the surface of cells/tissues include electrochemical and optical techniques. A wealth of knowledge was gained using these tools for quantifying real-time physiology. Fiber-optic microprobes (optrodes) have recently been developed for measuring oxygen in a variety of biomedical and environmental applications. We have adopted the use of these optical microsensors for plant physiology applications, and used the microsensors in an advanced sensing modality known as self-referencing. Self-referencing is a non-invasive microsensor technique used for measuring real-time flux of analytes. This paper demonstrates the use of optical microsensors for non-invasively measuring rhizosphere oxygen flux associated with respiration in plant roots, as well as boundary layer oxygen flux in phytoplankton mats. Highly sensitive/selective optrodes had little to no hysteresis/calibration drift during experimentation, and an extremely high signal-to-noise ratio. We have used this new tool to compare various aspects of rhizosphere oxygen flux for roots of Glycine max, Zea mays, and Phaseolus vulgaris, and also mapped developmentally relevant profiles and distinct temporal patterns. We also characterized real-time respiratory patterns during inhibition of cytochrome and alternative oxidase pathways via pharmacology. Boundary layer oxygen flux was also measured for a phytoplankton mat during dark:light cycling and exposure to pharamacological inhibitors. This highly sensitive technology enables non-invasive study of oxygen transport in plant systems under physiologically relevant conditions.

  8. Real-Time Propagation Measurement System and Scattering Object Identification by 3D Visualization by Using VRML for ETC System

    Directory of Open Access Journals (Sweden)

    Ando Tetsuo

    2009-01-01

    Full Text Available In the early deployment of electric toll collecting (ETC system, multipath interference has caused the malfunction of the system. Therefore, radio absorbers are installed in the toll gate to suppress the scattering effects. This paper presents a novel radio propagation measurement system using the beamforming with 8-elmenet antenna array to examine the power intensity distribution of the ETC gate in real time without closing the toll gates that are already open for traffic. In addition, an identification method of the individual scattering objects with 3D visualization by using virtual reality modeling language will be proposed and the validity is also demonstrated by applying to the measurement data.

  9. Comparison of real-time instruments and gravimetric method when measuring particulate matter in a residential building.

    Science.gov (United States)

    Wang, Zuocheng; Calderón, Leonardo; Patton, Allison P; Sorensen Allacci, MaryAnn; Senick, Jennifer; Wener, Richard; Andrews, Clinton J; Mainelis, Gediminas

    2016-11-01

    This study used several real-time and filter-based aerosol instruments to measure PM 2.5 levels in a high-rise residential green building in the Northeastern US and compared performance of those instruments. PM 2.5 24-hr average concentrations were determined using a Personal Modular Impactor (PMI) with 2.5 µm cut (SKC Inc., Eighty Four, PA) and a direct reading pDR-1500 (Thermo Scientific, Franklin, MA) as well as its filter. 1-hr average PM 2.5 concentrations were measured in the same apartments with an Aerotrak Optical Particle Counter (OPC) (model 8220, TSI, Inc., Shoreview, MN) and a DustTrak DRX mass monitor (model 8534, TSI, Inc., Shoreview, MN). OPC and DRX measurements were compared with concurrent 1-hr mass concentration from the pDR-1500. The pDR-1500 direct reading showed approximately 40% higher particle mass concentration compared to its own filter (n = 41), and 25% higher PM 2.5 mass concentration compared to the PMI 2.5 filter. The pDR-1500 direct reading and PMI 2.5 in non-smoking homes (self-reported) were not significantly different (n = 10, R 2 = 0.937), while the difference between measurements for smoking homes was 44% (n = 31, R 2 = 0.773). Both OPC and DRX data had substantial and significant systematic and proportional biases compared with pDR-1500 readings. However, these methods were highly correlated: R 2 = 0.936 for OPC versus pDR-1500 reading and R 2 = 0.863 for DRX versus pDR-1500 reading. The data suggest that accuracy of aerosol mass concentrations from direct-reading instruments in indoor environments depends on the instrument, and that correction factors can be used to reduce biases of these real-time monitors in residential green buildings with similar aerosol properties. This study used several real-time and filter-based aerosol instruments to measure PM 2.5 levels in a high-rise residential green building in the northeastern United States and compared performance of those instruments. The data show that while the use of real-time

  10. Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization in Iris. lactea var. chinensis Roots under Cadmium, Lead, and Salt Stress Conditions

    Directory of Open Access Journals (Sweden)

    Chun-Sun Gu

    2014-01-01

    Full Text Available Quantitative real time PCR (RT-qPCR has emerged as an accurate and sensitive method to measure the gene expression. However, obtaining reliable result depends on the selection of reference genes which normalize differences among samples. In this study, we assessed the expression stability of seven reference genes, namely, ubiquitin-protein ligase UBC9 (UBC, tubulin alpha-5 (TUBLIN, eukaryotic translation initiation factor (EIF-5A, translation elongation factor EF1A (EF1α, translation elongation factor EF1B (EF1b, actin11 (ACTIN, and histone H3 (HIS, in Iris. lactea var. chinensis (I. lactea var. chinensis root when the plants were subjected to cadmium (Cd, lead (Pb, and salt stress conditions. All seven reference genes showed a relatively wide range of threshold cycles (Ct values in different samples. GeNorm and NormFinder algorithms were used to assess the suitable reference genes. The results from the two software units showed that EIF-5A and UBC were the most stable reference genes across all of the tested samples, while TUBLIN was unsuitable as internal controls. I. lactea var. chinensis is tolerant to Cd, Pb, and salt. Our results will benefit future research on gene expression in response to the three abiotic stresses.

  11. Real-time quantitative PCR with SYBR Green I detection for estimating copy numbers of nine drug resistance candidate genes in Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Cravo Pedro VL

    2006-01-01

    Full Text Available Abstract Background Evaluating copy numbers of given genes in Plasmodium falciparum parasites is of major importance for laboratory-based studies or epidemiological surveys. For instance, pfmdr1 gene amplification has been associated with resistance to quinine derivatives and several genes involved in anti-oxidant defence may play an important role in resistance to antimalarial drugs, although their potential involvement has been overlooked. Methods The ΔΔCt method of relative quantification using real-time quantitative PCR with SYBR Green I detection was adapted and optimized to estimate copy numbers of three genes previously indicated as putative candidates of resistance to quinolines and artemisinin derivatives: pfmdr1, pfatp6 (SERCA and pftctp, and in six further genes involved in oxidative stress responses. Results Using carefully designed specific RT-qPCR oligonucleotides, the methods were optimized for each gene and validated by the accurate measure of previously known number of copies of the pfmdr1 gene in the laboratory reference strains P. falciparum 3D7 and Dd2. Subsequently, Standard Operating Procedures (SOPs were developed to the remaining genes under study and successfully applied to DNA obtained from dried filter blood spots of field isolates of P. falciparum collected in São Tomé & Principe, West Africa. Conclusion The SOPs reported here may be used as a high throughput tool to investigate the role of these drug resistance gene candidates in laboratory studies or large scale epidemiological surveys.

  12. Real-time quantitative PCR with SYBR Green I detection for estimating copy numbers of nine drug resistance candidate genes in Plasmodium falciparum.

    Science.gov (United States)

    Ferreira, Isabel D; Rosário, Virgílio E do; Cravo, Pedro V L

    2006-01-18

    Evaluating copy numbers of given genes in Plasmodium falciparum parasites is of major importance for laboratory-based studies or epidemiological surveys. For instance, pfmdr1 gene amplification has been associated with resistance to quinine derivatives and several genes involved in anti-oxidant defence may play an important role in resistance to antimalarial drugs, although their potential involvement has been overlooked. The DeltaDeltaCt method of relative quantification using real-time quantitative PCR with SYBR Green I detection was adapted and optimized to estimate copy numbers of three genes previously indicated as putative candidates of resistance to quinolines and artemisinin derivatives: pfmdr1, pfatp6 (SERCA) and pftctp, and in six further genes involved in oxidative stress responses. Using carefully designed specific RT-qPCR oligonucleotides, the methods were optimized for each gene and validated by the accurate measure of previously known number of copies of the pfmdr1 gene in the laboratory reference strains P. falciparum 3D7 and Dd2. Subsequently, Standard Operating Procedures (SOPs) were developed to the remaining genes under study and successfully applied to DNA obtained from dried filter blood spots of field isolates of P. falciparum collected in São Tomé & Principe, West Africa. The SOPs reported here may be used as a high throughput tool to investigate the role of these drug resistance gene candidates in laboratory studies or large scale epidemiological surveys.

  13. Implementation of a data processing platform for real-time distance measurement with dual-comb lasers

    Science.gov (United States)

    Ni, Kai; Xu, Mingfei; Zhou, Qian; Dong, Hao; Li, Xinghui; Wu, Guanhao

    2015-08-01

    Absolute distance measurement with dual femtosecond comb lasers has advantages of wide-range, high-accuracy and fast speed. It combines time-of-flight and interferometric measurement. The novelty of ranging method leads to new challenges in designing the data acquisition and processing hardware system. Currently there are no available real-time data processing system for dual-comb ranging. This paper introduces our recent progress on designing and implementing such a platform. Our platform mainly contains four different function modules. First, a clock module that accept a 250MHz maximum reference clock input was introduced to generate the sample clock for A/D converter, and the module's output clock can be delayed up to 20ns with a resolution of 714ps. Second, a high-speed data acquisition module with a 14-bit resolution and a 125 MSPS maximum sample rate was designed to convert the analog laser pulse signal to digital signal. Third, we built a real-time data processing module that allows an input of 16-bit data in the FPGA to calculate the distance from the digital signal within 83us. Finally, a data transmission module based on a 128MB DDR SDRAM and USB2.0 was added so that we can easily debug the platform in the PC. The performance of our system is evaluated in real-time. The test bench consists of two femtosecond laser sources, an optical fiber interferometer and our data processing system. The repetition frequencies of the two combs are around 50MHz, with frequency difference of 2.5kHz. The center wavelength of laser pulses is 1560nm. The target distance is from 0m to 3m. The experimental results show that our system can output measurement results at the rate of 2500 pts/s, and the measurement deviation is less than 10um.

  14. A dynamic approach to real-time performance measurement in design projects

    DEFF Research Database (Denmark)

    Skec, Stanko; Cash, Philip; Storga, Mario

    2017-01-01

    and teamwork performance in engineering design projects. This integrates multiple, previously disparate, aspects of design management and performance measurement theory in a single framework. Further, a fully realised performance measurement approach is developed, which complements existing management...

  15. Analysis of Gene and Protein Expression in Atherosclerotic Mouse Aorta by Western Blot and Quantitative Real-Time PCR.

    Science.gov (United States)

    Rivera-Torres, José

    2015-01-01

    Atherosclerosis involves changes in gene and protein expression patterns in affected arteries. Quantification of these alterations is essential for understanding the molecular mechanisms underlying this pathology. Western blot and real-time PCR-used to quantify protein and messenger RNA levels, respectively-are invaluable molecular biology tools, particularly when material is limited. The availability of many genetically modified mouse models of atherosclerosis makes the mouse aorta an ideal tissue in which to carry out these expression pattern analyses. In this chapter, protocols are presented for mRNA and protein extraction from mouse aorta and for the accurate quantification of mRNA expression by RT-PCR and of proteins by western blot.

  16. Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. niveum in soil.

    Science.gov (United States)

    Peng, Jun; Zhan, Yuanfeng; Zeng, Fanyun; Long, Haibo; Pei, Yuelin; Guo, Jianrong

    2013-12-01

    Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is one of the major limiting factors for watermelon production worldwide. Rapid and accurate detection of the causal pathogen is the cornerstone of integrated disease management. In this paper, a real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay was developed for the rapid and quantitative detection of Fon in soil. Positive products were amplified only from Fon isolates and not from any other species or formae speciales of F. oxysporum tested, showing a high specificity of the primer sets. The detection limit of the RealAmp assay was 1.2 pg μL(-1) genomic DNA or 10(3) spores g(-1) of artificially inoculated soil, whereas real-time PCR could detect as low as 12 fg μL(-1) or 10(2) spores g(-1). The RealAmp assay was further applied to detect eight artificially inoculated and 85 field soil samples. No significant differences were found between the results tested by the RealAmp and real-time PCR assays. The RealAmp assay is a simple, rapid and effective technique for the quantitative detection and monitoring of Fon in soil under natural conditions. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  17. Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts

    Directory of Open Access Journals (Sweden)

    Galli Cesare

    2009-12-01

    Full Text Available Abstract Background Application of reverse transcription quantitative real-time polymerase chain reaction is very well suited to reveal differences in gene expression between in vivo and in vitro produced embryos. Ultimately, this may lead to optimized equine assisted reproductive techniques. However, for a correct interpretation of the real-time PCR results, all data must be normalized, which is most reliably achieved by calculating the geometric mean of the most stable reference genes. In this study a set of reliable reference genes was identified for equine in vivo and fresh and frozen-thawed in vitro embryos. Findings The expression stability of 8 candidate reference genes (ACTB, GAPDH, H2A/I, HPRT1, RPL32, SDHA, TUBA4A, UBC was determined in 3 populations of equine blastocysts (fresh in vivo, fresh and frozen-thawed in vitro embryos. Application of geNorm indicated UBC, GAPDH, ACTB and HPRT1 as the most stable genes in the in vivo embryos and UBC, RPL32, GAPDH and ACTB in both in vitro populations. When in vivo and in vitro embryos were combined, UBC, ACTB, RPL32 and GAPDH were found to be the most stable. SDHA and H2A/I appeared to be highly regulated. Conclusions Based on these results, the geometric mean of UBC, ACTB, RPL32 and GAPDH is to be recommended for accurate normalization of quantitative real-time PCR data in equine in vivo and in vitro produced blastocysts.

  18. Real-Time Measurements and Modelling on Dynamic Behaviour of SonoVue Bubbles Based on Light Scattering Technology

    Science.gov (United States)

    Tu, Juan; Guan F., J.; Matula J., T.; Crum A., L.; Wei, Rongjue

    2008-01-01

    The dynamic behaviour of SonoVue microbubbles, a new generation ultrasound contrast agent, is investigated in real time with light scattering method. Highly diluted SonoVue microbubbles are injected into a diluted gel made of xanthan gum and water. The responses of individual SonoVue bubbles to driven ultrasound pulses are measured. Both linear and nonlinear bubble oscillations are observed and the results suggest that SonoVue microbubbles can generate strong nonlinear responses. By fitting the experimental data of individual bubble responses with Sarkar's model, the shell coating parameter of the bubbles and dilatational viscosity is estimated to be 7.0 nm.s.Pa.

  19. Real-Time, Single-Shot Temporal Measurements of Short Electron Bunches, Terahertz CSR and FEL Radiation

    CERN Document Server

    Berden, G; Van der Meer, A F G

    2005-01-01

    Electro-optic detection of the Coulomb field of electron bunches is a promising technique for single-shot measurements of the bunch length and shape in the sub-picosecond time domain. This technique has been applied to the measurement of 50 MeV electron bunches in the FELIX free electron laser, showing the longitudinal profile of single bunches of around 650 fs FWHM [Phys. Rev. Lett. 93, 114802 (2004)]. The method is non-destructive and real-time, and therefore ideal for online monitoring of the longitudinal shape of single electron bunches. At FELIX we have used it for real-time optimization of sub-picosecond electron bunches. Electro-optic detection has also been used to measure the electric field profiles of far-infrared (or terahertz) optical pulses generated by the relativistic electrons. We have characterised the far-infrared output of the free electron laser, and more recently, we have measured the temporal profile of terahertz optical pulses generated at one of the bending magnets.

