Science.gov

Sample records for device-independent real-time identification

  1. Real-time flutter identification

    NASA Technical Reports Server (NTRS)

    Roy, R.; Walker, R.

    1985-01-01

    The techniques and a FORTRAN 77 MOdal Parameter IDentification (MOPID) computer program developed for identification of the frequencies and damping ratios of multiple flutter modes in real time are documented. Physically meaningful model parameterization was combined with state of the art recursive identification techniques and applied to the problem of real time flutter mode monitoring. The performance of the algorithm in terms of convergence speed and parameter estimation error is demonstrated for several simulated data cases, and the results of actual flight data analysis from two different vehicles are presented. It is indicated that the algorithm is capable of real time monitoring of aircraft flutter characteristics with a high degree of reliability.

  2. Species identification in meat products using real-time PCR.

    PubMed

    Jonker, K M; Tilburg, J J H C; Hagele, G H; de Boer, E

    2008-05-01

    One of the most convenient methods for the identification of animal species in processed meat products is the examination of DNA sequences. Real-time polymerase chain reaction (qPCR) techniques are particularly suitable because even small fragments of DNA formed during heat processing of the meat can be amplified and identified. A real-time PCR method has been developed and evaluated for the identification of processed meat products. In test mixtures containing beef, pork, horse, mutton, chicken and turkey, it was possible to identify these species down to a level of 0.05%. By adjusting the number of cycles, it was possible to detect levels as low as 0.01% of these species. Cross-reactivity between these species was not found, except for pure horsemeat (250 ng DNA) in the assay for turkey meat. Cross-reactivity of deer, roe, ostrich, kangaroo, goat, domestic duck, mallard, goose, pigeon, guinea fowl, quail and pheasant was also investigated and it was found that amounts as high as 250 ng DNA of these species in the reaction vial did not result in (false) positive signals except for amounts higher than 125 ng deer DNA and higher than 50 ng pigeon DNA in the determination of chicken and beef, respectively. More than 150 meat samples were examined using DNA hybridization and real-time PCR. A comparison of the results showed a better performance of the real-time procedure compared to DNA hybridization.

  3. Real-time bioacoustics monitoring and automated species identification

    PubMed Central

    Corrada-Bravo, Carlos; Campos-Cerqueira, Marconi; Milan, Carlos; Vega, Giovany; Alvarez, Rafael

    2013-01-01

    Traditionally, animal species diversity and abundance is assessed using a variety of methods that are generally costly, limited in space and time, and most importantly, they rarely include a permanent record. Given the urgency of climate change and the loss of habitat, it is vital that we use new technologies to improve and expand global biodiversity monitoring to thousands of sites around the world. In this article, we describe the acoustical component of the Automated Remote Biodiversity Monitoring Network (ARBIMON), a novel combination of hardware and software for automating data acquisition, data management, and species identification based on audio recordings. The major components of the cyberinfrastructure include: a solar powered remote monitoring station that sends 1-min recordings every 10 min to a base station, which relays the recordings in real-time to the project server, where the recordings are processed and uploaded to the project website (arbimon.net). Along with a module for viewing, listening, and annotating recordings, the website includes a species identification interface to help users create machine learning algorithms to automate species identification. To demonstrate the system we present data on the vocal activity patterns of birds, frogs, insects, and mammals from Puerto Rico and Costa Rica. PMID:23882441

  4. Real-time MJO Identification and Subseasonal Predictions

    NASA Astrophysics Data System (ADS)

    Berry, E. K.; Weickmann, K.

    2012-12-01

    There is evidence that the Madden-Julian Oscillation (MJO) can enhance predictability of sensible weather possibly out to lead times of ~40-50 days. This includes high impact events such as extreme surface air temperatures and extended periods of severe storms/excessive precipitation. However, identification of MJOs in real-time is challenging because the weather-climate system is dominated by noise. In fact, stochastic extratropical dynamics can organize large scale subtropical wind fields and envelopes of tropical rainfall that can be misrepresented as MJOs especially when using combined wind and outgoing longwave radiation indices (Wheeler and Hendon, 2004). These mixed global wind-tropical convective variations (Weickmann and Berry, 2009) partially reflect the Global Wind Oscillation (GWO), which is non-oscillatory and does not provide useful forecast information beyond ~15 days. An analysis of outgoing longwave radiation (OLR) and global circulation data sets is performed for the 2006-07 through 2011-12 boreal cold seasons (which included the DYNAMO field experiment). During this roughly six year period nine MJOs were identified that had the potential to extend the range of skillful or useful prediction. The breakdown of these events and their interactions with ENSO are discussed. Examples of red noise dominated variations and predictability ramifications will also be given. These include the premature ending of the 2006-07 El-Niño, extratropical feedbacks during 2010-11 leading to a strong jet stream not consistent with La-Niña, and constructive interference of an MJO and La-Niña that contributed to the March 2012 Midwest-eastern USA "heat wave". The criticality to distinguish in real time between "sustained, coherent MJOs" and other types of coherent or noisy tropical-extratropical variability is emphasized. The fast, moderate MJOs that initiated during DYNAMO might provide clues about MJO initiation and succession.

  5. Real-Time PCR Identification of Unique Bacillus anthracis Sequences.

    PubMed

    Cieślik, P; Knap, J; Kolodziej, M; Mirski, T; Joniec, J; Graniak, G; Zakowska, D; Winnicka, I; Bielawska-Drózd, A

    2015-01-01

    Bacillus anthracis is a spore-forming, Gram-positive microorganism. It is a causative agent of anthrax, a highly infectious disease. It belongs to the "Bacillus cereus group", which contains other closely related species, including Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus weihenstephanensis, and Bacillus pseudomycoides. B. anthracis naturally occurs in soil environments. The BA5345 genetic marker was used for highly specific detection of B. anthracis with TaqMan probes. The detection limit of a real-time PCR assay was estimated at the level of 16.9 copies (CI95% - 37.4 to 37.86, SD = 0.2; SE = 0.118). Oligonucleotides designed for the targeted sequences (within the tested locus) revealed 100 % homology to B. anthracis strain reference sequences deposited in the database (NCBI) and high specificity to all tested B. anthracis strains. Additional in silico analysis of plasmid markers pag and cap genes with B. anthracis strains included in the database was carried out. Our study clearly indicates that the BA5345 marker can be used with success as a chromosomal marker in routine identification of B. anthracis; moreover, detection of plasmid markers indicates virulence of the examined strains.

  6. Genus identification of toxic plant by real-time PCR.

    PubMed

    Matsuyama, Shuji; Nishi, Katsuji

    2011-03-01

    Some plants have toxicities that are dangerous for humans. In the case of poisoning by toxic plants, a rapid and easy screening test is required for accurate medical treatment or forensic investigation. In this study, we designed specific primer pairs for identification of toxic plants, such as subgenus Aconitum, genus Ricinus, genus Illicium, and genus Scopolia, by internal transcribed spacer sequences of nuclear ribosomal DNA. Allied species of target plants, foods, and human DNA were not detected, but each primer pair provided a specific PCR product from the target plant using real-time PCR. This method can detect the subgenus Aconitum, genus Ricinus, and genus Scopolia with template DNA of 10 pg, respectively, and genus Illicium with 1 pg. Furthermore, each primer pair provided the exact PCR product from digested target plants in artificial gastric fluid. When a trace unknown plant sample in forensic investigation is collected from stomach contents, this PCR assay may be useful for screening toxic plants.

  7. Real-time machine tool chatter identification and control system

    NASA Astrophysics Data System (ADS)

    Zhang, Shilong

    1997-05-01

    Chatter in machining processes is one of the most important factors limiting production rates. In order to suppress machine tool chatter during orthogonal cutting processes, a real time active chatter controller is designed and implemented that is able to adopt to the continuously changing machining parameters. An electro-hydraulic servo system is used to control the movement of the cutting tool. The cutting force, workpiece acceleration, and tool displacement are measured in real time. The transfer function of the workpiece is estimated by using the cutting force and the acceleration of the workpiece. All the digital signal acquisition and processing tasks are performed by a digital signal processor (MicroStar DAP3200a/415). The digital controller is designed such that the servo/actuator dynamics is adjusted to match the workpiece dynamics to suppress chatter. To make the controller adaptive to the changing dynamics of the workpiece, a recursive least square technique is used to identify the workpiece dynamics in real time. The estimated workpiece dynamics parameters are then used in the digital controller to calculate a new servo output, thus controlling the tool movement. Simulations show that chatter can be suppressed successfully by using this method. Experiments agree well with simulations.

  8. Real-time flutter identification with close mode resolution

    NASA Technical Reports Server (NTRS)

    Roy, R. H.; Walker, R. A.; Gilyard, G. B.

    1986-01-01

    Real-time flutter prediction including close modes can be effectively estimated from turbulence or on-board excitation with an Extended Kalman Filter (EKF) approach. A physically based model form enables prediction of the damping rate as well as damping, giving a time to instability estimate with its variance. The approach is recursive and can operate asynchronously to drop data outliers and hence is quite robust. Its speed is reasonable for on-line application but can also be used effectively as an off-line analysis tool for application to any modal testing situation.

  9. Real-time Algorithms for Sparse Neuronal System Identification.

    PubMed

    Sheikhattar, Alireza; Babadi, Behtash

    2016-08-01

    We consider the problem of sparse adaptive neuronal system identification, where the goal is to estimate the sparse time-varying neuronal model parameters in an online fashion from neural spiking observations. We develop two adaptive filters based on greedy estimation techniques and regularized log-likelihood maximization. We apply the proposed algorithms to simulated spiking data as well as experimentally recorded data from the ferret's primary auditory cortex during performance of auditory tasks. Our results reveal significant performance gains achieved by the proposed algorithms in terms of sparse identification and trackability, compared to existing algorithms.