  20. HIV-1 viral load measurement in venous blood and fingerprick blood using Abbott RealTime HIV-1 DBS assay.

    Science.gov (United States)

    Tang, Ning; Pahalawatta, Vihanga; Frank, Andrea; Bagley, Zowie; Viana, Raquel; Lampinen, John; Leckie, Gregor; Huang, Shihai; Abravaya, Klara; Wallis, Carole L

    2017-07-01

    HIV RNA suppression is a key indicator for monitoring success of antiretroviral therapy. From a logistical perspective, viral load (VL) testing using Dried Blood Spots (DBS) is a promising alternative to plasma based VL testing in resource-limited settings. To evaluate the analytical and clinical performance of the Abbott RealTime HIV-1 assay using a fully automated one-spot DBS sample protocol. Limit of detection (LOD), linearity, lower limit of quantitation (LLQ), upper limit of quantitation (ULQ), and precision were determined using serial dilutions of HIV-1 Virology Quality Assurance stock (VQA Rush University), or HIV-1-containing armored RNA, made in venous blood. To evaluate correlation, bias, and agreement, 497 HIV-1 positive adult clinical samples were collected from Ivory Coast, Uganda and South Africa. For each HIV-1 participant, DBS-fingerprick, DBS-venous and plasma sample results were compared. Correlation and bias values were obtained. The sensitivity and specificity were analyzed at a threshold of 1000 HIV-1 copies/mL generated using the standard plasma protocol. The Abbott HIV-1 DBS protocol had an LOD of 839 copies/mL, a linear range from 500 to 1×107 copies/mL, an LLQ of 839 copies/mL, a ULQ of 1×107 copies/mL, and an inter-assay SD of ≤0.30 log copies/mL for all tested levels within this range. With clinical samples, the correlation coefficient (r value) was 0.896 between DBS-fingerprick and plasma and 0.901 between DBS-venous and plasma, and the bias was -0.07 log copies/mL between DBS-fingerprick and plasma and -0.02 log copies/mL between DBS-venous and plasma. The sensitivity of DBS-fingerprick and DBS-venous was 93%, while the specificity of both DBS methods was 95%. The results demonstrated that the Abbott RealTime HIV-1 assay with DBS sample protocol is highly sensitive, specific and precise across a wide dynamic range and correlates well with plasma values. The Abbott RealTime HIV-1 assay with DBS sample protocol provides an

  1. Real-time measurement of aerosol particle concentration at high temperatures; Hiukkaspitoisuuden reaaliaikainen mittaaminen korkeassa laempoetilassa

    Energy Technology Data Exchange (ETDEWEB)

    Keskinen, J.; Hautanen, J.; Laitinen, A. [Tampere Univ. of Technology (Finland). Physics

    1997-10-01

    The aim of this project is to develop a new method for continuous aerosol particle concentration measurement at elevated temperatures (up to 800-1000 deg C). The measured property of the aerosol particles is the so called Fuchs surface area. This quantity is relevant for diffusion limited mass transfer to particles. The principle of the method is as follows. First, aerosol particles are charged electrically by diffusion charging process. The charging takes place at high temperature. After the charging, aerosol is diluted and cooled. Finally, aerosol particles are collected and the total charge carried by the aerosol particles is measured. Particle collection and charge measurement take place at low temperature. Benefits of this measurement method are: particles are charged in-situ, charge of the particles is not affected by the temperature and pressure changes after sampling, particle collection and charge measurement are carried out outside the process conditions, and the measured quantity is well defined. The results of this study can be used when the formation of the fly ash particles is studied. Another field of applications is the study and the development of gasification processes. Possibly, the method can also be used for the monitoring the operation of the high temperature particle collection devices. (orig.)

  2. Extreme Design Loads Calibration of Offshore Wind Turbine Blades through Real Time Measurements

    DEFF Research Database (Denmark)

    Natarajan, Anand; Vesth, Allan; Lamata, Rebeca Rivera

    2014-01-01

    Blade Root flap and Edge moments are measured on the blades of a 3.6MW offshore wind turbine in normal operation. Ten minute maxima of the measurements are sampled to determine the extreme blade root flap moment, edge moment and resultant moment over six month duration. A random subset of the mea...... moment is extrapolated. This is found to possess smaller scaling factors in measurements over six months as compared to both the flap and edge moments, indicating that the contemporaneous load component of an extrapolated load should possess much smaller magnitude than its maxima....

  3. Extreme Design Loads Calibration of Offshore Wind Turbine Blades through Real Time Measurements

    DEFF Research Database (Denmark)

    Natarajan, Anand; Vesth, Allan; Lamata, Rebeca Rivera

    Blade Root flap and Edge moments are measured on the blades of a 3.6MW offshore wind turbine in normal operation. Ten minute maxima of the measurements are sampled to determine the extreme blade root flap moment, edge moment and resultant moment over six month duration. A random subset of the mea...... moment is extrapolated. This is found to possess smaller scaling factors in measurements over six months as compared to both the flap and edge moments, indicating that the contemporaneous load component of an extrapolated load should possess much smaller magnitude than its maxima....

  4. Combining remotely-sensed snow water equivalent with in-situ measurements to produce a real-time SWE product

    Science.gov (United States)

    Schneider, D.; Molotch, N. P.

    2013-12-01

    Snowmelt is the primary water source in the Western United States and mountainous regions globally. Forecasts of streamflow and water supply rely heavily on snow measurements from sparse observation networks that may not provide adequate information during abnormal climatic conditions. To address this issue we developed a real-time spatially distributed snow water equivalent (SWE) product for the Upper Colorado River Basin that merges previous years SWE patterns derived from a reconstruction model, with interpolations of real-time situ measurements. The approach uses a multiple linear regression to model SWE in which physiography and reconstructed SWE are treated as independent variables and observed SNOTEL SWE the dependent variable. Using a drop-1 approach, the years 2000 - 2011 (Mar 1, Apr 1, and May 1) consistently find reconstructed SWE to be a significant predictor and the explained variability of the model is improved between 0.1 and 0.5 (mean= 0.23) compared to a model just based on physiographics. Independent validation in the Front Range, CO produces mean absolute errors (MAE) between 0.13 and 0.18 m, with significant improvements between 0.03 and 0.09 m over both reconstructed SWE and physiographic SWE (psurface to incorporate regional effects within the modeling domain. However, MAE is only marginally reduced (~0.01) with blending. Improved analysis of past SWE distribution can provide valuable information for modeling efforts to predict, e.g. hydrologic impacts due to climate change and disturbances. Future validation is planned in additional locations within the modeling domain and a real-time product is in development that uses this ensemble of past patterns of SWE to estimate SWE in the current water year.

  5. A two-domain real-time algorithm for optimal data reduction: A case study on accelerator magnet measurements

    CERN Document Server

    Arpaia, P; Inglese, V

    2010-01-01

    A real-time algorithm of data reduction, based on the combination a two lossy techniques specifically optimized for high-rate magnetic measurements in two domains (e.g. time and space), is proposed. The first technique exploits an adaptive sampling rule based on the power estimation of the flux increments in order to optimize the information to be gathered for magnetic field analysis in real time. The tracking condition is defined by the target noise level in the Nyquist band required by post-processing procedure of magnetic analysis. The second technique uses a data reduction algorithm in order to improve the compression ratio while preserving the consistency of the measured signal. The allowed loss is set equal to the random noise level in the signal in order to force the loss and the noise to cancel rather than to add, by improving the signal-to-noise ratio. Numerical analysis and experimental results of on-field performance characterization and validation for two case studies of magnetic measurement syste...

  6. Evaluation of a wearable monitor for measuring real-time diesel particulate matter concentrations in several underground mines.

    Science.gov (United States)

    Noll, J D; Janisko, S

    2013-01-01

    The standard method for determining diesel particulate matter (DPM) exposures in underground metal/nonmetal mines provides the average exposure concentration for an entire working shift, and it can take weeks to obtain results. This approach is problematic because, although it reports that an overexposure has occurred, it fails to provide critical information about cause or prevention. Conversely, real-time measurement would provide miners with timely information to identify the major factors contributing to overexposures and would allow engineering controls to be deployed immediately. Due to these potential benefits, the National Institute for Occupational Safety and Health (NIOSH) developed a wearable instrument that measures real-time elemental carbon (EC) concentrations (EC is a DPM surrogate) via laser extinction. This instrument was later constructed into a commercial version (Airtec). This article evaluates the Airtec's performance in several underground metal/nonmetal mines by comparing it to the standard method for determining DPM exposures (NIOSH method 5040). The instrument was found to meet the NIOSH accuracy criteria and to show no statistical difference from NIOSH method 5040 results. In addition, the instrument's measurements were found to be unaffected by dust and humidity.

  7. Linking Near Real-Time Water Quality Measurements to Fecal Coliforms and Trace Organic Pollutants in Urban Streams

    Science.gov (United States)

    Henjum, M.; Wennen, C.; Hondzo, M.; Hozalski, R. M.; Novak, P. J.; Arnold, W. A.

    2009-05-01

    Anthropogenic pollutants, including pesticides, herbicides, pharmaceuticals, and estrogens are detected in urban water bodies. Effective examination of dilute organic and microbial pollutant loading rates within surface waters is currently prohibitively expensive and labor intensive. Effort is being placed on the development of improved monitoring methodologies to more accurately assess surface water quality and evaluate the effectiveness of water quality management practices. Throughout the summer and fall of 2008 a "real-time" wireless network equipped with high frequency fundamental water quality parameter sensors measured turbidity, conductivity, pH, depth, temperature, dissolved oxygen and nitrate above and below stormwater inputs at two urban stream locations. At each location one liter grab samples were concurrently collected by ISCO automatic samplers at two hour intervals for 24 hour durations during three dry periods and five rain events. Grab samples were analyzed for fecal coliforms, atrazine (agricultural herbicide), prometon (residential herbicide) and caffeine (wastewater indicator). Surrogate relationships between easy-to-measure water quality parameters and difficult-to-measure pollutants were developed, subsequently facilitating monitoring of these pollutants without the development of new, and likely costly, technologies. Additionally, comparisons were made between traditional grab sampling techniques and the "real-time" monitoring to assess the accuracy of Total Maximum Daily Load (TMDL) calculations.

  8. Quantitative assessment of Wilms tumor 1 expression by real-time quantitative polymerase chain reaction in patients with acute myeloblastic leukemia

    Directory of Open Access Journals (Sweden)

    Hossein Ayatollahi

    2017-01-01

    Full Text Available Background: The Wilms tumor 1 (WT1 gene is originally defined as a tumor suppressor gene and a transcription factor that overexpressed in leukemic cells. It is highly expressed in more than 80% of acute myeloid leukemia (AML patients, both in bone marrow (BM and in peripheral blood (PB, and it is used as a powerful and independent marker of minimal residual disease (MRD; we have determined the expression levels of the WT1 by real-time quantitative polymerase chain reaction (RQ-PCR in PB and BM in 126 newly diagnosed AML patients. Materials and Methods: This study was done in molecular pathology and cancer research center from April 2014 to June 2015, RQ-PCR method was used to determine the WT1 gene expression in BM and/or PB samples from 126 patients of AML, we cloned both WT1 and ABL genes for creating a standard curve, and we calculate copy number of WT1 genes in patients. Results: A total of 126 AML patients consist of 70 males (55.6% and 56 females (44.4%, with a median age of 26 years; 104 (81% patients out of 126 show overexpression of WT1 gene. We also concomitant monitoring of fusion transcripts (PML RARa, AML1-ETO, MLL-MLL, CBFb-MYH11, or DEK-CAN in our patients, the AML1-ETO group showing remarkably low levels of WT1 compared with other fusion transcript and the CBFB-MYH11 showing high levels of WT1. Conclusion: We conclude that WT1 expression by RQ-PCR in AML patients may be employed as an independent tool to detect MRD in the majority of normal karyotype AML patients.

  9. Development of Real-Time Measurement of Effective Dose for High Dose Rate Neutron Fields

    CERN Document Server

    Braby, L A; Reece, W D

    2003-01-01

    Studies of the effects of low doses of ionizing radiation require sources of radiation which are well characterized in terms of the dose and the quality of the radiation. One of the best measures of the quality of neutron irradiation is the dose mean lineal energy. At very low dose rates this can be determined by measuring individual energy deposition events, and calculating the dose mean of the event size. However, at the dose rates that are normally required for biology experiments, the individual events can not be separated by radiation detectors. However, the total energy deposited in a specified time interval can be measured. This total energy has a random variation which depends on the size of the individual events, so the dose mean lineal energy can be calculated from the variance of repeated measurements of the energy deposited in a fixed time. We have developed a specialized charge integration circuit for the measurement of the charge produced in a small ion chamber in typical neutron irradiation exp...

  10. Nonintrusive performance measurement of a gas turbine engine in real time

    Energy Technology Data Exchange (ETDEWEB)

    DeSilva, Upul P.; Claussen, Heiko

    2017-08-29

    Performance of a gas turbine engine is monitored by computing a mass flow rate through the engine. Acoustic time-of-flight measurements are taken between acoustic transmitters and receivers in the flow path of the engine. The measurements are processed to determine average speeds of sound and gas flow velocities along those lines-of-sound. A volumetric flow rate in the flow path is computed using the gas flow velocities together with a representation of the flow path geometry. A gas density in the flow path is computed using the speeds of sound and a measured static pressure. The mass flow rate is calculated from the gas density and the volumetric flow rate.

  11. Real-Time GNSS-Based Attitude Determination in the Measurement Domain.

    Science.gov (United States)

    Zhao, Lin; Li, Na; Li, Liang; Zhang, Yi; Cheng, Chun

    2017-02-05

    A multi-antenna-based GNSS receiver is capable of providing high-precision and drift-free attitude solution. Carrier phase measurements need be utilized to achieve high-precision attitude. The traditional attitude determination methods in the measurement domain and the position domain resolve the attitude and the ambiguity sequentially. The redundant measurements from multiple baselines have not been fully utilized to enhance the reliability of attitude determination. A multi-baseline-based attitude determination method in the measurement domain is proposed to estimate the attitude parameters and the ambiguity simultaneously. Meanwhile, the redundancy of attitude resolution has also been increased so that the reliability of ambiguity resolution and attitude determination can be enhanced. Moreover, in order to further improve the reliability of attitude determination, we propose a partial ambiguity resolution method based on the proposed attitude determination model. The static and kinematic experiments were conducted to verify the performance of the proposed method. When compared with the traditional attitude determination methods, the static experimental results show that the proposed method can improve the accuracy by at least 0.03° and enhance the continuity by 18%, at most. The kinematic result has shown that the proposed method can obtain an optimal balance between accuracy and reliability performance.

  12. In situ transmissiometer measurements for real-time monitoring of dust discharge during orchard nut harvesting.

    Science.gov (United States)

    Downey, D; Giles, D K; Thompson, J F

    2008-01-01

    Rapid assessments of operating conditions and field preparation on dust discharge from nut harvesters are needed to guide improved equipment design and grower practices for dust reduction. An industrial opacity sensor, typically used for industrial stack monitoring, was adapted for use on a nut harvester to measure relative dust intensity during nut pick-up operations in almond orchards. Due to the high volume of discharge air and the presence of large debris such as leaves, additional components were coupled with the sensor to enable subsampling of the air. Pre-harvest windrow preparation conditions were evaluated. Results indicated that relative dust intensity decreased by 32% during harvest activities after windrow preparation with proper nut sweeper adjustment. Conventional harvesting results indicated that under typical operating conditions, reducing the separation fan speed could reduce relative dust intensity by 54%. Ground speed also had a strong effect; reducing speed from 4.8 to 2.4 km h(-1) reduced opacity of discharged air by 50%. The measurement system was also mounted on a separate vehicle and used as a tool for comparing modifications in harvest machine designs where direct measurement of discharge may not be feasible due to mechanical constraints. A comparison between a conventional harvester and one modification in the harvester design found that the machine modification decreased relative dust intensity by 73%. The measurement tools described in this work can be used to provide rapid feedback on harvester operating conditions, orchard cultural practices, and machine design modifications.

  13. Nanoparticles in cigarette smoke; real-time undiluted measurements by a scanning mobility particle sizer

    NARCIS (Netherlands)

    Dijk, W.D. van; Gopal, S.R.; Scheepers, P.T.J.

    2011-01-01

    Cigarette smoke is a complex mixture of smoke constituents, often characterised by size-resolved particle distributions. Since descriptions of ultrafine particles <50 nm are absent, our aim was to explore the existence of these nanoparticles in fresh and undiluted cigarette smoke. We measured

  14. Earthquake source parameters from GPS-measured static displacements with potential for real-time application

    NARCIS (Netherlands)

    O'Toole, T.B.; Valentine, A.P.; Woodhouse, J.H.

    2013-01-01

    We describe a method for determining an optimal centroid– moment tensor solution of an earthquake from a set of static displacements measured using a network of Global Positioning System receivers. Using static displacements observed after the 4 April 2010, MW 7.2 El Mayor-Cucapah, Mexico,

  15. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.

    Science.gov (United States)

    Vandesompele, Jo; De Preter, Katleen; Pattyn, Filip; Poppe, Bruce; Van Roy, Nadine; De Paepe, Anne; Speleman, Frank

    2002-06-18

    Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem. We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data. The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.