  10. Identification of Aedes aegypti and its Respective Life Stages by Real-Time PCR

    DTIC Science & Technology

    2004-06-01

    RTO-MP-HFM-108 22 - 1 Identification of Aedes aegypti and its Respective Life Stages by Real - Time PCR James C. McAvin1*; Major David E...Stages by Real - Time PCR 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK...grade water Identification of Aedes aegypti and its Respective Life Stages by Real - Time PCR RTO-MP-HFM-108 22 - 3 for no template controls

  11. Quantitative Real-Time PCR Fecal Source Identification in the ...

    EPA Pesticide Factsheets

    Rivers in the Tillamook Basin play a vital role in supporting a thriving dairy and cheese-making industry, as well as providing a safe water resource for local human and wildlife populations. Historical concentrations of fecal bacteria in these waters are at times too high to allow for safe use leading to economic loss, endangerment of local wildlife, and poor conditions for recreational use. In this study, we employ host-associated qPCR methods for human (HF183/BacR287 and HumM2), ruminant (Rum2Bac), cattle (CowM2 and CowM3), canine (DG3 and DG37), and avian (GFD) fecal pollution combined with high-resolution geographic information system (GIS) land use data and general indicator bacteria measurements to elucidatewater quality spatial and temporal trends. Water samples (n=584) were collected over a 1-year period at 29 sites along the Trask, Kilchis, and Tillamook rivers and tributaries (Tillamook Basin, OR). A total of 16.6% of samples (n=97) yielded E. coli levels considered impaired based on Oregon Department of Environmental Quality bacteria criteria (406 MPN/100mL). Hostassociated genetic indicators were detected at frequencies of 39.2% (HF183/BacR287), 16.3% (HumM2), 74.6% (Rum2Bac), 13.0% (CowM2), 26.7% (CowM3), 19.8% (DG3), 3.2% (DG37), and 53.4% (GFD) across all water samples (n=584). Seasonal trends in avian, cattle, and human fecal pollution sources were evident over the study area. On a sample site basis, quantitative fecal source identification and

  12. Real-time identification method of driver model with steering manipulation

    NASA Astrophysics Data System (ADS)

    Tokutake, Hiroshi; Sugimoto, Youichi; Shirakata, Tetsuro

    2013-01-01

    This study proposes a method for real-time identification of a driver model. The proposed method requires only the yaw rate sensor, the steering angle sensor, and velocity sensors that are usually installed in the production car. The identification algorithm involves the division of the recorded data, prefiltering of the divided data, estimation of the driver's desired response, and identification. The prefilter extracts the driver's involuntary response that can be modelled in a simple form. The ideal car response that the driver attempts to track is estimated from the recorded data, and this response is provided to the identification algorithm of the feedback driver model for error tracking. These newly developed methods enable real-time identification under actual driving conditions. The driving simulator experiments and the actual driving tests were performed, and the proposed method was validated. The results show that the time history of the variation in the driver's characteristics can be realised in real time using the proposed method.

  13. Rapid species identification of cooked poisonous mushrooms by using real-time PCR.

    PubMed

    Maeta, Kazuhiko; Ochi, Tomoya; Tokimoto, Keisuke; Shimomura, Norihiro; Maekawa, Nitaro; Kawaguchi, Nobuhisa; Nakaya, Makoto; Kitamoto, Yutaka; Aimi, Tadanori

    2008-05-01

    Species-specific identification of the major cooked and fresh poisonous mushrooms in Japan was performed using a real-time PCR system. Specific fluorescence signals were detected, and no nonspecific signals were detected. Therefore, we succeeded in developing a species-specific test for the identification of poisonous mushrooms within 1.5 h.

  14. Real time identification of large space structures. Ph.D. Thesis - MIT

    NASA Technical Reports Server (NTRS)

    Voss, Janice E.

    1987-01-01

    Identification of frequencies, damping ratios, and mode shapes of large space structures (LSSs) are examined in real time. Real time processing allows for quick updates of model processing after a reconfiguration of structural failure. Recursive lattice least squares (RLLS) was selected as the baseline algorithm for the identification. Simulation results on a one dimensional LSS demonstrated that it provides good estimates, was not ill-conditioned in the presence of under-excited modes, allowed activity by a supervisory control system which prevented damage to the LSS or excessive drift, and was capable of real-time processing for typical LSS models. A suboptimal version of RLLS, which is equivalent to simulated parallel processing, was derived. A NASTRAN model of the dual keel U.S. space station was used to demonstrate the input/identification algorithm package in a more realistic simulation. Because the first eight flexible modes were very close together, the identification was much more difficult than in the simple examples. Even so, the model was accurately identified in real time.

  15. Towards real-time identification of cosmic rays with LOw-Frequency ARray radio antennas

    NASA Astrophysics Data System (ADS)

    Bonardi, Antonio; Buitink, Stijn; Corstanje, Arthur; Enriquez, J. Emilio; Falcke, Heino; Hörandel, Jörg R.; Mitra, Pragati; Mulrey, Katie; Nelles, Anna; Rachen, Jörg Paul; Rossetto, Laura; Schellart, Pim; Scholten, Olaf; Thoudam, Satyendra; Trinh, Gia; ter Veen, Sander; Winchen, Tobias

    2017-03-01

    Cosmic rays entering the Earth's atmosphere produce Extensive Air Showers, which emit a radio signal through Geo-magnetic radiation and Askaryan emission. At the present time, one of the biggest challenges for assessing the Radio detection as a valuable technique for Cosmic-ray observation is to identify in real-time the very short (less than 100 ns) radio signals over the background noise. In this work, we present the latest updates on the real-time identification of radio signals from Extensive Air Showers by using the data from LOFAR Low Band Antenna stations, which are sensitive in the 30-80 MHz region.

  16. Multiplex real-time PCR for identification of canine parvovirus antigenic types.

    PubMed

    Kaur, Gurpreet; Chandra, Mudit; Dwivedi, P N; Narang, Deepti

    2016-07-01

    Canine parvovirus (CPV) is an important disease causing gastroenteritis and/or haemorrhagic gastroenteritis in dogs. There are four antigenic types of CPV reported worldwide viz. CPV 2, CPV 2a, CPV 2b and CPV 2c. The diagnosis of CPV with the identification of the antigen type responsible remains problematic. In the present study, identification as well as antigenic typing of CPV was done using a de novo multiplex real time PCR to combat the problem of antigenic type identification. From the study it could be concluded that the here developed multiplex real time PCR assay could be used for rapid detection of CPV as well as typing of its three antigenic types.

  17. Development of a real time magnetic island identification system for HL-2A tokamak

    NASA Astrophysics Data System (ADS)

    Chen, Chao; Sun, Shan; Ji, Xiaoquan; Yin, Zejie

    2017-08-01

    A novel real time magnetic island identification system for HL-2A is introduced. The identification method is based on the measurement of Mirnov probes and the equilibrium flux constructed by the equilibrium fit (EFIT) code. The system consists of an analog front board and a digital processing board connected by a shield cable. Four octal-channel analog-to-digital convertors are utilized for 100 KHz simultaneous sampling of all the probes, and the applications of PCI extensions for Instrumentation platform and reflective memory allow the system to receive EFIT results simultaneously. A high performance field programmable gate array (FPGA) is used to realize the real time identification algorithm. Based on the parallel and pipeline processing of the FPGA, the magnetic island structure can be identified with a cycle time of 3 ms during experiments.

  18. Flight Investigation of Prescribed Simultaneous Independent Surface Excitations for Real-Time Parameter Identification

    NASA Technical Reports Server (NTRS)

    Moes, Timothy R.; Smith, Mark S.; Morelli, Eugene A.

    2003-01-01

    Near real-time stability and control derivative extraction is required to support flight demonstration of Intelligent Flight Control System (IFCS) concepts being developed by NASA, academia, and industry. Traditionally, flight maneuvers would be designed and flown to obtain stability and control derivative estimates using a postflight analysis technique. The goal of the IFCS concept is to be able to modify the control laws in real time for an aircraft that has been damaged in flight. In some IFCS implementations, real-time parameter identification (PID) of the stability and control derivatives of the damaged aircraft is necessary for successfully reconfiguring the control system. This report investigates the usefulness of Prescribed Simultaneous Independent Surface Excitations (PreSISE) to provide data for rapidly obtaining estimates of the stability and control derivatives. Flight test data were analyzed using both equation-error and output-error PID techniques. The equation-error PID technique is known as Fourier Transform Regression (FTR) and is a frequency-domain real-time implementation. Selected results were compared with a time-domain output-error technique. The real-time equation-error technique combined with the PreSISE maneuvers provided excellent derivative estimation in the longitudinal axis. However, the PreSISE maneuvers as presently defined were not adequate for accurate estimation of the lateral-directional derivatives.

  19. Floor Covering and Surface Identification for Assistive Mobile Robotic Real-Time Room Localization Application

    PubMed Central

    Gillham, Michael; Howells, Gareth; Spurgeon, Sarah; McElroy, Ben

    2013-01-01

    Assistive robotic applications require systems capable of interaction in the human world, a workspace which is highly dynamic and not always predictable. Mobile assistive devices face the additional and complex problem of when and if intervention should occur; therefore before any trajectory assistance is given, the robotic device must know where it is in real-time, without unnecessary disruption or delay to the user requirements. In this paper, we demonstrate a novel robust method for determining room identification from floor features in a real-time computational frame for autonomous and assistive robotics in the human environment. We utilize two inexpensive sensors: an optical mouse sensor for straightforward and rapid, texture or pattern sampling, and a four color photodiode light sensor for fast color determination. We show how data relating floor texture and color obtained from typical dynamic human environments, using these two sensors, compares favorably with data obtained from a standard webcam. We show that suitable data can be extracted from these two sensors at a rate 16 times faster than a standard webcam, and that these data are in a form which can be rapidly processed using readily available classification techniques, suitable for real-time system application. We achieved a 95% correct classification accuracy identifying 133 rooms' flooring from 35 classes, suitable for fast coarse global room localization application, boundary crossing detection, and additionally some degree of surface type identification. PMID:24351647

  20. Real-time PCR-based identification of bacterial and fungal pathogens from blood samples.

    PubMed

    Mai, Madeleine; Müller, Iris; Maneg, Daniela; Lohr, Benedikt; Haecker, Achim; Haberhausen, Gerd; Hunfeld, Klaus-Peter

    2015-01-01

    Latest major contributions in the field of sepsis diagnostics result from advances in PCR technologies permitting new standards in speed and quality, given the fact that a timely diagnosis is the decisive factor to the survival of patients with bloodstream infections.Multiplex real-time PCR is a quantitative method for simultaneous amplification and detection of different targeted DNA molecules within hours. Nevertheless, various studies have shown a number of technical shortcomings as well as a high heterogeneity in sensitivity.The present method allows the standardized and rapid detection and identification of 25 common bacteria and fungi responsible for bloodstream infections from whole blood samples by using LightCycler(®) SeptiFast (LC-SF) test, based on real-time PCR.

  1. Field-based species identification of closely-related plants using real-time nanopore sequencing.

    PubMed

    Parker, Joe; Helmstetter, Andrew J; Devey, Dion; Wilkinson, Tim; Papadopulos, Alexander S T

    2017-08-21

    Advances in DNA sequencing and informatics have revolutionised biology over the past four decades, but technological limitations have left many applications unexplored. Recently, portable, real-time, nanopore sequencing (RTnS) has become available. This offers opportunities to rapidly collect and analyse genomic data anywhere. However, generation of datasets from large, complex genomes has been constrained to laboratories. The portability and long DNA sequences of RTnS offer great potential for field-based species identification, but the feasibility and accuracy of these technologies for this purpose have not been assessed. Here, we show that a field-based RTnS analysis of closely-related plant species (Arabidopsis spp.) has many advantages over laboratory-based high-throughput sequencing (HTS) methods for species level identification and phylogenomics. Samples were collected and sequenced in a single day by RTnS using a portable, "al fresco" laboratory. Our analyses demonstrate that correctly identifying unknown reads from matches to a reference database with RTnS reads enables rapid and confident species identification. Individually annotated RTnS reads can be used to infer the evolutionary relationships of A. thaliana. Furthermore, hybrid genome assembly with RTnS and HTS reads substantially improved upon a genome assembled from HTS reads alone. Field-based RTnS makes real-time, rapid specimen identification and genome wide analyses possible.