  16. Noninvasive, real-time measurements of plasma parameters via optical emission spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Wang Shicong; Wendt, Amy E. [Department of Electrical and Computer Engineering, University of Wisconsin, Madison, Wisconsin 53706 (United States); Boffard, John B.; Lin, Chun C. [Department of Physics, University of Wisconsin, Madison, Wisconsin 53706 (United States); Radovanov, Svetlana; Persing, Harold [Varian Semiconductor Equipment, Applied Materials Inc., Gloucester, Massachusetts 01939 (United States)

    2013-03-15

    Plasma process control applications require acquisition of diagnostic data at a rate faster than the characteristic timescale of perturbations to the plasma. Diagnostics based on optical emission spectroscopy of intense emission lines permit rapid noninvasive measurements with low-resolution ({approx}1 nm), fiber-coupled spectrographs, which are included on many plasma process tools for semiconductor processing. Here the authors report on rapid analysis of Ar emissions with such a system to obtain electron temperatures, electron densities, and metastable densities in argon and argon/mixed-gas (Ar/N{sub 2}, Ar/O{sub 2}, Ar/H{sub 2}) inductively coupled plasmas. Accuracy of the results (compared to measurements made by Langmuir probe and white-light absorption spectroscopy) are typically better than {+-}15% with a time resolution of 0.1 s, which is more than sufficient to capture the transient behavior of many processes, limited only by the time response of the spectrograph used.

  17. Impact of real-time measurements for data assimilation in reservoir simulation

    Energy Technology Data Exchange (ETDEWEB)

    Schulze-Riegert, R.; Krosche, M. [Scandpower Petroleum Technology GmbH, Hamburg (Germany); Pajonk, O. [TU Braunschweig (Germany). Inst. fuer Wissenschaftliches Rechnen; Myrland, T. [Morges Teknisk-Naturvitenskapelige Univ. (NTNU), Trondheim (Germany)

    2008-10-23

    This paper gives an overview on the conceptual background of data assimilation techniques. The framework of sequential data assimilation as described for the ensemble Kalman filter implementation allows a continuous integration of new measurement data. The initial diversity of ensemble members will be critical for the assimilation process and the ability to successfully assimilate measurement data. At the same time the initial ensemble will impact the propagation of uncertainties with crucial consequences for production forecasts. Data assimilation techniques have complimentary features compared to other optimization techniques built on selection or regression schemes. Specifically, EnKF is applicable to real field cases and defines an important perspective for facilitating continuous reservoir simulation model updates in a reservoir life cycle. (orig.)

  18. Innovative real-time and non-destructive method of beam profile measurement under large beam current irradiation for BNCT

    Science.gov (United States)

    Takada, M.; Kamada, S.; Suda, M.; Fujii, R.; Nakamura, M.; Hoshi, M.; Sato, H.; Endo, S.; Hamano, T.; Arai, S.; Higashimata, A.

    2012-10-01

    We developed a real-time and non-destructive method of beam profile measurement on a target under large beam current irradiation, and without any complex radiation detectors or electrical circuits. We measured the beam profiles on a target by observing the target temperature using an infrared-radiation thermometer camera. The target temperatures were increased and decreased quickly by starting and stopping the beam irradiation within 1 s in response speed. Our method could trace beam movements rapidly. The beam size and position were calibrated by measuring O-ring heat on the target. Our method has the potential to measure beam profiles at beam current over 1 mA for proton and deuteron with the energy around 3 MeV and allows accelerator operators to adjust the beam location during beam irradiation experiments without decreasing the beam current.

  19. Innovative real-time and non-destructive method of beam profile measurement under large beam current irradiation for BNCT

    Energy Technology Data Exchange (ETDEWEB)

    Takada, M., E-mail: m_takada@nirs.go.jp [National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Kamada, S.; Suda, M. [National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Fujii, R.; Nakamura, M. [Cancer Intelligence Care Systems, Inc., 3-5-7 Ariake, Koto-ku, Tokyo 135-0063 (Japan); Hoshi, M. [Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 kasumi, Minami-ku, Hiroshima 734-8553 (Japan); Sato, H. [Ibaraki Prefectural University of Health Sciences, 4669-2, Ami Ami-Cho, Inashiki-gun, Ibaraki 300-0394 (Japan); Endo, S. [Quantum Energy Applications, Graduate School of Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima 739-8527 (Japan); Hamano, T. [National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Arai, S.; Higashimata, A. [Sanki Industry Co., 318-6, Sannoh, Inage-ku, Chiba 263-0002 (Japan)

    2012-10-11

    We developed a real-time and non-destructive method of beam profile measurement on a target under large beam current irradiation, and without any complex radiation detectors or electrical circuits. We measured the beam profiles on a target by observing the target temperature using an infrared-radiation thermometer camera. The target temperatures were increased and decreased quickly by starting and stopping the beam irradiation within 1 s in response speed. Our method could trace beam movements rapidly. The beam size and position were calibrated by measuring O-ring heat on the target. Our method has the potential to measure beam profiles at beam current over 1 mA for proton and deuteron with the energy around 3 MeV and allows accelerator operators to adjust the beam location during beam irradiation experiments without decreasing the beam current.

  20. Real-time specific surface area measurements via laser-induced breakdown spectroscopy

    Science.gov (United States)

    Washburn, Kathryn E.; Birdwell, Justin E.; Howard, James E.

    2017-01-01

    From healthcare to cosmetics to environmental science, the specific surface area (SSA) of micro- and mesoporous materials or products can greatly affect their chemical and physical properties. SSA results are also widely used to examine source rocks in conventional and unconventional petroleum resource plays. Despite its importance, current methods to measure SSA are often cumbersome, time-consuming, or require cryogenic consumables (e.g., liquid nitrogen). These methods are not amenable to high-throughput environments, have stringent sample preparation requirements, and are not practical for use in the field. We present a new application of laser-induced breakdown spectroscopy for rapid measurement of SSA. This study evaluates geological samples, specifically organic-rich oil shales, but the approach is expected to be applicable to many other types of materials. The method uses optical emission spectroscopy to examine laser-generated plasma and quantify the amount of argon adsorbed to a sample during an inert gas purge. The technique can accommodate a wide range of sample sizes and geometries and has the potential for field use. These advantages for SSA measurement combined with the simultaneous acquisition of composition information make this a promising new approach for characterizing geologic samples and other materials.

  1. Toward real-time measurement of atmospheric mercury concentrations using cavity ring-down spectroscopy

    Directory of Open Access Journals (Sweden)

    X. Faïn

    2010-03-01

    Full Text Available Cavity ring-down spectroscopy (CRDS is a direct absorption technique that utilizes path lengths up to multiple kilometers in a compact absorption cell and has a significantly higher sensitivity than conventional absorption spectroscopy. This tool opens new prospects for study of gaseous elemental mercury (Hg0 because of its high temporal resolution and reduced sample volume requirements (<0.5 l of sample air. We developed a new sensor based on CRDS for measurement of (Hg0 mass concentration. Sensor characteristics include sub-ng m−3 detection limit and high temporal resolution using a frequency-doubled, tuneable dye laser emitting pulses at ~253.65 nm with a pulse repetition frequency of 50 Hz. The dye laser incorporates a unique piezo element attached to its tuning grating allowing it to tune the laser on and off the Hg0 absorption line on a pulse-to-pulse basis to facilitate differential absorption measurements. Hg0 absorption measurements with this CRDS laboratory prototype are highly linearly related to Hg0 concentrations determined by a Tekran 2537B analyzer over an Hg0 concentration range from 0.2 ng m−3 to 573 ng m−3, implying excellent linearity of both instruments. The current CRDS instrument has a sensitivity of 0.10 ng Hg0 m−3 at 10-s time resolution. Ambient-air tests showed that background Hg0 levels can be detected at low temporal resolution (i.e., 1 s, but also highlight a need for high-frequency (i.e., pulse-to-pulse differential on/off-line tuning of the laser wavelength to account for instabilities of the CRDS system and variable background absorption interferences. Future applications may include ambient Hg0 flux measurements with eddy covariance techniques, which require measurements of Hg0 concentrations with sub-ng m−3 sensitivity and sub-second time

  2. Real-time estimate of body kinematics during a planar squat task using a single inertial measurement unit.

    Science.gov (United States)

    Bonnet, Vincent; Mazzà, Claudia; Fraisse, Philippe; Cappozzo, Aurelio

    2013-07-01

    This study aimed at the real-time estimation of the lower-limb joint and torso kinematics during a squat exercise, performed in the sagittal plane, using a single inertial measurement unit placed on the lower back. The human body was modeled with a 3-DOF planar chain. The planar IMU orientation and vertical displacement were estimated using one angular velocity and two acceleration components and a weighted Fourier linear combiner. The ankle, knee, and hip joint angles were thereafter obtained through a novel inverse kinematic module based on the use of a Jacobian pseudoinverse matrix and null-space decoupling. The aforementioned algorithms were validated on a humanoid robot for which the mechanical model used and the measured joint angles virtually exhibited no inaccuracies. Joint angles were estimated with a maximal error of 1.5°. The performance of the proposed analytical and experimental methodology was also assessed by conducting an experiment on human volunteers and by comparing the relevant results with those obtained through the more conventional photogrammetric approach. The joint angles provided by the two methods displayed differences equal to 3±1°. These results, associated with the real-time capability of the method, open the door to future field applications in both rehabilitation and sport.

  3. A Novel Low-Cost Real-Time Power Measurement Platform for LoWPAN IoT Devices

    Directory of Open Access Journals (Sweden)

    Yang Liu

    2017-01-01

    Full Text Available With the rapid development of technology and application for Internet of Things (IoT, Low-Power Wireless Personal Area Network (LoWPAN devices are more popularly applied. Evaluation of power efficiency is important to LoWPAN applications. Conventional method to evaluate the power efficiency of different LoWPAN devices is as follows: first measure the current of the devices under working/idle/sleep state and then make an average and estimation of the lifetime of batteries, which deeply relied on the accuracy of testing equipment and is not that accurate and with high cost. In this work, a low-cost, real-time power measurement platform called PTone is proposed, which can be used to detect the real-time power of LoWPAN devices (above 99.63% and be able to determine the state of each module of DUT system. Based on the PTone, a novel abnormal status diagnosis mechanism is proposed. The mechanism can not only judge abnormal status but also find accurate abnormal status locating and classify abnormal status accurately. According to the method, each state of Device Under Test (DUT during wireless transmission is estimated, different abnormal status can be classified, and thus specific location of abnormal module can be found, which will significantly shorten the development process for LoWPAN devices and thus reduce costs.

  4. Quantitative analysis of aortic regurgitation: real-time 3-dimensional and 2-dimensional color Doppler echocardiographic method--a clinical and a chronic animal study

    Science.gov (United States)

    Shiota, Takahiro; Jones, Michael; Tsujino, Hiroyuki; Qin, Jian Xin; Zetts, Arthur D.; Greenberg, Neil L.; Cardon, Lisa A.; Panza, Julio A.; Thomas, James D.

    2002-01-01

    BACKGROUND: For evaluating patients with aortic regurgitation (AR), regurgitant volumes, left ventricular (LV) stroke volumes (SV), and absolute LV volumes are valuable indices. AIM: The aim of this study was to validate the combination of real-time 3-dimensional echocardiography (3DE) and semiautomated digital color Doppler cardiac flow measurement (ACM) for quantifying absolute LV volumes, LVSV, and AR volumes using an animal model of chronic AR and to investigate its clinical applicability. METHODS: In 8 sheep, a total of 26 hemodynamic states were obtained pharmacologically 20 weeks after the aortic valve noncoronary (n = 4) or right coronary (n = 4) leaflet was incised to produce AR. Reference standard LVSV and AR volume were determined using the electromagnetic flow method (EM). Simultaneous epicardial real-time 3DE studies were performed to obtain LV end-diastolic volumes (LVEDV), end-systolic volumes (LVESV), and LVSV by subtracting LVESV from LVEDV. Simultaneous ACM was performed to obtain LVSV and transmitral flows; AR volume was calculated by subtracting transmitral flow volume from LVSV. In a total of 19 patients with AR, real-time 3DE and ACM were used to obtain LVSVs and these were compared with each other. RESULTS: A strong relationship was found between LVSV derived from EM and those from the real-time 3DE (r = 0.93, P clinically applicable.

  5. REAL-TIME STABILITY AND PROFILE COMPARISON MEASUREMENTS BETWEEN TWO DIFFERENT LTPS.

    Energy Technology Data Exchange (ETDEWEB)

    QIAN, S.; WANG, D.J.

    2005-07-31

    The Long Trace Profiler (LTP) is a precise angle measurement instrument, with a sensitivity and accuracy that can be in the sub-micron radian range. LTP characteristics depend on the particular LTP system schematic design, and the quality of components and assembly. The conditions of temperature, alignment, and mirror support during the measurement process vary between different laboratories, which influences significantly the test repeatability and accuracy. In this paper we introduce a direct comparison method to test the same object at the same point in the same environment at the same time by using two LTPs, which significantly increases the reliability of the comparison. A compact, portable LTP (PTLTP), which can be carried to different laboratories around the world, is used for comparison testing. Stability Comparison experiments between the LTP II at the National Synchrotron Radiation Research Center (NSRRC), and the PTLTP of Brookhaven National Laboratory (BNL) reveal significant differences in performance between the instruments. The experiment is set up so that each optical head simultaneously records both its own sample probe beam and also the probe beam from the other optical head. The two probe beams are reflected from same point on the mirror. Tests show that the stability of the PTLTP with a monolithic beam splitter is 10 times better than the stability of the LTP II which has a separated beam splitter unit. A scheme for comparing scanning measurements of a mirror is introduced. Experimental results show a significant difference between the two LTPs due mainly to distortions in the optical components inside the optical head. A new scheme is proposed for further mirror comparison scanning tests.

  6. RTX Correction Accuracy and Real-Time Data Processing of the New Integrated SeismoGeodetic System with Real-Time Acceleration and Displacement Measurements for Earthquake Characterization Based on High-Rate Seismic and GPS Data

    Science.gov (United States)

    Zimakov, L. G.; Raczka, J.; Barrientos, S. E.

    2016-12-01

    We will discuss and show the results obtained from an integrated SeismoGeodetic System, model SG160-09, installed in the Chile (Chilean National Network), Italy (University of Naples Network), and California. The SG160-09 provides the user high rate GNSS and accelerometer data, full epoch-by-epoch measurement integrity and the ability to create combined GNSS and accelerometer high-rate (200Hz) displacement time series in real-time. The SG160-09 combines seismic recording with GNSS geodetic measurement in a single compact, ruggedized case. The system includes a low-power, 220-channel GNSS receiver powered by the latest Trimble-precise Maxwell™6 technology and supports tracking GPS, GLONASS and Galileo signals. The receiver incorporates on-board GNSS point positioning using Real-Time Precise Point Positioning (PPP) technology with satellite clock and orbit corrections delivered over IP networks. The seismic recording includes an ANSS Class A, force balance accelerometer with the latest, low power, 24-bit A/D converter, producing high-resolution seismic data. The SG160-09 processor acquires and packetizes both seismic and geodetic data and transmits it to the central station using an advanced, error-correction protocol providing data integrity between the field and the processing center. The SG160-09 has been installed in three seismic stations in different geographic locations with different Trimble global reference stations coverage The hardware includes the SG160-09 system, external Zephyr Geodetic-2 GNSS antenna, both radio and high-speed Internet communication media. Both acceleration and displacement data was transmitted in real-time to the centralized Data Acquisition Centers for real-time data processing. Command/Control of the field station and real-time GNSS position correction are provided via the Pivot platform. Data from the SG160-09 system was used for seismic event characterization along with data from traditional seismic and geodetic stations

  7. Phasor Measurement Unit and Phasor Data Concentrator test with Real Time Digital Simulator

    DEFF Research Database (Denmark)

    Diakos, Konstantinos; Wu, Qiuwei; Nielsen, Arne Hejde

    2014-01-01

    network to a more reliable, secure and economic operation. The implementation of these devices though, demands the warranty of a secure operation and high-accuracy performance. This paper describes the procedure of establishing a PMU (Phasor Measurement Unit)–PDC (Phasor Data Concentrator) platform......The main focus of the electrical engineers nowadays, is to develop a smart grid that is able to monitor, evaluate and control the power system operation. The integration of Intelligent Electronic Devices (IED s) to the power network, is a strong indication of the inclination to lead the power...

  8. Physical processes and real-time chemical measurement of the insect olfactory environment.

    Science.gov (United States)

    Riffell, Jeffrey A; Abrell, Leif; Hildebrand, John G

    2008-07-01

    Odor-mediated insect navigation in airborne chemical plumes is vital to many ecological interactions, including mate finding, flower nectaring, and host locating (where disease transmission or herbivory may begin). After emission, volatile chemicals become rapidly mixed and diluted through physical processes that create a dynamic olfactory environment. This review examines those physical processes and some of the analytical technologies available to characterize those behavior-inducing chemical signals at temporal scales equivalent to the olfactory processing in insects. In particular, we focus on two areas of research that together may further our understanding of olfactory signal dynamics and its processing and perception by insects. First, measurement of physical atmospheric processes in the field can provide insight into the spatiotemporal dynamics of the odor signal available to insects. Field measurements in turn permit aspects of the physical environment to be simulated in the laboratory, thereby allowing careful investigation into the links between odor signal dynamics and insect behavior. Second, emerging analytical technologies with high recording frequencies and field-friendly inlet systems may offer new opportunities to characterize natural odors at spatiotemporal scales relevant to insect perception and behavior. Characterization of the chemical signal environment allows the determination of when and where olfactory-mediated behaviors may control ecological interactions. Finally, we argue that coupling of these two research areas will foster increased understanding of the physicochemical environment and enable researchers to determine how olfactory environments shape insect behaviors and sensory systems.