  2. New Products for Near Real-Time Enhanced Landslide Identification and Precipitation Monitoring

    NASA Astrophysics Data System (ADS)

    Roberts-Pierel, J.; Ahamed, A.; Fayne, J.; Rumsey, A.

    2015-12-01

    Nepal and the Himalayan region are hotspots for landslide activity due to mountainous topography, complex terrain, and monsoon rains. Current research in landslide modeling and detection generally requires high resolution imagery with software aided classification or manual digitization by analysts. These methods are plagued by low spatial and temporal accuracy. Addressing issues in conventional measurement, this study combined optical data from Landsat 8, a Digital Elevation Model (DEM) generated from Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER), and precipitation data from the Global Precipitation Measurement Mission (GPM) to create two products. The Sudden Landslide Identification Product (SLIP) uses Landsat 8 and the ASTER DEM to identify landslides in near real-time, and provides damage assessments by mapping landslides triggered by precipitation. Detecting Real-time Increased Precipitation (DRIP) monitors precipitation levels extracted from the GPM-IMERG 30-minute product to create alerts in near real-time when current rainfall levels exceed regional threshold values. After a landslide detection is made by SLIP, historical rainfall data from DRIP is analyzed to estimate a date for the detected landslide. Together, DRIP and SLIP will be used by local and regional organizations in Nepal such as the International Centre for Integrated Mountain Development (ICIMOD), as well as the international scientific community to protect lives, preserve infrastructure, and manage local ecosystems.

  3. Real-Time Near-Infrared Fluorescence-Guided Identification of the Ureters using Methylene Blue

    PubMed Central

    Matsui, Aya; Tanaka, Eiichi; Choi, Hak Soo; Kianzad, Vida; Gioux, Sylvain; Lomnes, Stephen J.; Frangioni, John V.

    2009-01-01

    Background The aim of this study was to determine whether the invisible near-infrared (NIR) fluorescence properties of methylene blue (MB), a dye already FDA-approved for other indications, could be exploited for real-time, intraoperative identification of the ureters. Methods The optical properties of MB were quantified in vitro. Open surgery and laparoscopic NIR fluorescence imaging systems were employed. Yorkshire pigs were injected intravenously with: 0.1 mg/kg MB (n = 8), 10 mg furosemide followed by 0.1 mg/kg MB (n = 6), or 0.5 mg/kg MB (n = 6). The contrast-to-background ratio (CBR) of the kidney and ureters, and MB concentration in urine, were quantified. Results Peak MB absorbance, emission, and intensity in urine occurred at 668 nm, 688 nm, and 20 μM, respectively. After intravenous injection, doses as low as 0.1 mg/kg MB provided prolonged imaging of the ureters, and a dose of 0.5 mg/kg provided statistically significant improvement of CBR. Pre-injection of furosemide increased urine volume but did not improve CBR. Laparoscopic identification of the ureter using MB NIR fluorescence was demonstrated. Conclusions Ureteral imaging using MB NIR fluorescence provides sensitive, real-time, intraoperative identification of the ureters during open and laparoscopic surgeries. PMID:20117811

  4. Real-time, near-infrared, fluorescence-guided identification of the ureters using methylene blue.

    PubMed

    Matsui, Aya; Tanaka, Eiichi; Choi, Hak Soo; Kianzad, Vida; Gioux, Sylvain; Lomnes, Stephen J; Frangioni, John V

    2010-07-01

    The aim of this study was to determine whether the invisible near-infrared (NIR) fluorescence properties of methylene blue (MB), a dye already approved by the U.S. Food and Drug Administration for other indications, could be exploited for real-time, intra-operative identification of the ureters. The optical properties of MB were quantified in vitro. Open surgery and laparoscopic NIR fluorescence imaging systems were employed. Yorkshire pigs were injected intravenously with 0.1-mg/kg MB (n = 8), 10-mg furosemide followed by 0.1-mg/kg MB (n = 6), or 0.5-mg/kg MB (n = 6). The contrast-to-background ratio (CBR) of the kidney and ureters, and the MB concentration in the urine, were quantified. Peak MB absorbance, emission, and intensity in urine occurred at 668 nm, 688 nm, and 20 mumol/L, respectively. After intravenous injection, doses as low as 0.1-mg/kg MB provided prolonged imaging of the ureters, and a dose of 0.5 mg/kg provided statistically significant improvement of CBR. The preinjection of furosemide increased urine volume but did not improve CBR. Laparoscopic identification of the ureter using MB NIR fluorescence was demonstrated. Ureteral imaging using MB NIR fluorescence provides sensitive, real-time, intra-operative identification of the ureters during open and laparoscopic surgeries. Copyright 2010 Mosby, Inc. All rights reserved.

  5. Status of FARTECH's Multi-Sensor Real-Time Resistive Wall Mode Identification Project

    NASA Astrophysics Data System (ADS)

    Kim, J. S.; Edgell, D. H.; Bogatu, I. N.; Kim, Y. B.; Humphreys, D. A.; Walker, M. L.; Leuer, J. A.; Turnbull, A. D.

    2002-11-01

    Early detection of resistive wall mode (RWM) identification (ID) is possible utilizing multiple sensors to enhance the signal-to-noise ratio, and mode structure recognition with a pre-modeled numerical structure, assuming similar equilibria to be reproduced. Magnetic probes, and internal and external saddle loops are currently used. The predicted structures are modeled via FARVAC(D.H. Edgell, FARTECH, Inc. Report FT020523, May (2002).) and VACUUM using the GATO linear RWM mode. The RWM structures are then matched to the experimental data in real-time. For better algorithm and understanding of the modes, other sensors such as soft x-ray data are being incorporated in the program. In addition, an equilibrium and stability code is being developed for basic understanding of the RWM physics such as RWM rotation and dissipation. Systematic implementation and communication of our mode identification to the DIII-D PCS are being developed and tested.

  6. Real-Time PCR Assay for the Identification of the Brown Marmorated Stink Bug (Halyomorpha halys)

    PubMed Central

    Dhami, Manpreet K.; Dsouza, Melissa; Waite, David W.; Anderson, Diane; Li, Dongmei

    2016-01-01

    The brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), is a gregarious crop pest that has rapidly spread across the world in the last two decades. It is an excellent hitchhiker species, especially as an over-wintering adult. During this period it is often associated with non-biological commodities such as shipping containers and machinery that travel long distances. Inadequate identification keys and similarity to common species has assisted its spread across Europe, while accurate identification from immature stages or eggs is not possible. We developed a real-time TaqMan PCR assay for the accurate and sensitive detection of the brown marmorated stink bug from all life stages. The assay performance against required diagnostic criterion and within a quarantine framework are described. PMID:26955631

  7. Real-time human identification using a pyroelectric infrared detector array and hidden Markov models.

    PubMed

    Fang, Jian-Shuen; Hao, Qi; Brady, David J; Guenther, Bob D; Hsu, Ken Y

    2006-07-24

    This paper proposes a real-time human identification system using a pyroelectric infrared (PIR) detector array and hidden Markov models (HMMs). A PIR detector array with masked Fresnel lens arrays is used to generate digital sequential data that can represent a human motion feature. HMMs are trained to statistically model the motion features of individuals through an expectation-maximization (EM) learning process. Human subjects are recognized by evaluating a set of new feature data against the trained HMMs using the maximum-likelihood (ML) criterion. We have developed a prototype system to verify the proposed method. Sensor modules with different numbers of detectors and different sampling masks were tested to maximize the identification capability of the sensor system.

  8. Identification of six Listeria species by real-time PCR assay.

    PubMed

    Hage, E; Mpamugo, O; Ohai, C; Sapkota, S; Swift, C; Wooldridge, D; Amar, C F L

    2014-06-01

    The Listeria genus comprises 10 recognized species. Listeria monocytogenes causes listeriosis in humans and other animals primarily via contaminated food or animal feed. Listeria ivanovii causes listeriosis in animals and on rare occasions in humans. The identification of nonpathogenic species of Listeria in foods indicates that conditions exist that support the growth of pathogenic strains and is used to facilitate the implementation of control and prevention measures. This study shows the development and evaluation of a 5'exonuclease real-time PCR assay for the rapid identification of Listeria seeligeri, Listeria welshimeri, L. monocytogenes, L. ivanovii, Listeria grayi and Listeria innocua. The assay consists of two triplexes that were evaluated using 53 cultures of Gram-positive bacteria, including 49 Listeria spp. from human, animal, food or food-processing environments. The assay was rapid, specific and reproducible and could identify each of the six species from a mixture of strains. The developed assay proved to be a powerful means of rapidly identifying Listeria species and could be usefully implemented in busy specialist reference laboratories. The identification of species of Listeria from foods is important to monitor pathogenic strains and facilitates the implementation of control measures. This study shows the development and evaluation of a 5'exonuclease real-time PCR assay for the rapid identification of L. seeligeri, L. welshimeri, L. monocytogenes and L. ivanovii, L. grayi, L. innocua. The developed assay proved to be specific, rapid and reproducible and therefore could be implemented in busy specialist reference laboratories. © 2014 The Society for Applied Microbiology.

  9. Real-time star identification using synthetic radial pattern and its hardware implementation

    NASA Astrophysics Data System (ADS)

    Zhao, Yang; Wei, Xinguo; Wang, Gangyi; Li, Jian

    2017-02-01

    Star identification algorithm has always been the core strategy of star sensors. For the time being, the identification speed is becoming the bottleneck of satisfying real-time requirements (e.g. high-speed maneuvering applications, power breakdown and so on) on the premise of the robustness. To solve this problem, this paper proposes a novel lost-in-space star identification algorithm based on the synthetic radial pattern, which is dedicated to the pipelined parallel architecture of FPGAs (Field Programmable Gate Arrays). The synthetic radial pattern consists of two single radial patterns connected by their two respective polestars. In the algorithm, the polestar-pair is firstly matched and optimum identification results can be obtained after the follow-up radial pattern filtering. Then, the number of spurious matches can be reduced to a much less level and a mathematical model is also developed to demonstrate the efficiency compared with conventional radial algorithms. Simulations show that the approach is very robust towards various noise conditions and the quantified identification time is less than 1 ms at a low resource cost when it is implemented on FPGA platforms.