  9. Real Time Microelectrode Measurement of Nitric Oxide in Kidney Tubular Fluid in vivo

    Directory of Open Access Journals (Sweden)

    Michelle Iacovitti

    2003-08-01

    Full Text Available In this review we summarize our experience using a microelectrode to measure nitric oxide concentrations [NO] in living rat kidney tubules. In the anaesthetized living rat, the abdomen can be opened, and the kidney can be placed in a cup such that one can puncture a surface single tubular segment, 1-2 mm long, connected to one of 30,000 filtering glomeruli. The tubular segment can be viewed with a stereo microscope and punctured using sophisticated micromanipulators. The segment, ranging in diameter from about 15 - 35 um contains freely flowing RBC-free fluid, electrolytes, O2, pCO2 and NO gas concentrations, and a host of other known and unknown substances. After a “pre” puncture with a 7-10 um beveled glass pipette, intratubular [NO] can be directly determined by inserting, into the tubular lumen, the tip of a specially modified amperometric integrated electrode (WPI P/N ISO-NOP007. We review our in vivo experience with this electrode, emphasizing optimal practice to ensure appropriate calibration, stability, and selectivity for in vivo use. The electrode is highly selective for NO, and, despite fragility, with appropriate precautions, it can provide reproducible and highly sensitive NO measurements in the 40-1000 nM range.

  10. Practical Testing and Performance Analysis of Phasor Measurement Unit Using Real Time Digital Simulator (RTDS)

    DEFF Research Database (Denmark)

    Liu, Leo; Rather, Zakir Hussain; Stearn, Nathen

    2012-01-01

    visualisation and post event analysis of power systems. It is expected however, that through integration with traditional Supervisory Control and Data Acquisition (SCADA) systems, closed loop control applications will be possible. Phasor Measurement Units (PMUs) are fundamental components of WAMS. Large WAMS...... for the testing of PMUs [2] there is a lack of specialised test equipment for performing such testing efficiently [3]. In this paper, RTDS based steady state and dynamic testing of the ALSTOM MiCOM P847 PMU using hardware in the loop is demonstrated. A correction algorithm supported by promising results is also...... proposed to realize highly precise phasoreasurements. Further a comparative study based on features of PMUs from different major manufacturers is presented. The selection of optimal parameters, such as phasor format and filter length is also discussed for various applications....

  11. Real-time measurement of inhaled and exhaled cigarette smoke: Implications for dose

    Energy Technology Data Exchange (ETDEWEB)

    McGrath, Conor; Warren, Nigel; Biggs, Philip; McAughey, John, E-mail: conor_mcgrath@bat.co [British American Tobacco, Group R and D Centre, Southampton, SO15 8TL (United Kingdom)

    2009-02-01

    Inhalation of tobacco smoke aerosol is a two-step process involving puffing followed by inhalation. Measured smoke deposition efficiencies in the lung (20-70%) are greater than expected for smoke particles of 150 -- 250 nm count median diameter (CMD). Various mechanisms have been put forward to explain this enhanced deposition pattern, including coagulation, hygroscopic growth, condensation and evaporation, changes in composition, or changes in inhalation behaviour. This paper represents one of a series of studies seeking to better quantify smoke chemistry, inhalation behaviour and cumulative particle growth. The studies have been conducted to better understand smoke dosimetry and links to disease as part of a wider programme defining risk and potential harm reduction. In this study, the average CMD of inhaled smoke was 160 nm while the average CMD of exhaled smoke was 239 nm with an average growth factor of 1.5.

  12. Non-contact Real-time heart rate measurements based on high speed circuit technology research

    Science.gov (United States)

    Wu, Jizhe; Liu, Xiaohua; Kong, Lingqin; Shi, Cong; Liu, Ming; Hui, Mei; Dong, Liquan; Zhao, Yuejin

    2015-08-01

    In recent years, morbidity and mortality of the cardiovascular or cerebrovascular disease, which threaten human health greatly, increased year by year. Heart rate is an important index of these diseases. To address this status, the paper puts forward a kind of simple structure, easy operation, suitable for large populations of daily monitoring non-contact heart rate measurement. In the method we use imaging equipment video sensitive areas. The changes of light intensity reflected through the image grayscale average. The light change is caused by changes in blood volume. We video the people face which include the sensitive areas (ROI), and use high-speed processing circuit to save the video as AVI format into memory. After processing the whole video of a period of time, we draw curve of each color channel with frame number as horizontal axis. Then get heart rate from the curve. We use independent component analysis (ICA) to restrain noise of sports interference, realized the accurate extraction of heart rate signal under the motion state. We design an algorithm, based on high-speed processing circuit, for face recognition and tracking to automatically get face region. We do grayscale average processing to the recognized image, get RGB three grayscale curves, and extract a clearer pulse wave curves through independent component analysis, and then we get the heart rate under the motion state. At last, by means of compare our system with Fingertip Pulse Oximeter, result show the system can realize a more accurate measurement, the error is less than 3 pats per minute.

  13. A Comprehensive Statistically-Based Method to Interpret Real-Time Flowing Measurements

    Energy Technology Data Exchange (ETDEWEB)

    Keita Yoshioka; Pinan Dawkrajai; Analis A. Romero; Ding Zhu; A. D. Hill; Larry W. Lake

    2007-01-15

    With the recent development of temperature measurement systems, continuous temperature profiles can be obtained with high precision. Small temperature changes can be detected by modern temperature measuring instruments such as fiber optic distributed temperature sensor (DTS) in intelligent completions and will potentially aid the diagnosis of downhole flow conditions. In vertical wells, since elevational geothermal changes make the wellbore temperature sensitive to the amount and the type of fluids produced, temperature logs can be used successfully to diagnose the downhole flow conditions. However, geothermal temperature changes along the wellbore being small for horizontal wells, interpretations of a temperature log become difficult. The primary temperature differences for each phase (oil, water, and gas) are caused by frictional effects. Therefore, in developing a thermal model for horizontal wellbore, subtle temperature changes must be accounted for. In this project, we have rigorously derived governing equations for a producing horizontal wellbore and developed a prediction model of the temperature and pressure by coupling the wellbore and reservoir equations. Also, we applied Ramey's model (1962) to the build section and used an energy balance to infer the temperature profile at the junction. The multilateral wellbore temperature model was applied to a wide range of cases at varying fluid thermal properties, absolute values of temperature and pressure, geothermal gradients, flow rates from each lateral, and the trajectories of each build section. With the prediction models developed, we present inversion studies of synthetic and field examples. These results are essential to identify water or gas entry, to guide flow control devices in intelligent completions, and to decide if reservoir stimulation is needed in particular horizontal sections. This study will complete and validate these inversion studies.

  14. Measuring and tracking eye movements of a behaving archer fish by real-time stereo vision.

    Science.gov (United States)

    Ben-Simon, Avi; Ben-Shahar, Ohad; Segev, Ronen

    2009-11-15

    The archer fish (Toxotes chatareus) exhibits unique visual behavior in that it is able to aim at and shoot down with a squirt of water insects resting on the foliage above water level and then feed on them. This extreme behavior requires excellent visual acuity, learning, and tight synchronization between the visual system and body motion. This behavior also raises many important questions, such as the fish's ability to compensate for air-water refraction and the neural mechanisms underlying target acquisition. While many such questions remain open, significant insights towards solving them can be obtained by tracking the eye and body movements of freely behaving fish. Unfortunately, existing tracking methods suffer from either a high level of invasiveness or low resolution. Here, we present a video-based eye tracking method for accurately and remotely measuring the eye and body movements of a freely moving behaving fish. Based on a stereo vision system and a unique triangulation method that corrects for air-glass-water refraction, we are able to measure a full three-dimensional pose of the fish eye and body with high temporal and spatial resolution. Our method, being generic, can be applied to studying the behavior of marine animals in general. We demonstrate how data collected by our method may be used to show that the hunting behavior of the archer fish is composed of surfacing concomitant with rotating the body around the direction of the fish's fixed gaze towards the target, until the snout reaches in the correct shooting position at water level.

  15. Real-Time Rain Rate Evaluation via Satellite Downlink Signal Attenuation Measurement.

    Science.gov (United States)

    Giannetti, Filippo; Reggiannini, Ruggero; Moretti, Marco; Adirosi, Elisa; Baldini, Luca; Facheris, Luca; Antonini, Andrea; Melani, Samantha; Bacci, Giacomo; Petrolino, Antonio; Vaccaro, Attilio

    2017-08-12

    We present the NEFOCAST project (named by the contraction of "Nefele", which is the Italian spelling for the mythological cloud nymph Nephele, and "forecast"), funded by the Tuscany Region, about the feasibility of a system for the detection and monitoring of precipitation fields over the regional territory based on the use of a widespread network of new-generation Eutelsat "SmartLNB" (smart low-noise block converter) domestic terminals. Though primarily intended for interactive satellite services, these devices can also be used as weather sensors, as they have the capability of measuring the rain-induced attenuation incurred by the downlink signal and relaying it on an auxiliary return channel. We illustrate the NEFOCAST system architecture, consisting of the network of ground sensor terminals, the space segment, and the service center, which has the task of processing the information relayed by the terminals for generating rain field maps. We discuss a few methods that allow the conversion of a rain attenuation measurement into an instantaneous rainfall rate. Specifically, we discuss an exponential model relating the specific rain attenuation to the rainfall rate, whose coefficients were obtained from extensive experimental data. The above model permits the inferring of the rainfall rate from the total signal attenuation provided by the SmartLNB and from the link geometry knowledge. Some preliminary results obtained from a SmartLNB installed in Pisa are presented and compared with the output of a conventional tipping bucket rain gauge. It is shown that the NEFOCAST sensor is able to track the fast-varying rainfall rate accurately with no delay, as opposed to a conventional gauge.

  16. Data acquisition and processing platform in the real-time distance measurement system with dual-comb lasers

    Science.gov (United States)

    Ni, Kai; Wang, Lanlan; Zhou, Qian; Li, Xinghui; Dong, Hao; Wang, Xiaohao

    2016-11-01

    The real-time distance measurement system with dual femtosecond comb lasers combines time-of-flight and interferometric measurement. It has advantages of wide-range, high-accuracy and fast speed at the rate about 10000 pts/s. Such a distance measurement system needs dedicated higher performance of the data acquisition and processing hardware platform to support. This paper introduces the dedicated platform of the developed absolute distance measurement system. This platform is divided into three parts according to their respective functions. First part is the data acquisition module, which function is mainly to realize the A/D conversion. In this part we designed a sampling clock adjustment module to assist the A/D conversion module to sample accurately. The sampling clock adjustment module accept a 250MHz maximum reference clock input, which from the same femtosecond laser source as the optical measurement system, then generate an output clock for the A/D converter that can be delayed up to 20ns with a resolution of 714ps. This data acquisition module can convert the analog laser pulse signal to digital signal with a 14 bits resolution and a 250 MSPS maximum sample rate. Second is the data processing and storage module consists of FPGA and DDR3 modules. The FPGA module calculates the test distance by the 16 bits digital sampling signal from the front data acquisition module. The DDR3 module implements sampling data caching. Finally part is the data transmission and peripheral interfaces module based on three DB9 and USB2.0. We can easily debug the platform in the PC and implement communication with upper machine. We tested our system used dedicate test bench in real-time. The scope of the measurement system range is 0 to 3 meters and the measurement deviation is less than 10um.

  17. Comparison between quantitative real-time reverse transcription PCR results for norovirus in oysters and self-reported gastroenteric illness in restaurant customers.

    Science.gov (United States)

    Lowther, James A; Avant, Justin M; Gizynski, Krzysztof; Rangdale, Rachel E; Lees, David N

    2010-02-01

    Norovirus is the principal agent of bivalve shellfish-associated gastroenteric illness worldwide. Numerous studies using PCR have demonstrated norovirus contamination in a significant proportion of both oyster and other bivalve shellfish production areas and ready-to-eat products. By comparison, the number of epidemiologically confirmed shellfish-associated outbreaks is relatively low. This study attempts to compare norovirus RNA detection in Pacific oysters (Crassostrea gigas) by quantitative real-time reverse transcription PCR (RT-PCR) and human health risk. Self-reported customer complaints of illness in a restaurant setting (screened for credible norovirus symptoms) were compared with presence and levels of norovirus as determined by real-time RT-PCR for the batch of oysters consumed. No illness was reported for batches consistently negative for norovirus by real-time RT-PCR. However, norovirus was detected in some batches for which no illness was reported. Overall presence or absence of norovirus showed a significant association with illness complaints. In addition, the batch with the highest norovirus RNA levels also resulted in the highest rate of reported illness, suggesting a linkage between virus RNA levels and health risks. This study suggests that detection of high levels of norovirus RNA in oysters is indicative of a significantly elevated health risk. However, illness may not necessarily be reported after detection of norovirus RNA at low levels.

  18. Selection of reference genes for gene expression studies in Siberian Apricot (Prunus sibirica L. Germplasm using quantitative real-time PCR.

    Directory of Open Access Journals (Sweden)

    Jun Niu

    Full Text Available Quantitative real time reverse transcription polymerase chain reaction has been applied in a vast range of studies of gene expression analysis. However, real-time PCR data must be normalized with one or more reference genes. In this study, eleven putative consistently expressed genes (ACT, TUA, TUB, CYP, DNAj, ELFA, F-box27, RPL12, GAPDH, UBC and UBQ in nine Siberian Apricot Germplasms (including much variability were evaluated for their potential as references for the normalization of gene expression by NormFinder and geNorm programs. From our studies, ACT, UBC, CYP, UBQ and RPL12 as suitable for normalization were identified by geNorm, while UBC and CYP as the best pair by NormFinder. Moreover, UBC was selected as the most stably expressed gene by both algorithms in different Siberian Apricot seed samples. We also detected that a set of three genes (ACT, CYP and UBC by geNorm as control for normalization could lead to accurate results. Furthermore, the expression levels of oleosin gene were analyzed to validate the suitability of the selected reference genes. These obtained experimental results could make an important contribution to normalize real-time PCR data for gene expression analysis in Siberian Apricot Germplasm.

  19. Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

    Energy Technology Data Exchange (ETDEWEB)

    Malik, Afshan N., E-mail: afshan.malik@kcl.ac.uk [King' s College London, Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Medicine (United Kingdom); Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil [King' s College London, Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Medicine (United Kingdom)

    2011-08-19

    Highlights: {yields} Mitochondrial dysfunction is central to many diseases of oxidative stress. {yields} 95% of the mitochondrial genome is duplicated in the nuclear genome. {yields} Dilution of untreated genomic DNA leads to dilution bias. {yields} Unique primers and template pretreatment are needed to accurately measure mitochondrial DNA content. -- Abstract: Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as {beta}-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a 'dilution bias' when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

  20. Development of quantitative real-time PCR assays for fathead minnow (Pimephales promelas) gonadotropin ß subunit mRNAs to support endocrine disruptor research.

    Energy Technology Data Exchange (ETDEWEB)

    Villeneuve, Daniel L.; Miracle, Ann L.; Jensen, Kathleen M.; Degitz, Sigmund J.; Kahl, Michael D.; Korte, Joseph J.; Greene, Katie J.; Blake, Lindsey S.; Linnum, Ann; Ankley, Gerald T.

    2007-03-01

    Fathead minnows (Pimephales promelas) are one of the most widely-used small fish models for regulatory ecotoxicology testing and research related to endocrine disrupting chemicals (EDCs). In this study, we isolated and sequenced cDNAs for fathead minnow follicle-stimulating hormone-like and luteinizing hormone-like β (FSHβ and LHβ) and glycoprotein α (GPα) subunits. Quantitative real-time PCR assays for measuring gonadotropin (GtH) β subunit transcripts were developed and used to examine “baseline” transcript levels over a range of age classes and reproductive states encompassed in EDC testing. In females, FSHβ and LHβ transcripts were greater in 4-5 month old than in younger fish and were significantly correlated with one another across all age classes examined. In males, FSHβ transcripts were greatest in 2-3 month old fish and were inversely correlated with various measures of testis development including, gonadal-somatic index (GSI), and histological stage. Overall, the pattern of GtHβ expression over age classes associated with gonad development was similar to that reported for other asynchronous-spawning fish. Despite significant changes in female GSI, gonad stage, and plasma vitellogenin within 24 h of spawning, GtHβ transcript levels in fish that had spawned within the preceding 24 h were not significantly different from those in fish that were 2-3 days post-spawn and expected to spawn within the next 24 h based on spawning history. Results of this study provide insights related to the role of GtHs in fathead minnow reproductive development and function. Additionally they provide useful “baseline” data needed to design and interpret effective experiments for studying direct and indirect effects of EDCs on GtH subunit mRNA expression, which will facilitate a greater understanding of integrated system-wide responses of the fathead minnow brain-pituitary-gonadal axis to stressors including EDCs.