  10. Real-time skin feature identification in a time-sequential video stream

    NASA Astrophysics Data System (ADS)

    Kramberger, Iztok

    2005-04-01

    Skin color can be an important feature when tracking skin-colored objects. Particularly this is the case for computer-vision-based human-computer interfaces (HCI). Humans have a highly developed feeling of space and, therefore, it is reasonable to support this within intelligent HCI, where the importance of augmented reality can be foreseen. Joining human-like interaction techniques within multimodal HCI could, or will, gain a feature for modern mobile telecommunication devices. On the other hand, real-time processing plays an important role in achieving more natural and physically intuitive ways of human-machine interaction. The main scope of this work is the development of a stereoscopic computer-vision hardware-accelerated framework for real-time skin feature identification in the sense of a single-pass image segmentation process. The hardware-accelerated preprocessing stage is presented with the purpose of color and spatial filtering, where the skin color model within the hue-saturation-value (HSV) color space is given with a polyhedron of threshold values representing the basis of the filter model. An adaptive filter management unit is suggested to achieve better segmentation results. This enables the adoption of filter parameters to the current scene conditions in an adaptive way. Implementation of the suggested hardware structure is given at the level of filed programmable system level integrated circuit (FPSLIC) devices using an embedded microcontroller as their main feature. A stereoscopic clue is achieved using a time-sequential video stream, but this shows no difference for real-time processing requirements in terms of hardware complexity. The experimental results for the hardware-accelerated preprocessing stage are given by efficiency estimation of the presented hardware structure using a simple motion-detection algorithm based on a binary function.

  11. Rapid Qualitative Urinary Tract Infection Pathogen Identification by SeptiFast® Real-Time PCR

    PubMed Central

    Malinka, Thomas; Klaschik, Sven; Weber, Stefan U.; Schewe, Jens-Christian; Stüber, Frank; Book, Malte

    2011-01-01

    Background Urinary tract infections (UTI) are frequent in outpatients. Fast pathogen identification is mandatory for shortening the time of discomfort and preventing serious complications. Urine culture needs up to 48 hours until pathogen identification. Consequently, the initial antibiotic regimen is empirical. Aim To evaluate the feasibility of qualitative urine pathogen identification by a commercially available real-time PCR blood pathogen test (SeptiFast®) and to compare the results with dipslide and microbiological culture. Design of study Pilot study with prospectively collected urine samples. Setting University hospital. Methods 82 prospectively collected urine samples from 81 patients with suspected UTI were included. Dipslide urine culture was followed by microbiological pathogen identification in dipslide positive samples. In parallel, qualitative DNA based pathogen identification (SeptiFast®) was performed in all samples. Results 61 samples were SeptiFast® positive, whereas 67 samples were dipslide culture positive. The inter-methodological concordance of positive and negative findings in the gram+, gram- and fungi sector was 371/410 (90%), 477/492 (97%) and 238/246 (97%), respectively. Sensitivity and specificity of the SeptiFast® test for the detection of an infection was 0.82 and 0.60, respectively. SeptiFast® pathogen identifications were available at least 43 hours prior to culture results. Conclusion The SeptiFast® platform identified bacterial DNA in urine specimens considerably faster compared to conventional culture. For UTI diagnosis sensitivity and specificity is limited by its present qualitative setup which does not allow pathogen quantification. Future quantitative assays may hold promise for PCR based UTI pathogen identification as a supplementation of conventional culture methods. PMID:21359187

  12. REAL-TIME IDENTIFICATION AND CHARACTERIZATION OF ASBESTOS AND CONCRETE MATERIALS WITH RADIOACTIVE CONTAMINATION

    SciTech Connect

    XU, X. George; Zhang, X.C.

    2002-05-10

    Concrete and asbestos-containing materials were widely used in DOE building construction in the 1940s and 1950s. Over the years, many of these porous materials have been contaminated with radioactive sources, on and below the surface. To improve current practice in identifying hazardous materials and in characterizing radioactive contamination, an interdisciplinary team from Rensselaer has conducted research in two aspects: (1) to develop terahertz time-domain spectroscopy and imaging system that can be used to analyze environmental samples such as asbestos in the field, and (2) to develop algorithms for characterizing the radioactive contamination depth profiles in real-time in the field using gamma spectroscopy. The basic research focused on the following: (1) mechanism of generating of broadband pulsed radiation in terahertz region, (2) optimal free-space electro-optic sampling for asbestos, (3) absorption and transmission mechanisms of asbestos in THz region, (4) the role of asbestos sample conditions on the temporal and spectral distributions, (5) real-time identification and mapping of asbestos using THz imaging, (7) Monte Carlo modeling of distributed contamination from diffusion of radioactive materials into porous concrete and asbestos materials, (8) development of unfolding algorithms for gamma spectroscopy, and (9) portable and integrated spectroscopy systems for field testing in DOE. Final results of the project show that the combination of these innovative approaches has the potential to bring significant improvement in future risk reduction and cost/time saving in DOE's D and D activities.

  13. A robust approach to battery fuel gauging, part I: Real time model identification

    NASA Astrophysics Data System (ADS)

    Balasingam, B.; Avvari, G. V.; Pattipati, B.; Pattipati, K. R.; Bar-Shalom, Y.

    2014-12-01

    In this paper, the first of a series of papers on battery fuel gauge (BFG), we present a real time parameter estimation strategy for robust state of charge (SOC) tracking. The proposed parameter estimation scheme has the following novel features: it models hysteresis as an error in the open circuit voltage (OCV) and employs a combination of real time, linear parameter estimation and SOC tracking technique to compensate for it. This obviates the need for modeling of hysteresis as a function of SOC and load current. We identify the presence of correlated noise that has been so far ignored in the literature and use it to enhance the accuracy of model identification. As a departure from the conventional "one model fits all" strategy, we identify four different equivalent models of the battery that represent four modes of typical battery operation and develop the framework for seamless SOC tracking by switching. The proposed parameter approach enables a robust initialization/re-initialization strategy for continuous operation of the BFG. The performance of the online parameter estimation scheme was first evaluated through simulated data. Then, the proposed algorithm was validated using hardware-in-the-loop (HIL) data collected from commercially available Li-ion batteries.

  14. Species identification of white false hellebore (Veratrum album subsp. oxysepalum) using real-time PCR.

    PubMed

    Kikkawa, Hitomi S; Tsuge, Kouichiro; Kubota, Satoshi; Aragane, Masako; Ohta, Hikoto; Sugita, Ritsuko

    2017-03-20

    Food poisoning is frequently caused by the accidental ingestion of toxic plants that possess strong morphological similarities to edible plants. False helleborine (Veratrum album) is one of the most common plants involved in such accidents. In cases of poisoning by toxic plants, rapid and accurate identification, usually based on the morphological or chemical analysis of plant parts, is required for appropriate medical treatment or forensic investigation. However, morphological examinations require experience in systematic botany because the samples are fragmentary, and chemical analysis of natural compounds can be difficult. In this study, we developed a TaqMan real-time PCR method using trnH-psbA and trnL-trnF that could be carried out in 30-60min. The lower detection limit was less than 10pg of DNA and the primer sets were specific to V. album and Veratrum stamineum. Mixed samples, cooked samples, and simulated gastric contents were successfully identified, and a multiplex assay of two regions was also possible. These results indicate that the TaqMan real-time PCR analysis is a very effective method to detect small samples of V. album and V. stamineum accurately and rapidly in poisoning cases.

  15. A novel algorithm for real-time adaptive signal detection and identification

    SciTech Connect

    Sleefe, G.E.; Ladd, M.D.; Gallegos, D.E.; Sicking, C.W.; Erteza, I.A.

    1998-04-01

    This paper describes a novel digital signal processing algorithm for adaptively detecting and identifying signals buried in noise. The algorithm continually computes and updates the long-term statistics and spectral characteristics of the background noise. Using this noise model, a set of adaptive thresholds and matched digital filters are implemented to enhance and detect signals that are buried in the noise. The algorithm furthermore automatically suppresses coherent noise sources and adapts to time-varying signal conditions. Signal detection is performed in both the time-domain and the frequency-domain, thereby permitting the detection of both broad-band transients and narrow-band signals. The detection algorithm also provides for the computation of important signal features such as amplitude, timing, and phase information. Signal identification is achieved through a combination of frequency-domain template matching and spectral peak picking. The algorithm described herein is well suited for real-time implementation on digital signal processing hardware. This paper presents the theory of the adaptive algorithm, provides an algorithmic block diagram, and demonstrate its implementation and performance with real-world data. The computational efficiency of the algorithm is demonstrated through benchmarks on specific DSP hardware. The applications for this algorithm, which range from vibration analysis to real-time image processing, are also discussed.

  16. Identification of pork in meat products using real-time loop-mediated isothermal amplification.

    PubMed

    Yang, Lixia; Fu, Shujun; Peng, Xinkai; Li, Le; Song, Taoping; Li, Lin

    2014-09-03

    In this study, a one-step, real-time, loop-mediated isothermal amplification (RealAmp) assay was developed, for the highly specific detection of pork DNA. For the assay, the mtDNA of cytochrome b (cytb) gene was amplified at 63 °C using SYBR Green I for 45 min with a Real-Time Polymerase Chain Reaction (PCR) System that measured the fluorescent signal at one-minute intervals. As little as 1 pg of template DNA could be detected, without any cross-reactivity with non-target species. Meat mixtures, heat-treated at 100 °C for 15 min, prepared by mixing pork meat with beef at different ratios (0.01%-10%) were tested, and the RealAmp assays allowed the detection of as little as 0.01% pork in the meat mixtures. Thus, this work showed that RealAmp could be used for specific identification and sensitive quantification of meat species, even for heat-treated meat products.

  17. Molecular detection and identification of Aspergillus spp. from clinical samples using real-time PCR.

    PubMed

    Ramírez, Mercedes; Castro, Carmen; Palomares, José Carlos; Torres, M José; Aller, Ana Isabel; Ruiz, Maite; Aznar, Javier; Martín-Mazuelos, Estrella

    2009-03-01

    The definite and rapid diagnosis of invasive aspergillosis is necessary because of the high mortality caused. The objective of this study was to evaluate a real-time PCR assay to detect Aspergillus spp. in clinical samples, based on the Light Cycler technology. Specificity was assessed by using DNA extracted from pathogenic and non-pathogenic bacteria/fungi from Spanish Collection including: two Aspergillus flavus, four Aspergillus fumigatus, two Aspergillus nidulans, two Aspergillus niger and two Aspergillus terreus isolates. The analytical sensitivity was evaluated with different inocula (10(1)-10(5) conidia ml(-1)), and serially diluted DNA of A. fumigatus. To assess clinical applicability, samples from patients at risk were analysed. Species identification was determined by analysing the melting curves. Reactions using genomic DNA from other species of different genera than Aspergillus were negative (specificity: 100%). Analytical sensitivity was 60 fg using DNA and 5-20 conidia using conidial suspensions. The linear range was from 60 to 6 x 10(7) fg. The Tm ranged from 67.34 to 70.7 degrees C for the different Aspergillus spp. studied. Nine hundred and forty-eight consecutive blood samples from 127 patients were processed. In total, 10 (1%) of 948 samples from blood samples were PCR-positive. The real-time PCR assay provides a high sensitivity and specificity for detection of fungal DNA and rapidly identifies most of clinically relevant Aspergillus species.

  18. Identification of pork in meat products using real-time loop-mediated isothermal amplification

    PubMed Central

    Yang, Lixia; Fu, Shujun; Peng, Xinkai; Li, Le; Song, Taoping; Li, Lin

    2014-01-01

    In this study, a one-step, real-time, loop-mediated isothermal amplification (RealAmp) assay was developed, for the highly specific detection of pork DNA. For the assay, the mtDNA of cytochrome b (cytb) gene was amplified at 63 °C using SYBR Green I for 45 min with a Real-Time Polymerase Chain Reaction (PCR) System that measured the fluorescent signal at one-minute intervals. As little as 1 pg of template DNA could be detected, without any cross-reactivity with non-target species. Meat mixtures, heat-treated at 100 °C for 15 min, prepared by mixing pork meat with beef at different ratios (0.01%–10%) were tested, and the RealAmp assays allowed the detection of as little as 0.01% pork in the meat mixtures. Thus, this work showed that RealAmp could be used for specific identification and sensitive quantification of meat species, even for heat-treated meat products. PMID:26019573

  19. Real-time aircraft structural damage identification with flight condition variations

    NASA Astrophysics Data System (ADS)

    Lew, Jiann-Shiun; Loh, Chin-Hsiung

    2012-04-01

    This paper presents a real-time structural damage identification method for aircraft with flight condition variations. The proposed approach begins by identifying the dynamic models under various test conditions from time-domain input/output data. A singular value decomposition technique is then used to characterize and quantify the parameter uncertainties from the identified models. The uncertainty coordinates, corresponding to the identified principal directions, of the identified models are computed, and the residual errors between the identified uncertainty coordinates and the estimated uncertainty coordinates of the health structure are used to identify damage status. A correlation approach is applied to identify damage type and intensity, based on the difference between the identified parameters and the estimated parameters of the healthy structure. The proposed approach is demonstrated by application to the Benchmark Active Controls Technology (BACT) wind-tunnel model.

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Development of a real-time PCR method for the identification of Atlantic mackerel (Scomber scombrus).

    PubMed

    Velasco, Amaya; Sánchez, Ana; Martínez, Icíar; Santaclara, Francisco J; Pérez-Martín, Ricardo I; Sotelo, Carmen G

    2013-12-01

    A Real Time-PCR method based on TaqMan technology for the identification of Scomber scombrus has been developed. A system of specific primers and a Minor Groove Binding (MGB) TaqMan probe based on sequences of the mitochondrial cytochrome b region was designed. The method was successfully tested in 81 specimens of S. scombrus and related species and validated in 26 different commercial samples. An average Threshold Cycle (Ct) value of 15.3 was obtained with S. scombrus DNA. With the other species tested fluorescence signal was not detected or Ct was significantly higher (P<0.001). The efficiency of the assay was estimated to be 92.41%, with 100% specificity, and no cross reactivity was detected with any other species. These results reveal that the developed method is a rapid and efficient tool to unequivocally identify S. scombrus and may aid in the prevention of fraud or mislabelling in mackerel products.

  2. Detection of dermcidin for sweat identification by real-time RT-PCR and ELISA.

    PubMed

    Sakurada, Koichi; Akutsu, Tomoko; Fukushima, Hisayo; Watanabe, Ken; Yoshino, Mineo

    2010-01-30

    We evaluated the performance of real-time RT-PCR and ELISA assays for detection of dermcidin (DCD) in sweat and body-fluid stains. DCD, a small antibiotic peptide secreted into human sweat, was detected by real-time RT-PCR in 7-day-old stains containing as small as 10 microL of sweat, and the assay showed high specificity when testing 7-day-old stains containing 30 microL of other body-fluid. ELISA using anti-human dermcidin mouse monoclonal antibody detected DCD sweat diluted up to approximately 10,000-fold and could specifically detect DCD in 10 microL of body-fluid stains. The performance of the two assays was tested during winter on samples that simulated forensic case samples: an undershirt and a sock worn for 20 h, a handkerchief used to wipe the brow several times within 12h, a cap and a cotton glove worn for 4h, and a white robe worn at intervals for 2 years. The result showed that the former assay detected DCD in all sites of the undershirt examined (armpit, back, and breast), and the latter gave a relatively high OD value in the armpit among the three sites. For the socks, although the latter assay gave very high OD values in both the center and toe of the foot sole, the former could not detect DCD in both of them. These results indicate that highly damp conditions, such as inside a shoe, might promote the degradation of mRNA in samples such as socks. In the other case samples, sweat was adequately detected by both assays. This study is the first demonstration of the use of real-time RT-PCR to sensitively identify sweat among body-fluid stains, and it confirmed that dermcidin was an excellent marker for sweat identification. In addition, the usefulness of ELISA was also verified. Positive sweat identification using these assays is expected to assist forensic practice. 2009 Elsevier Ireland Ltd. All rights reserved.

  3. Identification of five highly priced tuna species by quantitative real-time polymerase chain reaction.

    PubMed

    Liu, Shasha; Xu, Kunhua; Wu, Zhigang; Xie, Xiao; Feng, Junli

    2016-09-01

    Tunas are economically important fishery worldwide, and are often used for commercial processed production. For effective fishery management and protection of consumers' rights, it is important to develop a molecular method to identify species in canned tuna products rapidly and reliably. Here, we have developed a duplex quantitative real-time PCR (qPCR) for identification of five highly priced tuna species (Thunnus maccoyii, Thunnus obesus, Thunnus albacares, Thunnus alalunga and Katsuwonus pelamis) from processed as well as fresh fish. After amplification and sequencing of seven genetic markers commonly used for species identification, 16S rDNA and control region (CR) of mitochondrial DNA were selected as the reference gene markers for genus Thunnus and tuna species identification, respectively. Subsequently, a 73 bp fragment of 16S rDNA and 85-99 bp fragment of CR were simultaneously amplified from each target species by qPCR. The qPCR efficiency of each reaction was calculated according to the standard curves, and the method was validated by amplification DNA extracted from single or mixed tuna specimen. The developed duplex qPCR system was applied to authenticate species of 14 commercial tuna products successfully, which demonstrated it was really a useful and academic technique to identify highly priced tuna species.

  4. Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR.

    PubMed

    Logan, J M; Edwards, K J; Saunders, N A; Stanley, J

    2001-06-01

    We describe rapid PCR-biprobe identification of Campylobacter spp. This is based on real-time PCR with product analysis in the same system. The assay identifies enteropathogenic campylobacters to the species level on the basis of their degree of hybridization to three 16S ribosomal DNA (rDNA) biprobes. First-round symmetric PCR is performed with genus-specific primers which selectively target and amplify a portion of the 16S rRNA gene common to all Campylobacter species. Second-round asymmetric PCR is performed in a LightCycler in the presence of one of three biprobes; the identity of an amplified DNA-biprobe duplex is established after determination of the species-specific melting peak temperature. The biprobe specificities were determined by testing 37 reference strains of Campylobacter, Helicobacter, and Arcobacter spp. and 59 Penner serotype reference strains of Campylobacter jejuni and C. coli. From the combination of melting peak profiles for each probe, an identification scheme was devised which accurately detected the five taxa pathogenic for humans (C. jejuni/C. coli, C. lari, C. upsaliensis, C. hyointestinalis, and C. fetus), as well as C. helveticus and C. lanienae. The assay was evaluated with 110 blind-tested field isolates; when the code was broken their previous phenotypic species identification was confirmed in every case. The PCR-biprobe assay also identified campylobacters directly from fecal DNA. PCR-biprobe testing of stools from 38 diarrheic subjects was 100% concordant with PCR-enzyme-linked immunosorbent assay identification (13, 20) and thus more sensitive than phenotypic identification following microaerobic culture.

  5. Rapid Identification of Campylobacter spp. by Melting Peak Analysis of Biprobes in Real-Time PCR

    PubMed Central

    Logan, J. M. J.; Edwards, K. J.; Saunders, N. A.; Stanley, J.

    2001-01-01

    We describe rapid PCR-biprobe identification of Campylobacter spp.. This is based on real-time PCR with product analysis in the same system. The assay identifies enteropathogenic campylobacters to the species level on the basis of their degree of hybridization to three 16S ribosomal DNA (rDNA) biprobes. First-round symmetric PCR is performed with genus-specific primers which selectively target and amplify a portion of the 16S rRNA gene common to all Campylobacter species. Second-round asymmetric PCR is performed in a LightCycler in the presence of one of three biprobes; the identity of an amplified DNA-biprobe duplex is established after determination of the species-specific melting peak temperature. The biprobe specificities were determined by testing 37 reference strains of Campylobacter, Helicobacter, and Arcobacter spp. and 59 Penner serotype reference strains of Campylobacter jejuni and C. coli. From the combination of melting peak profiles for each probe, an identification scheme was devised which accurately detected the five taxa pathogenic for humans (C. jejuni/C. coli, C. lari, C. upsaliensis, C. hyointestinalis, and C. fetus), as well as C. helveticus and C. lanienae. The assay was evaluated with 110 blind-tested field isolates; when the code was broken their previous phenotypic species identification was confirmed in every case. The PCR-biprobe assay also identified campylobacters directly from fecal DNA. PCR-biprobe testing of stools from 38 diarrheic subjects was 100% concordant with PCR–enzyme-linked immunosorbent assay identification (13, 20) and thus more sensitive than phenotypic identification following microaerobic culture. PMID:11376061

  6. Identification of optimal operating point of PV modules using neural network for real time maximum power tracking control

    SciTech Connect

    Hiyama, Takashi, Kouzuma, Shinichi; Imakubo, Tomofumi

    1995-06-01

    This paper presents an application of a neutral network for the identification of the optimal operating point of PV modules for the real time maximum power tracking control. The output power from the modules depends on the environmental factors such as insolation, cell temperature, and so on. Therefore, accurate identification of optimal operating point and real time continuous control are required to achieve the maximum output efficiency. The proposed neural network has a quite simple structure and provides a highly accurate identification of the optimal operating point and also a highly accurate estimation of the maximum power from the PV modules.

  7. Dynamic fuel cell stack model for real-time simulation based on system identification

    NASA Astrophysics Data System (ADS)

    Meiler, M.; Schmid, O.; Schudy, M.; Hofer, E. P.

    The authors have been developing an empirical mathematical model to predict the dynamic behaviour of a polymer electrolyte membrane fuel cell (PEMFC) stack. Today there is a great number of models, describing steady-state behaviour of fuel cells by estimating the equilibrium voltage for a certain set of operating parameters, but models capable of predicting the transient process between two steady-state points are rare. However, in automotive applications round about 80% of operating situations are dynamic. To improve the reliability of fuel cell systems by model-based control for real-time simulation dynamic fuel cell stack model is needed. Physical motivated models, described by differential equations, usually are complex and need a lot of computing time. To meet the real-time capability the focus is set on empirical models. Fuel cells are highly nonlinear systems, so often used auto-regressive (AR), output-error (OE) or Box-Jenkins (BJ) models do not accomplish satisfying accuracy. Best results are achieved by splitting the behaviour into a nonlinear static and a linear dynamic subsystem, a so-called Uryson-Model. For system identification and model validation load steps with different amplitudes are applied to the fuel cell stack at various operation points and the voltage response is recorded. The presented model is implemented in MATLAB environment and has a computing time of less than 1 ms per step on a standard desktop computer with a 2.8 MHz CPU and 504 MB RAM. Lab tests are carried out at DaimlerChrysler R&D Centre with DaimlerChrysler PEMFC hardware and a good agreement is found between model simulations and lab tests.