  1. Real-Time Measurement of Face Recognition in Rapid Serial Visual Presentation

    Science.gov (United States)

    Touryan, Jon; Gibson, Laurie; Horne, James H.; Weber, Paul

    2011-01-01

    Event-related potentials (ERPs) have been used extensively to study the processes involved in recognition memory. In particular, the early familiarity component of recognition has been linked to the FN400 (mid-frontal negative deflection between 300 and 500 ms), whereas the recollection component has been linked to a later positive deflection over the parietal cortex (500–800 ms). In this study, we measured the ERPs elicited by faces with varying degrees of familiarity. Participants viewed a continuous sequence of faces with either low (novel faces), medium (celebrity faces), or high (faces of friends and family) familiarity while performing a separate face-identification task. We found that the level of familiarity was significantly correlated with the magnitude of both the early and late recognition components. Additionally, by using a single-trial classification technique, applied to the entire evoked response, we were able to distinguish between familiar and unfamiliar faces with a high degree of accuracy. The classification of high versus low familiarly resulted in areas under the curve of up to 0.99 for some participants. Interestingly, our classifier model (a linear discriminant function) was developed using a completely separate object categorization task on a different population of participants. PMID:21716601

  2. FluxPro: Real time monitoring and simulation system for eddy covariance flux measurement

    Science.gov (United States)

    Kim, W.; Seo, H.; Mano, M.; Ono, K.; Miyata, A.; Yokozawa, M.

    2010-12-01

    To cope with unusual weather changes on crop cultivation in a field level, prompt and precise monitoring of photosynthesis and evapotranspiration, and those fast and reliable forecasting are indispensable. So we have developed FluxPro which is simultaneous operating system of the monitoring and the forecasting. The monitoring subsystem provides vapor and CO2 fluxes with uncertainty to understand the live condition of photosynthesis and evapotranspiration by open-path eddy covariance flux measurement (EC) system and self-developed EC tolerance analysis scheme. The forecasting subsystem serves the predicted fluxes with anomaly based on model parameter assimilation to estimate the hourly or daily water consumption and carbon assimilation during a week by multi-simulation package consisting of various models from simple to complicate. FluxPro is helpful not only to detect a critical condition of growing crop in terms of photosynthesis and evapotranspiration but also to decide time and amount of launching control for keeping those optimization condition when an unusual weather event is arisen. In our presentation, we will demonstrate the FluxPro operated at tangerine orchard in Jeju, Korea.

  3. Measuring Sea Level Rise-Induced Shoreline Changes and Inundation in Real Time

    Science.gov (United States)

    Shilling, F.; Waetjen, D.; Grijalva, E.

    2016-12-01

    We describe a method to monitor shoreline inundation and changes in response to sea level rise (SLR) using a network of time-lapse cameras. We found for coastal tidal marshes that this method was sensitive to vertical changes in sea level of 20 cm has occurred in the San Francisco Bay and other US coastal areas and is likely to rise by another 30-45 cm by mid-century, which will flood and erode many coastal ecosystems, highways, and urban areas. This rapid degree of rise means that it is imperative to co-plan for natural and built systems. Many public facilities are adjacent to shoreline ecosystems, which both protect infrastructure from wave and tide energy and are home to regulated species and habitats. Accurate and timely information about the actual extent of SLR impacts to shorelines will be critical during built-system adaptation. Currently, satellite-sourced imagery cannot provide the spatial or temporal resolution necessary to investigate fine-scale shoreline changes, leaving a gap between predictive models and knowing how, where and when these changes are occurring. The method described is feasible for near-term (1 to 10 years) to long-term application and can be used for measuring fine-resolution shoreline changes (mashup. This information could be used to validate models predicting shoreline inundation and loss, inform SLR-adaptation planning, and to visualize SLR impacts to the public.

  4. A Field-Portable Membrane Introduction Mass Spectrometer for Real-time Quantitation and Spatial Mapping of Atmospheric and Aqueous Contaminants

    Science.gov (United States)

    Bell, Ryan J.; Davey, Nicholas G.; Martinsen, Morten; Collin-Hansen, Christian; Krogh, Erik T.; Gill, Christopher G.

    2015-02-01

    Environmental concentrations of volatile and semivolatile organic compounds (VOC/SVOCs) can vary dramatically in time and space under the influence of environmental conditions. In an industrial setting, multiple point and diffuse sources can contribute to fugitive emissions. Assessments and monitoring programs using periodic grab sampling provide limited information, often with delay times of days or weeks. We report the development and use of a novel, portable membrane introduction mass spectrometry (MIMS) system capable of resolving and quantifying VOC and SVOCs with high spatial and temporal resolution, in the field, in real-time. An electron impact ionization cylindrical ion trap mass spectrometer modified with a capillary hollow fiber polydimethylsiloxane membrane interface was used for continuous air and water sampling. Tandem mass spectrometry and selected ion monitoring scans performed in series allowed for the quantitation of target analytes, and full scan mode was used to survey for unexpected analytes. Predeployment and in-field external calibrations were combined with a continuously infused internal standard to enable real-time quantitation and monitor instrument performance. The system was operated in a moving vehicle with internet-linked data processing and storage. Software development to integrate MIMS and relevant meta-data for visualization and geospatial presentation in Google Earth is presented. Continuous quantitation enables the capture of transient events that may be missed or under-represented by traditional grab sampling strategies. Real-time geospatial maps of chemical concentration enable adaptive sampling and in-field decision support. Sample datasets presented in this work were collected in Northern Alberta in 2010-2012.

  5. Real-time quantitative PCR of telomerase mRNA is useful for the differentiation of benign and malignant pancreatic disorders.

    Science.gov (United States)

    Büchler, P; Conejo-Garcia, J R; Lehmann, G; Müller, M; Emrich, T; Reber, H A; Büchler, M W; Friess, H

    2001-05-01

    The presence of telomerase activity has been proposed as a specific and sensitive marker for malignant tissue, and positivity rates of up to 95% have been reported in pancreatic cancer. In the present study telomerase activity analysis was reevaluated in 29 pancreatic cancer tissues compared with 36 chronic pancreatitis tissues and 21 normal controls, and a study was made of whether malignant and benign pancreatic disorders can be better differentiated using a novel technique real-time quantitative PCR analysis-analyzing telomerase mRNA expression. Telomerase activity was present in 35% (10 of 29) of pancreatic cancer samples, 3% (one of 36) of chronic pancreatitis samples, and none of the normal pancreatic tissue samples in the TRAP assay. Real-time quantitative PCR analysis revealed the presence of telomerase mRNA expression in 50% (10 of 20) of normal, 86% (31 of 36) of chronic pancreatitis, and 90% (26 of 29) of pancreatic cancer samples. However, quantification of the expression data revealed that the relative increase above normal was 5.5 (range, 3.5-8.6) for chronic pancreatitis and 23.9 (range, 18.6-30.7) for pancreatic cancer samples (p data indicate that detection of telomerase activity using the TRAP assay has limitations in differentiating benign and malignant pancreatic disorders. However, telomerase mRNA analysis by real-time quantitative PCR analysis allows a highly sensitive detection and differentiation of pancreatic cancer from normal pancreas and chronic pancreatitis and thereby may serve as a new reliable, easy, and effective diagnostic tool for cancer diagnosis.

  6. Quantitation of Epstein-Barr virus mRNA using reverse transcription and real-time PCR.

    Science.gov (United States)

    Weinberger, Birgit; Plentz, Annelie; Weinberger, Klaus M; Hahn, Joachim; Holler, Ernst; Jilg, Wolfgang

    2004-12-01

    Monitoring of Epstein-Barr virus (EBV) infection and reactivation in immunocompromized patients (e.g., after organ or bone-marrow transplantation) is based mainly on serological assays and detection of viral DNA. For further characterization of virus reactivation and monitoring of viral transcription we established real-time RT-PCR assays using TaqMan technology to sensitively quantify viral transcripts expressed at different times of the lytic cycle: for BZLF1, an immediate early transactivator initiating the transition from latency to lytic replication, for the DNA-polymerase BALF5 and for the major viral glycoprotein gp350/220 (BLLF1). RNA-isolation was optimized to eliminate contaminating DNA. Preparations were shown to be virtually DNA-free for up to 10(6) copies of RNA. With our PCR systems, it is possible to detect 10 copies of DNA or 100 copies of RNA per reaction as shown with serial dilutions of DNA-plasmids or in vitro transcribed RNA, respectively. This corresponds to a detection limit of 8 x 10(2) copies/10(6) peripheral blood mononuclear cells (PBMCs). Evaluation of this system showed that even in healthy carriers borderline levels of BLLF1 mRNA were sometimes detectable. In patients with acute infectious mononucleosis (IM) viral transcripts were regularly found in varying concentrations. Extremely high levels of all three mRNA species could be seen in a patient after bone-marrow transplantation monitored during an episode of lymphoproliferation which regressed during treatment with acyclovir and transfusion of donor T-cells. This sensitive and reproducible method to detect and quantify different transcripts of EBV can be used to closely monitor reactivation of EBV, e.g., in immunocompromized patients. 2004 Wiley-Liss, Inc.

  7. A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences.

    Science.gov (United States)

    Iraola, Gregorio; Pérez, Ruben; Betancor, Laura; Marandino, Ana; Morsella, Claudia; Méndez, Alejandra; Paolicchi, Fernando; Piccirillo, Alessandra; Tomás, Gonzalo; Velilla, Alejandra; Calleros, Lucía

    2016-12-15

    Campylobacter fetus is a pathogen of major concern for animal and human health. The species shows a great intraspecific variation, with three subspecies: C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. testudinum. Campylobacter fetus fetus affects a broad range of hosts and induces abortion in sheep and cows. Campylobacter fetus venerealis is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus testudinum has been proposed recently based on genetically divergent strains isolated from reptiles and humans. Both C. fetus fetus and C. fetus testudinum are opportunistic pathogens for immune-compromised humans. Biochemical tests remain as the gold standard for identifying C. fetus but the fastidious growing requirements and the lack of reliability and reproducibility of some biochemical tests motivated the development of molecular diagnostic tools. These methods have been successfully tested on bovine isolates but fail to detect some genetically divergent strains isolated from other hosts. The aim of the present study was to develop a highly specific molecular assay to identify and quantify C. fetus strains. We developed a highly sensitive real-time PCR assay that targets a unique region of the 16S rRNA gene. This assay successfully detected all C. fetus strains, including those that were negative for the cstA gene-based assay used as a standard for molecular C. fetus identification. The assay showed high specificity and absence of cross-reactivity with other bacterial species. The analytical testing of the assay was determined using a standard curve. The assay demonstrated a wide dynamic range between 10 2 and 107 genome copies per reaction, and a good reproducibility with small intra- and inter-assay variability. The possibility to characterize samples in a rapid, sensitive and reproducible way makes this assay

  8. Utility of microscopic techniques and quantitative real-time polymerase chain reaction for the diagnosis of vaginal microflora alterations.

    Science.gov (United States)

    Rumyantseva, Tatiana A; Bellen, Gert; Romanuk, Tatiana N; Shipulina, Olga Iu; Guschin, Alexander E; Shipulin, German A; Donders, Gilbert G G

    2015-04-01

    The aim of the study was to evaluate the diagnostic value of Nugent score, wet mount microscopy, and polymerase chain reaction (PCR) test developed in Russia for bacterial vaginosis (BV) diagnosis. One hundred Caucasian women were enrolled in this study. Three vaginal samples were taken from each participant: 1 for PCR analysis, 1 for Nugent score evaluation, and 1 for wet mount microscopy. The smears for microscopy were air-dried and sent to Femicare, Tienen, Belgium, for blinded analysis by microscopy. Multiplex real-time PCR was performed using primers and probes targeting Gardnerella vaginalis, Atopobium vaginae, Lactobacillus species, and total quantity of bacterial DNA (16SrRNA gene). Agreement among the 3 methods was 72 (73.5%) of 98 samples. Agreement between Nugent score and PCR results was 77 (78.6%) of 98 samples; between wet mount microscopy and PCR, 81 (82.65%) of 98 samples; between wet mount microscopy and Nugent score, 84 (85.7%) of 98 samples. The sensitivity and specificity of the methods studied were as follows: 75% (21/28) and 97.1% (68/70) for Nugent score, 96.4% (27/28) and 94.3% (66/70) for wet mount microscopy, 92.8% (26/28) and 85.7% (60/70) for PCR, respectively. This study demonstrated that wet mount microscopy is a superior method for BV diagnosis. The PCR test under study showed a high sensitivity and can be used for discrimination between normal flora and BV.

  9. Quantitative analysis of aortic regurgitation: real-time 3-dimensional and 2-dimensional color Doppler echocardiographic method--a clinical and a chronic animal study

    Science.gov (United States)

    Shiota, Takahiro; Jones, Michael; Tsujino, Hiroyuki; Qin, Jian Xin; Zetts, Arthur D.; Greenberg, Neil L.; Cardon, Lisa A.; Panza, Julio A.; Thomas, James D.

    2002-01-01

    BACKGROUND: For evaluating patients with aortic regurgitation (AR), regurgitant volumes, left ventricular (LV) stroke volumes (SV), and absolute LV volumes are valuable indices. AIM: The aim of this study was to validate the combination of real-time 3-dimensional echocardiography (3DE) and semiautomated digital color Doppler cardiac flow measurement (ACM) for quantifying absolute LV volumes, LVSV, and AR volumes using an animal model of chronic AR and to investigate its clinical applicability. METHODS: In 8 sheep, a total of 26 hemodynamic states were obtained pharmacologically 20 weeks after the aortic valve noncoronary (n = 4) or right coronary (n = 4) leaflet was incised to produce AR. Reference standard LVSV and AR volume were determined using the electromagnetic flow method (EM). Simultaneous epicardial real-time 3DE studies were performed to obtain LV end-diastolic volumes (LVEDV), end-systolic volumes (LVESV), and LVSV by subtracting LVESV from LVEDV. Simultaneous ACM was performed to obtain LVSV and transmitral flows; AR volume was calculated by subtracting transmitral flow volume from LVSV. In a total of 19 patients with AR, real-time 3DE and ACM were used to obtain LVSVs and these were compared with each other. RESULTS: A strong relationship was found between LVSV derived from EM and those from the real-time 3DE (r = 0.93, P <.001, mean difference (3D - EM) = -1.0 +/- 9.8 mL). A good relationship between LVSV and AR volumes derived from EM and those by ACM was found (r = 0.88, P <.001). A good relationship between LVSV derived from real-time 3DE and that from ACM was observed (r = 0.73, P <.01, mean difference = 2.5 +/- 7.9 mL). In patients, a good relationship between LVSV obtained by real-time 3DE and ACM was found (r = 0.90, P <.001, mean difference = 0.6 +/- 9.8 mL). CONCLUSION: The combination of ACM and real-time 3DE for quantifying LV volumes, LVSV, and AR volumes was validated by the chronic animal study and was shown to be clinically applicable.

  10. An electrospray chemical ionization source for real-time measurement of atmospheric organic and inorganic vapors

    Science.gov (United States)

    Zhao, Yue; Chan, Jeremy K.; Lopez-Hilfiker, Felipe D.; McKeown, Megan A.; D'Ambro, Emma L.; Slowik, Jay G.; Riffell, Jeffrey A.; Thornton, Joel A.

    2017-10-01

    We present an electrospray ion source coupled to an orthogonal continuous-flow atmospheric pressure chemical ionization region. The source can generate intense and stable currents of several specific reagent ions using a range of salt solutions prepared in methanol, thereby providing both an alternative to more common radioactive ion sources and allowing for the generation of reagent ions that are not available in current chemical ionization mass spectrometry (CIMS) techniques, such as alkaline cations. We couple the orthogonal electrospray chemical ionization (ESCI) source to a high-resolution time-of-flight mass spectrometer (HR-ToF-MS), and assess instrument performance through calibrations using nitric acid (HNO3), formic acid (HCOOH), and isoprene epoxydiol (trans-β-IEPOX) gas standards, and through measurements of oxidized organic compounds formed from ozonolysis of α-pinene in a continuous-flow reaction chamber. When using iodide as the reagent ion, the HR-ToF-ESCIMS prototype has a sensitivity of 11, 2.4, and 10 cps pptv-1 per million counts per second (cps) of reagent ions and a detection limit (3σ, 5 s averaging) of 4.9, 12.5, and 1.4 pptv to HNO3, HCOOH, and IEPOX, respectively. These values are comparable to those obtained using an iodide-adduct HR-ToF-CIMS with a radioactive ion source and low-pressure ion-molecule reaction region. Applications to the α-pinene ozonolysis system demonstrates that HR-ToF-ESCIMS can generate multiple reagent ions (e.g., I-, NO3-, acetate, Li+, Na+, K+, and NH4+) having different selectivity to provide a comprehensive molecular description of a complex organic system.