  8. Toward real-time tumor margin identification in image-guided robotic brain tumor resection

    NASA Astrophysics Data System (ADS)

    Hu, Danying; Jiang, Yang; Belykh, Evgenii; Gong, Yuanzheng; Preul, Mark C.; Hannaford, Blake; Seibel, Eric J.

    2017-03-01

    For patients with malignant brain tumors (glioblastomas), a safe maximal resection of tumor is critical for an increased survival rate. However, complete resection of the cancer is hard to achieve due to the invasive nature of these tumors, where the margins of the tumors become blurred from frank tumor to more normal brain tissue, but in which single cells or clusters of malignant cells may have invaded. Recent developments in fluorescence imaging techniques have shown great potential for improved surgical outcomes by providing surgeons intraoperative contrast-enhanced visual information of tumor in neurosurgery. The current near-infrared (NIR) fluorophores, such as indocyanine green (ICG), cyanine5.5 (Cy5.5), 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX), are showing clinical potential to be useful in targeting and guiding resections of such tumors. Real-time tumor margin identification in NIR imaging could be helpful to both surgeons and patients by reducing the operation time and space required by other imaging modalities such as intraoperative MRI, and has the potential to integrate with robotically assisted surgery. In this paper, a segmentation method based on the Chan-Vese model was developed for identifying the tumor boundaries in an ex-vivo mouse brain from relatively noisy fluorescence images acquired by a multimodal scanning fiber endoscope (mmSFE). Tumor contours were achieved iteratively by minimizing an energy function formed by a level set function and the segmentation model. Quantitative segmentation metrics based on tumor-to-background (T/B) ratio were evaluated. Results demonstrated feasibility in detecting the brain tumor margins at quasi-real-time and has the potential to yield improved precision brain tumor resection techniques or even robotic interventions in the future.

  9. Real-time instrument detection in minimally invasive surgery using radiofrequency identification technology.

    PubMed

    Kranzfelder, Michael; Schneider, Armin; Fiolka, Adam; Schwan, Elena; Gillen, Sonja; Wilhelm, Dirk; Schirren, Rebecca; Reiser, Silvano; Jensen, Brian; Feussner, Hubertus

    2013-12-01

    A key part of surgical workflow recording is recognition of the instrument in use. We present a radiofrequency identification (RFID)-based approach for real-time tracking of laparoscopic instruments. The system consists of RFID-tagged instruments and an antenna unit positioned on the Mayo stand. For reliability analysis, RFID tracking data were compared with the assessment of the perioperative video data of instrument changes (the reference standard for instrument application detection) in 10 laparoscopic cholecystectomies. When the tagged instrument was on the Mayo stand, it was referred to as "not in use." Once it was handed to the surgeon, it was considered to be "in use." Temporal miscounts (incorrect number of instruments "in use") were analyzed. The surgeons and scrub nurses completed a questionnaire after each operation for individual system evaluation. A total of 110 distinct instrument applications ("in use" versus "not in use") were eligible for analysis. No RFID tag failure occurred. The RFID detection rates were consistent with the period of effective instrument application. The delay in instrument detection was 4.2 ± 1.7 s. The highest percentage of temporal miscounts occurred during phases with continuous application of coagulation current. Surgeons generally rated the system better than the scrub nurses (P = 0.54). The feasibility of RFID-based real-time instrument detection was successfully proved in our study, with reliable detection results during laparoscopic cholecystectomy. Thus, RFID technology has the potential to be a valuable additional tool for surgical workflow recognition that could enable a situation dependent assistance of the surgeon in the future. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. The development of a near-real time hail damage swath identification algorithm for vegetation

    NASA Astrophysics Data System (ADS)

    Bell, Jordan R.

    The central United States is primarily covered in agricultural lands with a growing season that peaks during the same time as the region's climatological maximum for severe weather. These severe thunderstorms can bring large hail that can cause extensive areas of crop damage, which can be difficult to survey from the ground. Satellite remote sensing can help with the identification of these damaged areas. This study examined three techniques for identifying damage using satellite imagery that could be used in the development of a near-real time algorithm formulated for the detection of damage to agriculture caused by hail. The three techniques: a short term Normalized Difference Vegetation Index (NDVI) change product, a modified Vegetation Health Index (mVHI) that incorporates both NDVI and land surface temperature (LST), and a feature detection technique based on NDVI and LST anomalies were tested on a single training case and five case studies. Skill scores were computed for each of the techniques during the training case and each case study. Among the best-performing case studies, the probability of detection (POD) for the techniques ranged from 0.527 - 0.742. Greater skill was noted for environments that occurred later in the growing season over areas where the land cover was consistently one or two types of uniform vegetation. The techniques struggled in environments where the land cover was not able to provide uniform vegetation, resulting in POD of 0.067 - 0.223. The feature detection technique was selected to be used for the near-real-time algorithm, based on the consistent performance throughout the entire growing season.

  11. Identification of four squid species by quantitative real-time polymerase chain reaction.

    PubMed

    Ye, Jian; Feng, Junli; Liu, Shasha; Zhang, Yanping; Jiang, Xiaona; Dai, Zhiyuan

    2016-02-01

    Squids are distributed worldwide, including many species of commercial importance, and they are often made into varieties of flavor foods. The rapid identification methods for squid species especially their processed products, however, have not been well developed. In this study, quantitative real-time PCR (qPCR) systems based on specific primers and TaqMan probes have been established for rapid and accurate identification of four common squid species (Ommastrephes bartramii, Dosidicus gigas, Illex argentinus, Todarodes pacificus) in Chinese domestic market. After analyzing mitochondrial genes reported in GenBank, the mitochondrial cytochrome b (Cytb) gene was selected for O. bartramii detection, cytochrome c oxidase subunit I (COI) gene for D. gigas and T. Pacificus detection, ATPase subunit 6 (ATPase 6) gene for I. Argentinus detection, and 12S ribosomal RNA (12S rDNA) gene for designing Ommastrephidae-specific primers and probe. As a result, all the TaqMan systems are of good performance, and efficiency of each reaction was calculated by making standard curves. This method could detect target species either in single or mixed squid specimen, and it was applied to identify 12 squid processed products successfully. Thus, it would play an important role in fulfilling labeling regulations and squid fishery control. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Water monitoring: automated and real time identification and classification of algae using digital microscopy.

    PubMed

    Coltelli, Primo; Barsanti, Laura; Evangelista, Valtere; Frassanito, Anna Maria; Gualtieri, Paolo

    2014-11-01

    Microalgae are unicellular photoautotrophs that grow in any habitat from fresh and saline water bodies, to hot springs and ice. Microalgae can be used as indicators to monitor water ecosystem conditions. These organisms react quickly and predictably to a broad range of environmental stressors, thus providing early signals of a changing environment. When grown extensively, microalgae may produce harmful effects on marine or freshwater ecology and fishery resources. Rapid and accurate recognition and classification of microalgae is one of the most important issues in water resource management. In this paper, a methodology for automatic and real time identification and enumeration of microalgae by means of image analysis is presented. The methodology is based on segmentation, shape feature extraction, pigment signature determination and neural network grouping; it attained 98.6% accuracy from a set of 53,869 images of 23 different microalgae representing the major algal phyla. In our opinion this methodology partly overcomes the lack of automated identification systems and is on the forefront of developing a computer-based image processing technique to automatically detect, recognize, identify and enumerate microalgae genera and species from all the divisions. This methodology could be useful for an appropriate and effective water resource management.

  13. Development of a Near-Real Time Hail Damage Swath Identification Algorithm for Vegetation

    NASA Technical Reports Server (NTRS)

    Bell, Jordan R.; Molthan, Andrew L.; Schultz, Lori A.; McGrath, Kevin M.; Burks, Jason E.

    2015-01-01

    The Midwest is home to one of the world's largest agricultural growing regions. Between the time period of late May through early September, and with irrigation and seasonal rainfall these crops are able to reach their full maturity. Using moderate to high resolution remote sensors, the monitoring of the vegetation can be achieved using the red and near-infrared wavelengths. These wavelengths allow for the calculation of vegetation indices, such as Normalized Difference Vegetation Index (NDVI). The vegetation growth and greenness, in this region, grows and evolves uniformly as the growing season progresses. However one of the biggest threats to Midwest vegetation during the time period is thunderstorms that bring large hail and damaging winds. Hail and wind damage to crops can be very expensive to crop growers and, damage can be spread over long swaths associated with the tracks of the damaging storms. Damage to the vegetation can be apparent in remotely sensed imagery and is visible from space after storms slightly damage the crops, allowing for changes to occur slowly over time as the crops wilt or more readily apparent if the storms strip material from the crops or destroy them completely. Previous work on identifying these hail damage swaths used manual interpretation by the way of moderate and higher resolution satellite imagery. With the development of an automated and near-real time hail swath damage identification algorithm, detection can be improved, and more damage indicators be created in a faster and more efficient way. The automated detection of hail damage swaths will examine short-term, large changes in the vegetation by differencing near-real time eight day NDVI composites and comparing them to post storm imagery from the Moderate Resolution Imaging Spectroradiometer (MODIS) aboard Terra and Aqua and Visible Infrared Imaging Radiometer Suite (VIIRS) aboard Suomi NPP. In addition land surface temperatures from these instruments will be examined as

  14. Validation of a real-time PCR assay for the molecular identification of Mycobacterium tuberculosis

    PubMed Central

    Sales, Mariana L.; Fonseca, Antônio Augusto; Orzil, Lívia; Alencar, Andrea Padilha; Silva, Marcio Roberto; Issa, Marina Azevedo; Filho, Paulo Martins Soares; Lage, Andrey Pereira; Heinemann, Marcos Bryan

    2014-01-01

    Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p < 0.001, CI = 0.84 – 1.0). The diagnostic sensitivity and specificity were 100% (CI = 95.94% – 100%) and 100% (CI = 93.98% – 100%). This qPCR was developed with the goal of diagnosing the bacillus M. tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method. PMID:25763042

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

    PubMed Central

    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. PMID:25798897

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

    PubMed

    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.