  11. An electrospray chemical ionization source for real-time measurement of atmospheric organic and inorganic vapors

    Directory of Open Access Journals (Sweden)

    Y. Zhao

    2017-10-01

    Full Text Available We present an electrospray ion source coupled to an orthogonal continuous-flow atmospheric pressure chemical ionization region. The source can generate intense and stable currents of several specific reagent ions using a range of salt solutions prepared in methanol, thereby providing both an alternative to more common radioactive ion sources and allowing for the generation of reagent ions that are not available in current chemical ionization mass spectrometry (CIMS techniques, such as alkaline cations. We couple the orthogonal electrospray chemical ionization (ESCI source to a high-resolution time-of-flight mass spectrometer (HR-ToF-MS, and assess instrument performance through calibrations using nitric acid (HNO3, formic acid (HCOOH, and isoprene epoxydiol (trans-β-IEPOX gas standards, and through measurements of oxidized organic compounds formed from ozonolysis of α-pinene in a continuous-flow reaction chamber. When using iodide as the reagent ion, the HR-ToF-ESCIMS prototype has a sensitivity of 11, 2.4, and 10 cps pptv−1 per million counts per second (cps of reagent ions and a detection limit (3σ, 5 s averaging of 4.9, 12.5, and 1.4 pptv to HNO3, HCOOH, and IEPOX, respectively. These values are comparable to those obtained using an iodide-adduct HR-ToF-CIMS with a radioactive ion source and low-pressure ion–molecule reaction region. Applications to the α-pinene ozonolysis system demonstrates that HR-ToF-ESCIMS can generate multiple reagent ions (e.g., I−, NO3−, acetate, Li+, Na+, K+, and NH4+ having different selectivity to provide a comprehensive molecular description of a complex organic system.

  12. Endonuclease Restriction-Mediated Real-Time Polymerase Chain Reaction: A Novel Technique for Rapid, Sensitive and Quantitative Detection of Nucleic-Acid Sequence

    Science.gov (United States)

    Wang, Yi; Wang, Yan; Zhang, Lu; Li, Machao; Luo, Lijuan; Liu, Dongxin; Li, Hua; Cao, Xiaolong; Hu, Shoukui; Jin, Dong; Xu, Jianguo; Ye, Changyun

    2016-01-01

    The article reported a novel methodology for real-time PCR analysis of nucleic acids, termed endonuclease restriction-mediated real-time polymerase chain reaction (ET-PCR). Just like PCR, ET-PCR only required one pair of primers. A short sequence, which was recognized by restriction enzyme BstUI, was attached to the 5′ end of the forward (F) or reverse (R) PCR primer, and the new F or R primer was named EF or ER. EF/ER was labeled at the 5′ end with a reporter dye and in the middle with a quenching dye. BstUI cleaves the newly synthesized double-stranded terminal sequences (5′ end recognition sequences and their complementary sequences) during the extension phase, which separates the reporter molecule from the quenching dye, leading to a gain of fluorescence signal. This process is repeated in each amplification cycle and unaffected the exponential synthesis of the PCR amplification. ET-PCR allowed real-time analysis of single or multiple targets in a single vessel, and provided the reproducible quantitation of nucleic acids. The analytical sensitivity and specificity of ET-PCR were successfully evaluated, detecting down to 250 fg of genomic DNA per tube of target pathogen DNA examined, and the positive results were generated in a relatively short period. Moreover, the practical application of ET-PCR for simultaneous detection of multiple target pathogens was also demonstrated in artificially contaminated blood samples. In conclusion, due to the technique’s simplicity of design, reproducible data and low contamination risk, ET-PCR assay is an appealing alternative to conventional approaches currently used for real-time nucleic acid analysis. PMID:27468284

  13. An evaluation of technologies for real-time measurement of rates of outdoor airflow into HVAC systems

    Energy Technology Data Exchange (ETDEWEB)

    Fisk, William J.; Faulkner, David; Sullivan, Douglas P.

    2004-09-01

    During the last few years, new technologies have been introduced for real-time continuous measurement of the flow rates of outdoor air (OA) into HVAC systems; however, an evaluation of these measurement technologies has not previously been published. This document describes a test system and protocols developed for a controlled evaluation of these measurement technologies. The results of tests of four commercially available measurement technologies and one prototype based on a new design are also summarized. The test system and protocol were judged practical and very useful. The series of tests identified three commercially available measurement technologies that should provide reasonably accurate measurements of OA flow rates as long as air velocities are maintained high enough to produce accurately measurable pressure signals. In HVAC systems with economizer controls, to maintain the required air velocities the OA intake will need to be divided into two sections in parallel, each with a separate OA damper. The errors in OA flow rates measured with the fourth commercially available measurement technology were 20% to 30% with horizontal probes but much larger with vertical probes. The new prototype measurement technology was the only one that appears suitable for measuring OA flow rates over their full range from 20% OA to 100% OA without using two separate OA dampers. All of the measurement devices had pressure drops that are likely to be judged acceptable. The influence of wind on the accuracy of these measurement technologies still needs to be evaluated.

  14. Sources and Characteristics of Brown Carbon Aerosols over North India through Real-time Measurements

    Science.gov (United States)

    Rastogi, N.; Satish, R. V.; Shamjad, P.; Thambam, N.; Tripathi, S. N.

    2016-12-01

    Recent studies have documented that a certain type of organic carbon (predominantly water soluble) significantly absorb light at near-UV (300-400) and visible regions, and termed as "Brown Carbon (BrC)". Recent global models estimate that light absorption by BrC in different regions of the world may be 30-70% of that due to black carbon (BC). To assess the role of BrC on regional and global level, it is important to understand their sources and characteristics on temporal and spatial scale, which is severely lacking in literature. The major focus of present study is to fill this gap over India. The study site, Kanpur (26.5°N, 80.3°E, 142 m amsl) located in North India, receives emissions majorly from industries, vehicles, biofuel and biomass burning. Semi-continuous measurements of water soluble organic carbon (WSOC), BrC, BC and chemical composition of organic and inorganic aerosols were performed during winter season (December, 2015-Dec to February, 2016) using state-of-the-art instruments. Diurnal variability in the absorption coefficient of BrC at 365 nm (babs_365) showed higher values during late evening through early morning and attributed to primary emissions from biomass burning (BB) and fossil fuel burning (FFB). The babs_365 reduces as day progresses, which is ascribed to photo bleaching/dissociation of BrC. Primary BrC, assessed based on H:C ratios from HR-ToF-AMS, dominates the total BrC abundance with higher babs_365. Secondary BrC, assessed based on O:C ratios, was abundant in the morning and afternoon with lower babs_365. Further, diurnal variability in ratios of babs_365 with babs_405 and babs_420 suggests that BrC composition is not uniform throughout the day. Using BC370/BC880 ratio as an indicator of BB vis-à-vis FFB derived component, BB derived BrC was found to be more absorbing than that derived from FFB. Fog processing of BrC was also found to be affecting babs_365 positively. Detailed results will be presented.

  15. Comparison of FilmArray and Quantitative Real-Time Reverse Transcriptase PCR for Detection of Zaire Ebolavirus from Contrived and Clinical Specimens

    OpenAIRE

    Southern, Timothy R.; Racsa, Lori D.; Albariño, César G.; Fey, Paul D.; Hinrichs, Steven H.; Murphy, Caitlin N.; Herrera, Vicki L.; Sambol, Anthony R.; Hill, Charles E.; Ryan, Emily L.; Kraft, Colleen S.; Campbell, Shelley; Sealy, Tara K.; Schuh, Amy; Ritchie, James C.

    2015-01-01

    Rapid, reliable, and easy-to-use diagnostic assays for detection of Zaire ebolavirus (ZEBOV) are urgently needed. The goal of this study was to examine the agreement among emergency use authorization (EUA) tests for the detection of ZEBOV nucleic acids, including the BioFire FilmArray BioThreat (BT) panel, the FilmArray BT-E panel, and the NP2 and VP40 quantitative real-time reverse transcriptase (qRT) PCR assays from the Centers for Disease Control and Prevention (CDC). Specimens used in thi...

  16. Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS).

    Science.gov (United States)

    Yang, Yao; Sebra, Robert; Pullman, Benjamin S; Qiao, Wanqiong; Peter, Inga; Desnick, Robert J; Geyer, C Ronald; DeCoteau, John F; Scott, Stuart A

    2015-05-06

    DNA methylation has essential roles in transcriptional regulation, imprinting, X chromosome inactivation and other cellular processes, and aberrant CpG methylation is directly involved in the pathogenesis of human imprinting disorders and many cancers. To address the need for a quantitative and highly multiplexed bisulfite sequencing method with long read lengths for targeted CpG methylation analysis, we developed single-molecule real-time bisulfite sequencing (SMRT-BS). Optimized bisulfite conversion and PCR conditions enabled the amplification of DNA fragments up to ~1.5 kb, and subjecting overlapping 625-1491 bp amplicons to SMRT-BS indicated high reproducibility across all amplicon lengths (r=0.972) and low standard deviations (≤0.10) between individual CpG sites sequenced in triplicate. Higher variability in CpG methylation quantitation was correlated with reduced sequencing depth, particularly for intermediately methylated regions. SMRT-BS was validated by orthogonal bisulfite-based microarray (r=0.906; 42 CpG sites) and second generation sequencing (r=0.933; 174 CpG sites); however, longer SMRT-BS amplicons (>1.0 kb) had reduced, but very acceptable, correlation with both orthogonal methods (r=0.836-0.897 and r=0.892-0.927, respectively) compared to amplicons less than ~1.0 kb (r=0.940-0.951 and r=0.948-0.963, respectively). Multiplexing utility was assessed by simultaneously subjecting four distinct CpG island amplicons (702-866 bp; 325 CpGs) and 30 hematological malignancy cell lines to SMRT-BS (average depth of 110X), which identified a spectrum of highly quantitative methylation levels across all interrogated CpG sites and cell lines. SMRT-BS is a novel, accurate and cost-effective targeted CpG methylation method that is amenable to a high degree of multiplexing with minimal clonal PCR artifacts. Increased sequencing depth is necessary when interrogating longer amplicons (>1.0 kb) and the previously reported bisulfite sequencing PCR bias towards

  17. Practical performance of real-time shot-noise measurement in continuous-variable quantum key distribution

    Science.gov (United States)

    Wang, Tao; Huang, Peng; Zhou, Yingming; Liu, Weiqi; Zeng, Guihua

    2018-01-01

    In a practical continuous-variable quantum key distribution (CVQKD) system, real-time shot-noise measurement (RTSNM) is an essential procedure for preventing the eavesdropper exploiting the practical security loopholes. However, the performance of this procedure itself is not analyzed under the real-world condition. Therefore, we indicate the RTSNM practical performance and investigate its effects on the CVQKD system. In particular, due to the finite-size effect, the shot-noise measurement at the receiver's side may decrease the precision of parameter estimation and consequently result in a tight security bound. To mitigate that, we optimize the block size for RTSNM under the ensemble size limitation to maximize the secure key rate. Moreover, the effect of finite dynamics of amplitude modulator in this scheme is studied and its mitigation method is also proposed. Our work indicates the practical performance of RTSNM and provides the real secret key rate under it.

  18. Selection of appropriate reference genes for quantitative real-time PCR in Oxytropis ochrocephala Bunge using transcriptome datasets under abiotic stress treatments.

    Science.gov (United States)

    Zhuang, Huihui; Fu, Yanping; He, Wei; Wang, Lin; Wei, Yahui

    2015-01-01

    Oxytropis ochrocephala Bunge, an indigenous locoweed species in China, poses great threats to livestock on grasslands. There is a need for further genetic study in the plants per se, for understanding the basis of its acclimation mechanism in various unfavorable environmental conditions and to implement effective control measures. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method for gene expression analysis. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable reference genes is required. The aim of this study was to select the most stable reference genes for transcriptional analysis in O. ochrocephala. We selected 12 candidate reference genes, 18S ribosomal RNA (18S RNA), actin2/7 (ACT7), β-actin (ACTB), actin101 (ACT101), actin11 (ACT11), β-tubulin (TUB), α-tubulin (TUA), glyceraldehyde-3-phosphate dehydrogenase-1 (GAPDH1), GAPDH2, metallothionein-like protein (MET), fructose-bisphosphate aldolase (FBA) and histone H3 (HIS), from the transcriptome datasets of O. ochrocephala and determined the suitability by analyzing their expression levels when exposed to a range of abiotic stress conditions. By employing software packages including geNorm, NormFinder and BestKeeper, HIS, ACT7, and ACT101 were assessed as the most suitable set for normalization in all samples. When normalized with the most stable reference genes, the expression patterns of the three target genes were in accordance with those in the transcriptome data, indicating that the reference genes selected in this study are suitable. The study provided appropriate reference genes for accurate normalization in qRT-PCR analysis in O. ochrocephala and emphasized the importance of validating reference genes for gene expression analysis under specific experimental condition. The usage of inappropriate reference gene would cause misinterpretation.

  19. Selection of Suitable Internal Control Genes for Accurate Normalization of Real-Time Quantitative PCR Data of Buffalo (Bubalus bubalis) Blastocysts Produced by SCNT and IVF.

    Science.gov (United States)

    Sood, Tanushri Jerath; Lagah, Swati Viviyan; Sharma, Ankita; Singla, Suresh Kumar; Mukesh, Manishi; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

    2017-10-01

    We evaluated the suitability of 10 candidate internal control genes (ICGs), belonging to different functional classes, namely ACTB, EEF1A1, GAPDH, HPRT1, HMBS, RPS15, RPS18, RPS23, SDHA, and UBC for normalizing the real-time quantitative polymerase chain reaction (qPCR) data of blastocyst-stage buffalo embryos produced by hand-made cloning and in vitro fertilization (IVF). Total RNA was isolated from three pools, each of cloned and IVF blastocysts (n = 50/pool) for cDNA synthesis. Two different statistical algorithms geNorm and NormFinder were used for evaluating the stability of these genes. Based on gene stability measure (M value) and pairwise variation (V value), calculated by geNorm analysis, the most stable ICGs were RPS15, HPRT1, and ACTB for cloned blastocysts, HMBS, UBC, and HPRT1 for IVF blastocysts and RPS15, GAPDH, and HPRT1 for both the embryo types analyzed together. RPS18 was the least stable gene for both cloned and IVF blastocysts. Following NormFinder analysis, the order of stability was RPS15 = HPRT1>GAPDH for cloned blastocysts, HMBS = UBC>RPS23 for IVF blastocysts, and HPRT1>GAPDH>RPS15 for cloned and IVF blastocysts together. These results suggest that despite overlapping of the three most stable ICGs between cloned and IVF blastocysts, the panel of ICGs selected for normalization of qPCR data of cloned and IVF blastocyst-stage embryos should be different.

  20. Selection of appropriate reference genes for quantitative real-time PCR in Oxytropis ochrocephala Bunge using transcriptome datasets under abiotic stress treatments

    Directory of Open Access Journals (Sweden)

    huihui ezhuang

    2015-06-01

    Full Text Available Background: Oxytropis ochrocephala Bunge, an indigenous locoweed species in China, poses great threats to livestock on grasslands. There is a need for further genetic study in the plants per se, for understanding the basis of its acclimation mechanism in various unfavorable environmental conditions and to implement effective control measures. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR is the most commonly used method for gene expression analysis. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable reference genes is required. The aim of this study was to select the most stable reference genes for transcriptional analysis in O. ochrocephala. Results: We selected 12 candidate reference genes, 18S ribosomal RNA (18S RNA, actin2/7 (ACT7, β-actin (ACTB, actin101 (ACT101, actin11 (ACT11, β-tubulin (TUB, α-tubulin (TUA, glyceraldehyde-3-phosphate dehydrogenase-1 (GAPDH1, GAPDH2, metallothionein-like protein (MET, fructose-bisphosphate aldolase (FBA and histone H3 (HIS, from the transcriptome datasets of O. ochrocephala and determined the suitability by analyzing their expression levels when exposed to a range of abiotic stress conditions. By employing software packages including geNorm, NormFinder and BestKeeper, HIS, ACT7 and ACT101 were assessed as the most suitable set for normalization in all samples. When normalized with the most stable reference genes, the expression patterns of the three target genes were in accordance with those in the transcriptome data, indicating that the reference genes selected in this study are suitable. Conclusions: The study provided appropriate reference genes for accurate normalization in qRT-PCR analysis in O. ochrocephala and emphasized the importance of validating reference genes for gene expression analysis under specific experimental condition. The usage of inappropriate reference gene would cause misinterpretation.