  17. Extensible multiplex real-time PCR for rapid bacterial identification with carbon nanotube composite microparticles.

    PubMed

    Jung, Seungwon; Kim, Jungmin; Kim, Junsun; Yang, Sang Hwa; Kim, Sang Kyung

    2017-03-01

    The early diagnosis of pathogenic bacteria is significant for bacterial identification and antibiotic resistance. Implementing rapid, sensitive, and specific detection, molecular diagnosis has been considered complementary to the conventional bacterial culture. Composite microparticles of a primer-immobilized network (cPIN) are developed for multiplex detection of pathogenic bacteria with real-time polymerase chain reaction (qPCR). A pair of specific primers are incorporated and stably conserved in a cPIN particle. One primer is crosslinked to the polymer network, and the other is bound to carbon nanotubes (CNTs) in the particle. At the initiation of qPCR, the latter primer is released from the CNTs and participates in the amplification. The amplification efficiency of this cPIN qPCR is estimated at more than 90% with suppressed non-specific signals from complex samples. In multiplexing, four infective pathogens are successfully discriminated using this cPIN qPCR. Multiplex qPCR conforms with the corresponding singleplex assays, proving independent amplification in each particle. Four bacterial targets from clinical samples are differentially analyzed in 30min of a single qPCR trial with multiple cPIN particles.

  18. RAPHIDOPHYCEAE [CHADEFAUD EX SILVA] SYSTEMATICS AND RAPID IDENTIFICATION: SEQUENCE ANALYSES AND REAL-TIME PCR ASSAYS

    PubMed Central

    Bowers, Holly A.; Tomas, Carmelo; Tengs, Torstein; Kempton, Jason W.; Lewitus, Alan J.; Oldach, David W.

    2010-01-01

    Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real-time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small-subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species. PMID:20411032

  19. Validation of a real-time PCR assay for the molecular identification of Mycobacterium tuberculosis.

    PubMed

    Sales, Mariana L; Fonseca Júnior, Antônio Augusto; Orzil, Lívia; Alencar, Andrea Padilha; Silva, Marcio Roberto; Issa, Marina Azevedo; Soares Filho, Paulo Martins; Lage, Andrey Pereira; Heinemann, Marcos Bryan

    2014-01-01

    Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p < 0.001, CI = 0.84 - 1.0). The diagnostic sensitivity and specificity were 100% (CI = 95.94% - 100%) and 100% (CI = 93.98% - 100%). This qPCR was developed with the goal of diagnosing the bacillus M. tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method.

  20. Rapid identification of Salmonella enterica subsp. arizonae and S. enterica subsp. diarizonae by real-time polymerase chain reaction.

    PubMed

    Hopkins, Katie L; Peters, Tansy M; Lawson, Andy J; Owen, Robert J

    2009-08-01

    Reptiles are popular as pets, leading to an increased risk of human infections due to uncommon Salmonella strains including the Arizona group (subspecies arizonae and diarizonae). We present a real-time Arizona-specific polymerase chain reaction demonstrating 100% specificity and 99.6% sensitivity, offering savings in time and labor over traditional identification methods.

  1. Multiplex real-time PCR assays for the identification of the potato cyst and tobacco cyst nematodes

    USDA-ARS?s Scientific Manuscript database

    TaqMan primer-probe sets were developed for the detection and identification of potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis using two-tube, multiplex real-time PCR. One tube contained a primer-probe set specific for G. pallida (pale cyst nematode) multiplexed with another prim...

  2. [Development of a real-time polymerase chain reaction method for the identification of Candida species].

    PubMed

    Ağca, Harun; Dalyan Cilo, Burcu; Özmerdiven, Gülşah Ece; Sağlam, Sezcan; Ener, Beyza

    2015-01-01

    Candida species are one of the major causes of nosocomial infections and are the fourth most common agent involved in bloodstream infections. The impact of non-albicans Candida species is increasing, however C.albicans is still the most common species. Since the antifungal susceptibility pattern among Candida spp. may be different, rapid diagnosis and identification of non-albicans Candida spp. are important for the determination of antifungal agents that will be used for treatment. The aim of the study was to describe a real-time polymerase chain reaction (Rt-PCR) assay that rapidly detects, identifies and quantitates Candida species from blood culture samples. A total of 50 consecutive positive blood culture bottles (BACTEC, Beckton Dickinson, USA) identified at our laboratory between June-November 2013, were included in the study. Reference strains of Candida spp. (C.albicans ATCC 10231, C.glabrata ATCC 90030, C.tropicalis ATCC 1021, C.krusei ATCC 6258, C.parapsilosis ATCC 22019 and C. dubliniensis CD36) grown on Sabouraud dextrose agar were used for quality control. BACTEC bottles that were positive for Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were also studied to search the cross-reactivity. A commercial kit (Zymo Research, USA) was used for DNA extraction. Real-time PCR was performed on LightCycler 480 (Roche, Germany) with primers and probes specific for 18S rRNA of Candida species. Twenty microlitres of the reaction mix contained 2 μl of extracted DNA, 2 μl of LightCycler Fast Start DNA Master Probe (Roche Diagnostics, Germany), 2 μl of MgCl(2) (5 mmol), 2 μl of 10x PCR buffer (Roche Diagnostics, Germany), 0.5 μl of each primer (0.01 nmol/μl) and 1 μl of each probe (0.1 μmol/μl) (TibMolBiol, Germany). Amplification was performed using the following conditions; 95°C for 10 mins and 50 cycles of denaturation at 95°C for 10 secs, annealing at 62°C for 10 secs and polymerisation at 72°C for 20 secs. A melting curve was

  3. Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

    PubMed Central

    Alam, Mohammad J.; Tisdel, Naradah L.; Shah, Dhara N.; Yapar, Mehmet; Lasco, Todd M.; Garey, Kevin W.

    2015-01-01

    Background The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. Methods The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. Results A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. Conclusions The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run. PMID:25932438

  4. SNSMIL, a real-time single molecule identification and localization algorithm for super-resolution fluorescence microscopy

    PubMed Central

    Tang, Yunqing; Dai, Luru; Zhang, Xiaoming; Li, Junbai; Hendriks, Johnny; Fan, Xiaoming; Gruteser, Nadine; Meisenberg, Annika; Baumann, Arnd; Katranidis, Alexandros; Gensch, Thomas

    2015-01-01

    Single molecule localization based super-resolution fluorescence microscopy offers significantly higher spatial resolution than predicted by Abbe’s resolution limit for far field optical microscopy. Such super-resolution images are reconstructed from wide-field or total internal reflection single molecule fluorescence recordings. Discrimination between emission of single fluorescent molecules and background noise fluctuations remains a great challenge in current data analysis. Here we present a real-time, and robust single molecule identification and localization algorithm, SNSMIL (Shot Noise based Single Molecule Identification and Localization). This algorithm is based on the intrinsic nature of noise, i.e., its Poisson or shot noise characteristics and a new identification criterion, QSNSMIL, is defined. SNSMIL improves the identification accuracy of single fluorescent molecules in experimental or simulated datasets with high and inhomogeneous background. The implementation of SNSMIL relies on a graphics processing unit (GPU), making real-time analysis feasible as shown for real experimental and simulated datasets. PMID:26098742

  5. Identification of nasal blood by real-time RT-PCR.

    PubMed

    Sakurada, Koichi; Akutsu, Tomoko; Watanabe, Ken; Yoshino, Mineo

    2012-07-01

    A new approach for the identification of body fluid stains by comparing specific mRNA expression levels has been extensively studied in recent years. Here, we examine whether nasal blood, which is regarded as one of the most difficult types of blood to identify, can be identified by comparing mRNA expression levels of target genes specific to saliva, nasal secretion, and blood. The saliva-specific statherin gene (STATH) was found to be expressed at high levels in not only saliva (dCt value: 1.32±1.39, n=5), but also nasal secretions (dCt value: 0.90±1.14, n=5), while the histatin gene (HTN3) was only expressed at high levels in saliva (dCt value: 1.08±2.35, n=5). We also confirmed that the hemoglobin-beta gene (HBB) showed high expression levels in blood (dCt value: -9.51±0.40, n=5). Four nasal blood stains were found to highly express STATH (dCt value: 5.65±3.98) and HBB (dCt value: -8.79±1.67) but not HTN3, suggesting that the stain samples contained both nasal secretions and blood and can therefore be identified as nasal blood stains. Although menstrual blood showed the same expression pattern as nasal blood, the menstrual blood-specific protein matrix metallopeptidase 7 (MMP7) was not expressed in all nasal blood stain samples. Therefore, its expression levels could be used to discriminate between nasal and menstrual blood. In conclusion, real-time RT-PCR was able to identify nasal blood, although the stability of gene expression in nasal blood stains was low over time, suggesting that this assay may not be effective for older stains. Future work should examine the usefulness of this assay under various environmental conditions.

  6. Real-time PCR for Leishmania species identification: Evaluation and comparison with classical techniques.

    PubMed

    de Morais, Rayana Carla Silva; da Costa Oliveira, Cintia Nascimento; de Albuquerque, Suênia da Cunha Gonçalves; Mendonça Trajano Silva, Lays Adrianne; Pessoa-E-Silva, Rômulo; Alves da Cruz, Heidi Lacerda; de Brito, Maria Edileuza Felinto; de Paiva Cavalcanti, Milena

    2016-06-01

    Cutaneous leishmaniasis (CL) is a parasitic disease caused by various Leishmania species. Several studies have shown that real time quantitative PCR (qPCR) can be used for Leishmania spp. identification by analyzing the melting temperature (Tm). Thus, the aim of this study was to evaluate the viability of qPCR for differentiating eight closely related Leishmania species that cause the same clinical form of the disease and to compare the results with classical techniques. qPCR assays for standardizing the Tm using reference strains were performed. After the CL diagnosis on blood samples of domestic animals, positive samples were analyzed by their Tm and qPCR products were purified and sequenced. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by Tm. A Restriction Fragment Length Polymorphism (RFLP) assay, a reference test, was also standardized, by using the reference strains. Through standardization of Tm for Leishmania spp., two Tm ranges were created for analysis: 1 (Tm = 78-79.99 °C) included Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis and Leishmania (V.) shawi; and 2 (Tm = 80-82.2 °C) included Leishmania (V.) naiffi, Leishmania (L.) amazonensis and Leishmania (L.) mexicana. A total of 223 positive blood samples were analyzed, with 58 included in range 1 and 165 in range 2. L. (V.) braziliensis, L. (V.) panamensis and L. (V.) guyanensis were identified by sequencing, while L. (V.) braziliensis, L. (L.) mexicana and L. (V.) panamensis were identified by RFLP analysis. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by qPCR Tm analysis; five were classified in range 1 and five in range 2. A concordance of 80% was calculated between qPCR and the gold-standard (MLEE) with no significant difference between the methods (p = 0.6499); a similar result was observed for sequencing

  7. Real Time Network Assessment

    DTIC Science & Technology

    2013-07-12

    Demonstrate a simple system Conduct a feasibility assessment of data storage, maintenance, and integration requirements Test a web-based data feed...Real Time Network Assessment Prototype We demonstrated the feasibility of linking near real time network analytics to mashups and web- based...combining similar concepts into single node) Stemmers Thesauri application Network position Statistical common patterns Pronoun identification

  8. Custom FPGA processing for real-time fetal ECG extraction and identification.

    PubMed

    Torti, E; Koliopoulos, D; Matraxia, M; Danese, G; Leporati, F

    2017-01-01

    Monitoring the fetal cardiac activity during pregnancy is of crucial importance for evaluating fetus health. However, there is a lack of automatic and reliable methods for Fetal ECG (FECG) monitoring that can perform this elaboration in real-time. In this paper, we present a hardware architecture, implemented on the Altera Stratix V FPGA, capable of separating the FECG from the maternal ECG and to correctly identify it. We evaluated our system using both synthetic and real tracks acquired from patients beyond the 20th pregnancy week. This work is part of a project aiming at developing a portable system for FECG continuous real-time monitoring. Its characteristics of reduced power consumption, real-time processing capability and reduced size make it suitable to be embedded in the overall system, that is the first proposed exploiting Blind Source Separation with this technology, to the best of our knowledge.