  1. Identification of Suitable Reference Genes for Normalization of Real-Time Quantitative Polymerase Chain Reaction in an Intestinal Graft-Versus-Host Disease Mouse Model.

    Science.gov (United States)

    Li, X; Qiao, J; Yang, N; Mi, H; Chu, P; Xia, Y; Yao, H; Liu, Y; Qi, K; Yan, Z; Zeng, L; Xu, K

    2015-01-01

    With the development of real-time quantitative polymerase chain reaction (RT-qPCR) and intensive research on acute graft-versus-host disease (GVHD), selecting the best reference gene for normalization of RT-qPCR analysis in a GVHD model becomes more and more important. In this study, we aimed to identify suitable reference genes for mRNA studies in an intestinal GVHD mouse model after bone marrow transplantation (BMT). BALB/c recipients received 7.5 Gy total body irradiation (TBI) followed by injection of 5 × 10(6) bone marrow cells, without infusion of spleen cells for BMT, with infusion of 5 × 10(5) or 2.5 × 10(6) spleen cells for mild or moderate GVHD, respectively. Healthy mice were chosen as normal control subjects. Duodenum, jejunum, ileum, colon, and small intestine were collected at days 7, 14, 21, and 28 after transplantation. Transcription levels of 9 candidate genes, B2M, SDHA, HPRT, ACTB, GAPDH, HMBS, TBP, YWHAZ, and RPLP0, in each tissue were measured with the use of RT-qPCR. Combined data from these tissues in each group were defined as all samples. The expression stability of these genes was analyzed with the use of Genorm, Normfinder, Bestkeeper, and ΔCt. Our results showed that in all samples, ACTB and HMBS displayed the highest and lowest expression levels, respectively. Genorm identified HRPT and SDHA as the most stable reference genes, whereas Normfinder and ΔCt method showed HPRT as the most stably expressed gene. Bestkeeper ranked YWHAZ and HPRT as the top 2 most suitable genes. In conclusion, HPRT was recommended as the most suitable reference gene after comprehensive ranking, suggesting that it could be used as an internal control for mRNA studies in intestinal GVHD after BMT. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. A tissue-based approach to selection of reference genes for quantitative real-time PCR in a sheep osteoporosis model.

    Science.gov (United States)

    Schulze, Felix; Malhan, Deeksha; El Khassawna, Thaqif; Heiss, Christian; Seckinger, Anja; Hose, Dirk; Rösen-Wolff, Angela

    2017-12-19

    In order to better understand the multifactorial nature of osteoporosis, animal models are utilized and compared to healthy controls. Female sheep are well established as a model for osteoporosis induced by ovariectomy, calcium and vitamin D low diet, application of steroids, or a combination of these treatments. Transcriptional studies can be performed by applying quantitative real time PCR (RT-qPCR). RT-qPCR estimates mRNA-levels of target genes in relation to reference genes. A chosen set of reference genes should not show variation under experimental conditions. Currently, no standard reference genes are accepted for all tissue types and experimental conditions. Studies examining reference genes for sheep are rare and only one study described stable reference in mandibular bone. However, this type of bone differs from trabecular bone where most osteoporotic fractures occur. The present study aimed at identifying a set of reference genes for relative quantification of transcriptional activity of ovine spine bone and ovine in vitro differentiated mesenchymal stromal cells (MSC) for reliable comparability. Twelve candidate reference genes belonging to different functional classes were selected and their expression was measured from cultured ovMSCs (n = 18) and ovine bone samples (n = 16), respectively. RefFinder was used to rank the candidate genes. We identified B2M, GAPDH, RPL19 and YWHAZ as the best combination of reference genes for normalization of RT-qPCR results for transcriptional analyses of these ovine samples. This study demonstrates the importance of applying a set of reference genes for RT-qPCR analysis in sheep. Based on our data we recommend using four identified reference genes for relative quantification of gene expression studies in ovine bone or for in vitro experiments with osteogenically differentiated ovine MSCs.

  3. [Usefulness of a real-time quantitative polymerase-chain reaction (PCR) assay for the diagnosis of congenital and postnatal cytomegalovirus infection].

    Science.gov (United States)

    Reina, J; Weber, I; Riera, E; Busquets, M; Morales, C

    2014-05-01

    Cytomegalovirus (CMV) is the main virus causing congenital and postnatal infections in the pediatric population. The aim of this study is to evaluate the usefulness of a quantitative real-time PCR in the diagnosis of these infections using urine as a single sample. We studied all the urine samples of newborns (< 7 days) with suspected congenital infection, and urine of patients with suspected postnatal infection (urine negative at birth). Urines were simultaneously studied by cell culture, qualitative PCR (PCRc), and quantitative real-time PCR (PCRq). We analyzed 332 urine samples (270 to rule out congenital infection and 62 postnatal infections). Of the first, 22 were positive in the PCRq, 19 in the PCRc, and 17 in the culture. PCRq had a sensitivity of 100%, on comparing the culture with the rest of the techniques. Using the PCRq as a reference method, culture had a sensitivity of 77.2%, and PCRc 86.3%. In cases of postnatal infection, PCRq detected 16 positive urines, the PCRq 12, and the cell culture 10. The urines showed viral loads ranging from 2,178 to 116,641 copies/ml. The genomic amplification technique PCRq in real time was more sensitive than the other techniques evaluated. This technique should be considered as a reference (gold standard), leaving the cell culture as a second diagnostic level. The low cost and the automation of PCRq would enable the screening for CMV infection in large neonatal and postnatal populations. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

  4. Biosensor cell assay for measuring real-time aldosterone-induced release of histamine from mesenteric arteries

    DEFF Research Database (Denmark)

    Dalgaard, Emil G; Andersen, Kenneth; Svenningsen, Per

    2017-01-01

    AIMS: The aims were to develop a method for real-time detection of histamine release and to test if incubation with aldosterone induces histamine release from isolated, perfused mice mesenteric arteries. METHODS: Fura-2 loaded HEK-293 cells transfected with the histamine H1 receptor was used...... as a sensitive biosensor assay for histamine release from isolated mouse mesenteric arteries. Activation of the H1 receptor by histamine was measured as an increased number of intracellular Ca(2+) transient peaks using fluorescence imaging RESULTS: The developed biosensor was sensitive to histamine...... by the histamine H1 blocker pyrilamine. Mesenteric arteries from mast cell deficient SASH mice induced similar pyrilamine-sensitive Ca(2+) transient response in the biosensor cells. Mesenteric arteries from wild type and SASH mice expressed histamine decarboxylase mRNA, indicating that mast cells are not the only...

  5. Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment.

    Science.gov (United States)

    Hossain, Faria; Ghosh, Prakash; Khan, Md Anik Ashfaq; Duthie, Malcolm S; Vallur, Aarthy C; Picone, Alessandro; Howard, Randall F; Reed, Steven G; Mondal, Dinesh

    2017-01-01

    Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19-100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16-94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55-89.16%) for VL samples, 100% (95%CI, 69.15-100%) for RVL samples, and 52.5% (95% CI, 36.13-68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98-100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L. donovani parasites

  6. Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment

    Science.gov (United States)

    Ghosh, Prakash; Khan, Md. Anik Ashfaq; Duthie, Malcolm S.; Vallur, Aarthy C.; Picone, Alessandro; Howard, Randall F.; Reed, Steven G.

    2017-01-01

    Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19–100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16–94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55–89.16%) for VL samples, 100% (95%CI, 69.15–100%) for RVL samples, and 52.5% (95% CI, 36.13–68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98–100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L

  7. Quantitative real-time PCR identifies a critical region of deletion on 22q13 related to prognosis in oral cancer

    DEFF Research Database (Denmark)

    Reis, Patricia P; Rogatto, Silvia R; Kowalski, Luiz P

    2002-01-01

    Quantitative real time PCR was performed on genomic DNA from 40 primary oral carcinomas and the normal adjacent tissues. The target genes ECGFB, DIA1, BIK, and PDGFB and the microsatellite markers D22S274 and D22S277, mapped on 22q13, were selected according to our previous loss of heterozygosity.......0018) for patients with DIA1 gene loss. Relative copy number losses detected in these sequences may be related to disease progression and a worse prognosis in patients with oral cancer.......Quantitative real time PCR was performed on genomic DNA from 40 primary oral carcinomas and the normal adjacent tissues. The target genes ECGFB, DIA1, BIK, and PDGFB and the microsatellite markers D22S274 and D22S277, mapped on 22q13, were selected according to our previous loss of heterozygosity...... of disease (P=0.025), family history of cancer (P=0.001), and death (P=0.021). Relative copy number loss involving the DIA1 gene was correlated to family history of cancer (P

  8. Real-time measurement of the intracellular pH of yeast cells during glucose metabolism using ratiometric fluorescent nanosensors.

    Science.gov (United States)

    Elsutohy, Mohamed M; Chauhan, Veeren M; Markus, Robert; Kyyaly, Mohammed Aref; Tendler, Saul J B; Aylott, Jonathan W

    2017-05-11

    Intracellular pH is a key parameter that influences many biochemical and metabolic pathways that can also be used as an indirect marker to monitor metabolic and intracellular processes. Herein, we utilise ratiometric fluorescent pH-sensitive nanosensors with an extended dynamic pH range to measure the intracellular pH of yeast (Saccharomyces cerevisiae) during glucose metabolism in real-time. Ratiometric fluorescent pH-sensitive nanosensors consisting of a polyacrylamide nanoparticle matrix covalently linked to two pH-sensitive fluorophores, Oregon green (OG) and 5(6)carboxyfluorescein (FAM), and a reference pH-insensitive fluorophore, 5(6)carboxytetramethylrhodamine (TAMRA), were synthesised. Nanosensors were functionalised with acrylamidopropyltrimethyl ammonium hydrochloride (ACTA) to confer a positive charge to the nanoparticle surfaces that facilitated nanosensor delivery to yeast cells, negating the need to use stress inducing techniques. The results showed that under glucose-starved conditions the intracellular pH of yeast population (n ≈ 200) was 4.67 ± 0.15. Upon addition of d-(+)-glucose (10 mM), this pH value decreased to pH 3.86 ± 0.13 over a period of 10 minutes followed by a gradual rise to a maximal pH of 5.21 ± 0.26, 25 minutes after glucose addition. 45 minutes after the addition of glucose, the intracellular pH of yeast cells returned to that of the glucose starved conditions. This study advances our understanding of the interplay between glucose metabolism and pH regulation in yeast cells, and indicates that the intracellular pH homestasis in yeast is highly regulated and demonstrates the utility of nanosensors for real-time intracellular pH measurements.

  9. Selection of internal control genes for real-time quantitative PCR in ovary and uterus of sows across pregnancy.

    Directory of Open Access Journals (Sweden)

    María Martínez-Giner

    Full Text Available BACKGROUND: Reproductive traits play a key role in pig production in order to reduce costs and increase economic returns. Among others, gene expression analyses represent a useful approach to study genetic mechanisms underlying reproductive traits in pigs. The application of reverse-transcription quantitative PCR requires the selection of appropriate reference genes, whose expression levels should not be affected by the experimental conditions, especially when comparing gene expression across different physiological stages. RESULTS: The gene expression stability of ten potential reference genes was studied by three different methods (geNorm, NormFinder and BestKeeper in ovary and uterus collected at five different physiological time points (heat, and 15, 30, 45 and 60 days of pregnancy. Although final ranking differed, the three algorithms gave very similar results. Thus, the most stable genes across time were TBP and UBC in uterus and TBP and HPRT1 in ovary, while HMBS and ACTB showed the less stable expression in uterus and ovary, respectively. When studied as a systematic effect, the reproductive stage did not significantly affect the expression of the candidate reference genes except at 30d and 60d of pregnancy, when a general drop in expression was observed in ovary. CONCLUSIONS: Based in our results, we propose the use of TBP, UBC and SDHA in uterus and TBP, GNB2L1 and HPRT1 in ovary for normalization of longitudinal expression studies using quantitative PCR in sows.

  10. Evaluation of a rapid, quantitative real-time PCR method for enumeration of pathogenic Candida cells in water

    Science.gov (United States)

    Brinkman, Nichole E.; Haugland, Richard A.; Wymer, Larry J.; Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Vesper, Stephen J.

    2003-01-01

    Quantitative PCR (QPCR) technology, incorporating fluorigenic 5′ nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

  11. Low incidence of minor BRAF V600 mutation-positive subclones in primary and metastatic melanoma determined by sensitive and quantitative real-time PCR

    DEFF Research Database (Denmark)

    Kielsgaard Kristensen, Thomas; Clemmensen, Ole; Hoejberg, Lise

    2013-01-01

    , sensitive and quantitative BRAF V600E and V600K mutation-specific real-time quantitative PCR was used to study the occurrence of small subsets of mutation-positive cells in primary melanomas and melanoma metastases. The BRAF V600E mutation was detected in 39 of 82 melanoma patients. We observed a highly......BRAF V600 mutation is an important biological marker for therapeutic guidance in melanoma, where mutation-positive cases are candidates for therapy targeting mutant B-Raf. Recent studies showing intratumor variation in BRAF mutation status have caused concern that sensitive mutation analysis can...... lead to mutation-positive results in patients with melanomas with small subsets of mutation-positive cells who may not benefit from therapy targeting mutant B-Raf. Mutation analysis with high analytical sensitivity is generally preferred, to reduce the risk of false-negative results. In this study...

  12. Identification of human intestinal parasites affecting an asymptomatic peri-urban Argentinian population using multi-parallel quantitative real-time polymerase chain reaction.

    Science.gov (United States)

    Cimino, Rubén O; Jeun, Rebecca; Juarez, Marisa; Cajal, Pamela S; Vargas, Paola; Echazú, Adriana; Bryan, Patricia E; Nasser, Julio; Krolewiecki, Alejandro; Mejia, Rojelio

    2015-07-17

    In resource-limited countries, stool microscopy is the diagnostic test of choice for intestinal parasites (soil-transmitted helminths and/or intestinal protozoa). However, sensitivity and specificity is low. Improved diagnosis of intestinal parasites is especially important for accurate measurements of prevalence and intensity of infections in endemic areas. The study was carried out in Orán, Argentina. A total of 99 stool samples from a local surveillance campaign were analyzed by concentration microscopy and McMaster egg counting technique compared to the analysis by multi-parallel quantitative real-time polymerase chain reaction (qPCR). This study compared the performance of qPCR assay and stool microscopy for 8 common intestinal parasites that infect humans including the helminths Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Trichuris trichiura, and the protozoa Giardia lamblia, Cryptosporidium parvum/hominis, and Entamoeba histolytica, and investigated the prevalence of polyparasitism in an endemic area. qPCR showed higher detection rates for all parasites as compared to stool microscopy except T. trichiura. Species-specific primers and probes were able to distinguish between A. duodenale (19.1%) and N. americanus (36.4%) infections. There were 48.6% of subjects co-infected with both hookworms, and a significant increase in hookworm DNA for A. duodenale versus N. americanus (119.6 fg/μL: 0.63 fg/μL, P < 0.001) respectively. qPCR outperformed microscopy by the largest margin in G. lamblia infections (63.6% versus 8.1%, P < 0.05). Polyparasitism was detected more often by qPCR compared to microscopy (64.7% versus 24.2%, P < 0.05). Multi-parallel qPCR is a quantitative molecular diagnostic method for common intestinal parasites in an endemic area that has improved diagnostic accuracy compared to stool microscopy. This first time use of multi-parallel qPCR in Argentina has demonstrated the high

  13. Establishment of real-time quantitative reverse transcription polymerase chain reaction assay for transcriptional analysis of duck enteritis virus UL55 gene

    Directory of Open Access Journals (Sweden)

    Zhang Shunchuan

    2011-06-01

    Full Text Available Abstract Background Real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR has become the benchmark for detection and quantification of target gene expression level and been utilized increasingly in detection of viral load and therapy monitoring. The dynamic transcription variation of duck enteritis virus UL55 gene during the life cycle of duck enteritis virus in infected cells has not been reported yet. Results The newly identified duck enteritis virus UL55 gene was amplified and cloned into pMD18-T vector after digestion to generate a recombinant plasmid pMD18-T/UL55 for the establishment of qRT-PCR as standard DNA. The results of agarose gel electrophoresis and melting curve analysis demonstrated the primers we designed for qRT-PCR were specific and available. We used β-actin as a reference gene for normalization and established two standard curves based on pMD18-T/UL55 and pMD18-T/β-actin successfully. Based on that, the transcriptional analysis of DEV UL55 gene was performed, and the result suggested the expression of UL55 mRNA was at a low level from 0 to 8 h post-infection(p.i., then accumulated quickly since 12 h p.i. and peaked at 36 h p.i., it can be detected till 60 h p.i.. Nucleic acid inhibition test was carried out for analyzing a temporal regulation condition of DEV UL55 gene, result revealed that it was sensitive to ganciclovir. Synthesis procedures of DEV UL55 gene can be inhibited by ganciclovir. Conclusions The method we established in this paper can provide quantitative values reflecting the amounts of measured mRNA in samples. It's available for detection and quantification, also can be used in DEV diagnosis. The DEV UL55 gene was produced most abundantly during the late phase of replication in DEV-infected cells and the transcription of it depended on the synthesized DNA. DEV UL55 gene is a γ2 gene which occurs last and have a strict requirement for viral DNA synthesis.

  14. Quantitative real-time PCR with automated sample preparation for diagnosis and monitoring of cytomegalovirus infection in bone marrow transplant patients.

    Science.gov (United States)

    Hong, Kyeong Man; Najjar, Hazim; Hawley, Mary; Press, Richard D

    2004-05-01

    In bone marrow and stem cell transplant patients, the widespread use of preemptive cytomegalovirus (CMV) antiviral therapy necessitates faster, more precise, and more sensitive quantitative laboratory methods for serial viral load monitoring. We developed a novel CMV viral load assay using real-time PCR of plasma DNA prepared by an automated robotic workstation. Fluorescent hybridization probes directed at the glycoprotein B (gB) gene (or EcoRI D region) of CMV were used to detect and quantify PCR products. The beta-globin gene was amplified in parallel to control for the efficiency of the extraction and PCR steps. The assay was linear (R = 0.999) from a lower detection limit of 125 copies/mL to 5 x 10(9) copies/mL with a PCR efficiency of 1.975 (gB) or 2.02 (EcoRI D). The viral loads determined by PCRs directed at these two different viral targets were no different (n = 53; R = 0.928). The interassay CV was 3.5%, and the intraassay CV was 1-4%. Compared with a commercially available quantitative competitive PCR assay (Roche MONITOR; R = 0.59), the mean CMV viral load by real-time PCR was 3.1 times higher (mean ratio; P = 0.002). The diagnostic sensitivity and specificity of the real-time assay were 96% and 100%, respectively (n = 147), compared with 74% and 98% for a qualitative PCR assay (Roche AMPLICOR). On a subset of samples, the diagnostic sensitivity of viral culture was no greater than 50% (n = 44). Of 1115 clinical referral samples from 252 patients, 10% of the samples and 18% of the patients had low-level CMV viremia (median, 500 copies/mL). In this predominantly (85%) bone marrow transplant testing cohort, serial CMV viral load results were the predominant clinical trigger for the initiation, monitoring, and cessation of preemptive antiviral therapy. The combination of automated DNA preparation and semiautomated real-time fluorescent PCR detection allows for a sensitive, precise, and accurate high-throughput assay of CMV viral load that can be used as

  15. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

    2002-01-01

    ) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect ... axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer....... In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical...

  16. The Smallest R/V: A Small-scale Ocean Exploration Demonstration of Real-time Bathymetric Measurements

    Science.gov (United States)

    Howell, S. M.; Boston, B.; Maher, S. M.; Sleeper, J. D.; Togia, H.; Tree, J. P.

    2014-12-01

    In October 2013, graduate student members of the University of Hawaii Geophysical Society designed a small-scale model research vessel (R/V) that uses sonar to create 3D maps of a model seafloor in real-time. This pilot project was presented to the public at the School of Ocean and Earth Science and Technology's (SOEST) Biennial Open House weekend. An estimated 7,600 people attended the two-day event, including children and teachers from Hawaii's schools, home school students, community groups, families, and science enthusiasts. Our exhibit demonstrated real-time sonar mapping of a cardboard volcano using a toy size research vessel on a fixed 2D model ship track suspended above a model seafloor. Sound wave travel times were recorded using an unltrasonic emitter/receiver attached to an Arduino microcontroller platform, while the same system measured displacement along the ship track. This data was streamed through a USB connection to a PC running MatLab, where a 3D model was updated as the ship collected data. Our exhibit demonstrates the practical use of complicated concepts, like wave physics and data processing, in a way that even the youngest elementary students are able to understand. It provides an accessible avenue to learn about sonar mapping, and could easily be adapted to talk about bat and marine mammal echolocation by replacing the model ship and volcano. The exhibit received an overwhelmingly positive response from attendees, and has inspired the group to develop a more interactive model for future exhibitions, using multiple objects to be mapped that participants could arrange, and a more robust ship movement system that participants could operate.

  17. Real time Faraday spectrometer

    Science.gov (United States)

    Smith, Jr., Tommy E.; Struve, Kenneth W.; Colella, Nicholas J.

    1991-01-01

    This invention uses a dipole magnet to bend the path of a charged particle beam. As the deflected particles exit the magnet, they are spatially dispersed in the bend-plane of the magnet according to their respective momenta and pass to a plurality of chambers having Faraday probes positioned therein. Both the current and energy distribution of the particles is then determined by the non-intersecting Faraday probes located along the chambers. The Faraday probes are magnetically isolated from each other by thin metal walls of the chambers, effectively providing real time current-versus-energy particle measurements.

  18. Continuous, real time microwave plasma element sensor

    Science.gov (United States)

    Woskov, Paul P.; Smatlak, Donna L.; Cohn, Daniel R.; Wittle, J. Kenneth; Titus, Charles H.; Surma, Jeffrey E.

    1995-01-01

    Microwave-induced plasma for continuous, real time trace element monitoring under harsh and variable conditions. The sensor includes a source of high power microwave energy and a shorted waveguide made of a microwave conductive, refractory material communicating with the source of the microwave energy to generate a plasma. The high power waveguide is constructed to be robust in a hot, hostile environment. It includes an aperture for the passage of gases to be analyzed and a spectrometer is connected to receive light from the plasma. Provision is made for real time in situ calibration. The spectrometer disperses the light, which is then analyzed by a computer. The sensor is capable of making continuous, real time quantitative measurements of desired elements, such as the heavy metals lead and mercury.

  19. LEMming: A Linear Error Model to Normalize Parallel Quantitative Real-Time PCR (qPCR Data as an Alternative to Reference Gene Based Methods.

    Directory of Open Access Journals (Sweden)

    Ronny Feuer

    Full Text Available Gene expression analysis is an essential part of biological and medical investigations. Quantitative real-time PCR (qPCR is characterized with excellent sensitivity, dynamic range, reproducibility and is still regarded to be the gold standard for quantifying transcripts abundance. Parallelization of qPCR such as by microfluidic Taqman Fluidigm Biomark Platform enables evaluation of multiple transcripts in samples treated under various conditions. Despite advanced technologies, correct evaluation of the measurements remains challenging. Most widely used methods for evaluating or calculating gene expression data include geNorm and ΔΔCt, respectively. They rely on one or several stable reference genes (RGs for normalization, thus potentially causing biased results. We therefore applied multivariable regression with a tailored error model to overcome the necessity of stable RGs.We developed a RG independent data normalization approach based on a tailored linear error model for parallel qPCR data, called LEMming. It uses the assumption that the mean Ct values within samples of similarly treated groups are equal. Performance of LEMming was evaluated in three data sets with different stability patterns of RGs and compared to the results of geNorm normalization. Data set 1 showed that both methods gave similar results if stable RGs are available. Data set 2 included RGs which are stable according to geNorm criteria, but became differentially expressed in normalized data evaluated by a t-test. geNorm-normalized data showed an effect of a shifted mean per gene per condition whereas LEMming-normalized data did not. Comparing the decrease of standard deviation from raw data to geNorm and to LEMming, the latter was superior. In data set 3 according to geNorm calculated average expression stability and pairwise variation, stable RGs were available, but t-tests of raw data contradicted this. Normalization with RGs resulted in distorted data contradicting

  20. LEMming: A Linear Error Model to Normalize Parallel Quantitative Real-Time PCR (qPCR) Data as an Alternative to Reference Gene Based Methods.

    Science.gov (United States)

    Feuer, Ronny; Vlaic, Sebastian; Arlt, Janine; Sawodny, Oliver; Dahmen, Uta; Zanger, Ulrich M; Thomas, Maria

    2015-01-01

    Gene expression analysis is an essential part of biological and medical investigations. Quantitative real-time PCR (qPCR) is characterized with excellent sensitivity, dynamic range, reproducibility and is still regarded to be the gold standard for quantifying transcripts abundance. Parallelization of qPCR such as by microfluidic Taqman Fluidigm Biomark Platform enables evaluation of multiple transcripts in samples treated under various conditions. Despite advanced technologies, correct evaluation of the measurements remains challenging. Most widely used methods for evaluating or calculating gene expression data include geNorm and ΔΔCt, respectively. They rely on one or several stable reference genes (RGs) for normalization, thus potentially causing biased results. We therefore applied multivariable regression with a tailored error model to overcome the necessity of stable RGs. We developed a RG independent data normalization approach based on a tailored linear error model for parallel qPCR data, called LEMming. It uses the assumption that the mean Ct values within samples of similarly treated groups are equal. Performance of LEMming was evaluated in three data sets with different stability patterns of RGs and compared to the results of geNorm normalization. Data set 1 showed that both methods gave similar results if stable RGs are available. Data set 2 included RGs which are stable according to geNorm criteria, but became differentially expressed in normalized data evaluated by a t-test. geNorm-normalized data showed an effect of a shifted mean per gene per condition whereas LEMming-normalized data did not. Comparing the decrease of standard deviation from raw data to geNorm and to LEMming, the latter was superior. In data set 3 according to geNorm calculated average expression stability and pairwise variation, stable RGs were available, but t-tests of raw data contradicted this. Normalization with RGs resulted in distorted data contradicting literature, while

  1. Identification of appropriate reference genes for human mesenchymal stem cell analysis by quantitative real-time PCR.

    Science.gov (United States)

    Li, Xiuying; Yang, Qiwei; Bai, Jinping; Xuan, Yali; Wang, Yimin

    2015-01-01

    Normalization to a reference gene is the method of choice for quantitative reverse transcription-PCR (RT-qPCR) analysis. The stability of reference genes is critical for accurate experimental results and conclusions. We have evaluated the expression stability of eight commonly used reference genes found in four different human mesenchymal stem cells (MSC). Using geNorm, NormFinder and BestKeeper algorithms, we show that beta-2-microglobulin and peptidyl-prolylisomerase A were the optimal reference genes for normalizing RT-qPCR data obtained from MSC, whereas the TATA box binding protein was not suitable due to its extensive variability in expression. Our findings emphasize the significance of validating reference genes for qPCR analyses. We offer a short list of reference genes to use for normalization and recommend some commercially-available software programs as a rapid approach to validate reference genes. We also demonstrate that the two reference genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase, are frequently used are not always successful in many cases.

  2. A de novo transcriptome and valid reference genes for quantitative real-time PCR in Colaphellus bowringi.

    Directory of Open Access Journals (Sweden)

    Qian-Qian Tan

    Full Text Available The cabbage beetle Colaphellus bowringi Baly is a serious insect pest of crucifers and undergoes reproductive diapause in soil. An understanding of the molecular mechanisms of diapause regulation, insecticide resistance, and other physiological processes is helpful for developing new management strategies for this beetle. However, the lack of genomic information and valid reference genes limits knowledge on the molecular bases of these physiological processes in this species.Using Illumina sequencing, we obtained more than 57 million sequence reads derived from C. bowringi, which were assembled into 39,390 unique sequences. A Clusters of Orthologous Groups classification was obtained for 9,048 of these sequences, covering 25 categories, and 16,951 were assigned to 255 Kyoto Encyclopedia of Genes and Genomes pathways. Eleven candidate reference gene sequences from the transcriptome were then identified through reverse transcriptase polymerase chain reaction. Among these candidate genes, EF1α, ACT1, and RPL19 proved to be the most stable reference genes for different reverse transcriptase quantitative polymerase chain reaction experiments in C. bowringi. Conversely, aTUB and GAPDH were the least stable reference genes.The abundant putative C. bowringi transcript sequences reported enrich the genomic resources of this beetle. Importantly, the larger number of gene sequences and valid reference genes provide a valuable platform for future gene expression studies, especially with regard to exploring the molecular mechanisms of different physiological processes in this species.

  3. [Development and application of TaqMan-MGB real-time quantitative PCR assay for detection of goat pox virus].

    Science.gov (United States)

    Cheng, Zhentao; Yue, Jun; Li, Yongming; Xu, Leren; Wang, Kaigong; Zhou, Bijun; Chen, Junyi; Li, Jun; Jiang, Nan

    2009-03-01

    The complete gene sequences of eight capripoxvirus strains in GenBank were aligned and analyzed with DNAStar software. We selected a size of 64 bp gene fragment that was located in gp064 region of goat pox virus (GPV) genome, and designed a pair of primers and a TaqMan-MGB probe against the gene fragment with Primer Express 2.0 software. Then, the fluorescence quantitative PCR (FQ-PCR) assay was developed and the standard curve of different dilution series was described. We extracted the DNA samples from clinical skin pox, scab and GPV infected materials of artificial challenge animals. The FQ-PCR assay has been performed for all kinds of DNA samples. The results showed that the FQ-PCR assay was sensitive, specific, stable and could be used for clinical diagnosis. This method provided an important tool for rapid diagnosis of goat pox clinically, and for study GPV pathogenesis in the course of disease occurrence, development and convalescence.

  4. Detection and quantification of Flavobacterium psychrophilum in water and fish tissue samples by quantitative real time PCR

    Science.gov (United States)

    2014-01-01

    Background Flavobacterium psychrophilum is the agent of Bacterial Cold Water Disease and Rainbow Trout Fry Syndrome, two diseases leading to high mortality. Pathogen detection is mainly carried out using cultures and more rapid and sensitive methods are needed. Results We describe a qPCR technique based on the single copy gene β’ DNA-dependent RNA polymerase (rpoC). Its detection limit was 20 gene copies and the quantification limit 103 gene copies per reaction. Tests on spiked spleens with known concentrations of F. psychrophilum (106 to 101 cells per reaction) showed no cross-reactions between the spleen tissue and the primers and probe. Screening of water samples and spleens from symptomless and infected fishes indicated that the pathogen was already present before the outbreaks, but F. psychrophilum was only quantifiable in spleens from diseased fishes. Conclusions This qPCR can be used as a highly sensitive and specific method to detect F. psychrophilum in different sample types without the need for culturing. qPCR allows a reliable detection and quantification of F. psychrophilum in samples with low pathogen densities. Quantitative data on F. psychrophilum abundance could be useful to investigate risk factors linked to infections and also as early warning system prior to potential devastating outbreak. PMID:24767577

  5. Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting the groEL Gene

    Science.gov (United States)

    Junick, Jana

    2012-01-01

    Quantitative real-time PCR assays targeting the groEL gene for the specific enumeration of 12 human fecal Bifidobacterium species were developed. The housekeeping gene groEL (HSP60 in eukaryotes) was used as a discriminative marker for the differentiation of Bifidobacterium adolescentis, B. angulatum, B. animalis, B. bifidum, B. breve, B. catenulatum, B. dentium, B. gallicum, B. longum, B. pseudocatenulatum, B. pseudolongum, and B. thermophilum. The bifidobacterial chromosome contains a single copy of the groEL gene, allowing the determination of the cell number by quantification of the groEL copy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a given Bifidobacterium species. Independent of the Bifidobacterium species tested, the proportion of groEL copies recovered from fecal samples spiked with 5 to 9 log10 cells/g feces was approximately 50%. The quantification limit was 5 to 6 log10 groEL copies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3 Bifidobacterium species and B. longum frequently detected. The predominant species in infant and adult fecal samples were B. breve and B. adolescentis, respectively. It was possible to distinguish B. catenulatum and B. pseudocatenulatum. We conclude that the groEL gene is a suitable molecular marker for the specific and accurate quantification of human fecal Bifidobacterium species by real-time PCR. PMID:22307308

  6. A two-stage method of quantitative flood risk analysis for reservoir real-time operation using ensemble-based hydrologic forecasts

    Science.gov (United States)

    Liu, P.

    2013-12-01

    Quantitative analysis of the risk for reservoir real-time operation is a hard task owing to the difficulty of accurate description of inflow uncertainties. The ensemble-based hydrologic forecasts directly depict the inflows not only the marginal distributions but also their persistence via scenarios. This motivates us to analyze the reservoir real-time operating risk with ensemble-based hydrologic forecasts as inputs. A method is developed by using the forec