  9. Development of a Near Real-Time Hail Damage Swath Identification Algorithm for Vegetation

    NASA Technical Reports Server (NTRS)

    Bell, Jordan R.; Molthan, Andrew L.; Schultz, Kori A.; McGrath, Kevin M.; Burks, Jason E.

    2015-01-01

    Every year in the Midwest and Great Plains, widespread greenness forms in conjunction with the latter part of the spring-summer growing season. This prevalent greenness forms as a result of the high concentration of agricultural areas having their crops reach their maturity before the fall harvest. This time of year also coincides with an enhanced hail frequency for the Great Plains (Cintineo et al. 2012). These severe thunderstorms can bring damaging winds and large hail that can result in damage to the surface vegetation. The spatial extent of the damage can relatively small concentrated area or be a vast swath of damage that is visible from space. These large areas of damage have been well documented over the years. In the late 1960s aerial photography was used to evaluate crop damage caused by hail. As satellite remote sensing technology has evolved, the identification of these hail damage streaks has increased. Satellites have made it possible to view these streaks in additional spectrums. Parker et al. (2005) documented two streaks using the Moderate Resolution Imaging Spectroradiometer (MODIS) that occurred in South Dakota. He noted the potential impact that these streaks had on the surface temperature and associated surface fluxes that are impacted by a change in temperature. Gallo et al. (2012) examined at the correlation between radar signatures and ground observations from storms that produced a hail damage swath in Central Iowa also using MODIS. Finally, Molthan et al. (2013) identified hail damage streaks through MODIS, Landsat-7, and SPOT observations of different resolutions for the development of a potential near-real time applications. The manual analysis of hail damage streaks in satellite imagery is both tedious and time consuming, and may be inconsistent from event to event. This study focuses on development of an objective and automatic algorithm to detect these areas of damage in a more efficient and timely manner. This study utilizes the

  10. [Identification of human pathogenic variola and monkeypox viruses by real-time polymerase chain reaction].

    PubMed

    Kostina, E V; Gavrilova, E V; Riabinin, V A; Shchelkunov, S N; Siniakov, A N

    2009-01-01

    A kit of specific oligonucleotide primers and hybridization probes has been proposed to detect orthopoxviruses (OPV) and to discriminate human pathogenic viruses, such as variola virus and monkey virus by real-time polymerase chain reaction (PCR). For real-time PCR, the following pairs of fluorophore and a fluorescence quencher were used: TAMRA-BHQ2 for genus-specific probes and FAM-BHQ1 for species-specific ones (variola virus, monkeypox virus, ectomelia virus). The specificity of this assay was tested on 38 strains of 6 OPV species and it was 100%.

  11. Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples.

    PubMed

    Irenge, Léonid M; Durant, Jean-François; Tomaso, Herbert; Pilo, Paola; Olsen, Jaran S; Ramisse, Vincent; Mahillon, Jacques; Gala, Jean-Luc

    2010-11-01

    A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

  12. Development of a real-time PCR method (Taqman) for rapid identification and quantification of Prorocentrum donghaiense

    NASA Astrophysics Data System (ADS)

    Yuan, Jian; Mi, Tiezhu; Zhen, Yu; Yu, Zhigang

    2012-09-01

    Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms (HABs). Therefore, it is necessary to study this dinoflagellate to monitor HABs. In this study, 13 pairs of primers specific to P. donghaiense (within its internal transcribed spacer (ITS) regions) were designed for SYBR Green I real-time PCR. As the SYBR Green I real-time PCR could not identify P. donghaiense in a specific manner, a Taqman real-time PCR method was developed by designing a set of specific primers and a Taqman probe. A 10-fold serial dilution of recombinant plasmid containing ITS regions of P. donghaiense was prepared as standard samples and the standard curve was established. Additionally, we quantified the genomic DNA in P. donghaiense cells and utilized this DNA to prepare another 10-fold serial dilution of standard sample and accordingly set up the standard curve. The mathematic correlation between the cell number and its corresponding plasmid copy number was also established. In order to test the efficiency of the real-time PCR method, laboratory samples and P. donghaiense HAB field samples were employed for identification and quantitative analysis. As to laboratory samples, as few as 102 cells of P. donghaiense could be quantified precisely utilizing both centrifugation and filtration techniques. The quantification results from field samples by real-time PCR were highly similar to those by light microscopy. In conclusion, the real-time PCR could be applied to identify and quantify P. donghaiense in HABs.

  13. Simple Real-Time PCR and Amplicon Sequencing Method for Identification of Plasmodium Species in Human Whole Blood.

    PubMed

    Lefterova, Martina I; Budvytiene, Indre; Sandlund, Johanna; Färnert, Anna; Banaei, Niaz

    2015-07-01

    Malaria is the leading identifiable cause of fever in returning travelers. Accurate Plasmodium species identification has therapy implications for P. vivax and P. ovale, which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay for Plasmodium species identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan-Plasmodium 18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% for P. falciparum (20/21 positives detected) and 100% for the Plasmodium genus (52/52), P. vivax (20/20), P. ovale (9/9), and P. malariae (6/6). The sensitivity of the P. knowlesi-specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% for P. vivax (49/52) and 100% for P. falciparum (51/51), P. ovale (62/62), P. malariae (69/69), and P. knowlesi (52/52). Thirty-three specimens were used to test species identification by sequencing the pan-Plasmodium 18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixed P. falciparum and P. ovale infection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitive Plasmodium species identification shortly after malaria diagnosis by microscopy.

  14. Simple Real-Time PCR and Amplicon Sequencing Method for Identification of Plasmodium Species in Human Whole Blood

    PubMed Central

    Budvytiene, Indre; Sandlund, Johanna; Färnert, Anna

    2015-01-01

    Malaria is the leading identifiable cause of fever in returning travelers. Accurate Plasmodium species identification has therapy implications for P. vivax and P. ovale, which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay for Plasmodium species identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan-Plasmodium 18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% for P. falciparum (20/21 positives detected) and 100% for the Plasmodium genus (52/52), P. vivax (20/20), P. ovale (9/9), and P. malariae (6/6). The sensitivity of the P. knowlesi-specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% for P. vivax (49/52) and 100% for P. falciparum (51/51), P. ovale (62/62), P. malariae (69/69), and P. knowlesi (52/52). Thirty-three specimens were used to test species identification by sequencing the pan-Plasmodium 18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixed P. falciparum and P. ovale infection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitive Plasmodium species identification shortly after malaria diagnosis by microscopy. PMID:25972416

  15. 3D surface real-time measurement using phase-shifted interference fringe technique for craniofacial identification

    NASA Astrophysics Data System (ADS)

    Levin, Gennady G.; Vishnyakov, Gennady N.; Naumov, Alexey V.; Abramov, Sergey

    1998-03-01

    We offer to use the 3D surface profile real-time measurement using phase-shifted interference fringe projection technique for the cranioficial identification. Our system realizes the profile measurement by projecting interference fringe pattern on the object surface and by observing the deformed fringe pattern at the direction different from the projection. Fringes are formed by a Michelson interferometer with one mirror mounted on a piezoelectric translator. Four steps self- calibration phase-shift method was used.

  16. Streaming algorithms for identification of pathogens and antibiotic resistance potential from real-time MinION(TM) sequencing.

    PubMed

    Cao, Minh Duc; Ganesamoorthy, Devika; Elliott, Alysha G; Zhang, Huihui; Cooper, Matthew A; Coin, Lachlan J M

    2016-07-26

    The recently introduced Oxford Nanopore MinION platform generates DNA sequence data in real-time. This has great potential to shorten the sample-to-results time and is likely to have benefits such as rapid diagnosis of bacterial infection and identification of drug resistance. However, there are few tools available for streaming analysis of real-time sequencing data. Here, we present a framework for streaming analysis of MinION real-time sequence data, together with probabilistic streaming algorithms for species typing, strain typing and antibiotic resistance profile identification. Using four culture isolate samples, as well as a mixed-species sample, we demonstrate that bacterial species and strain information can be obtained within 30 min of sequencing and using about 500 reads, initial drug-resistance profiles within two hours, and complete resistance profiles within 10 h. While strain identification with multi-locus sequence typing required more than 15x coverage to generate confident assignments, our novel gene-presence typing could detect the presence of a known strain with 0.5x coverage. We also show that our pipeline can process over 100 times more data than the current throughput of the MinION on a desktop computer.

  17. Protocol for real-time PCR identification of anthrax spores from nasal swabs after broth enrichment.

    PubMed

    Oggioni, Marco R; Meacci, Francesca; Carattoli, Alessandra; Ciervo, Alessandra; Orru, Germano; Cassone, Antonio; Pozzi, Gianni

    2002-11-01

    A mass-screening protocol for the diagnosis of anthrax from nasal swabs based on an enrichment step in liquid medium was devised. Incubation for growth was performed in autoclavable vials and racks which allow real-time PCR analysis of sterilized cultures. A dual-color PCR was set up with primers and probes for the chromosomal marker rpoB and the plasmid marker lef. Specific primer and probe sets were designed for the differentiation of Bacillus anthracis from B. cereus and for the differentiation of the Sterne vaccine strain from field isolates and the Ames strain, which was used in the recent anthrax bioterrorist attack. The present protocol thus combines the high specificity and sensitivity of real-time PCR with excellent biosafety and the low hands-on time necessary for the processing of large numbers of samples, which is extremely important during control programs involving the processing of large numbers of samples.

  18. Protocol for Real-Time PCR Identification of Anthrax Spores from Nasal Swabs after Broth Enrichment

    PubMed Central

    Oggioni, Marco R.; Meacci, Francesca; Carattoli, Alessandra; Ciervo, Alessandra; Orru, Germano; Cassone, Antonio; Pozzi, Gianni

    2002-01-01

    A mass-screening protocol for the diagnosis of anthrax from nasal swabs based on an enrichment step in liquid medium was devised. Incubation for growth was performed in autoclavable vials and racks which allow real-time PCR analysis of sterilized cultures. A dual-color PCR was set up with primers and probes for the chromosomal marker rpoB and the plasmid marker lef. Specific primer and probe sets were designed for the differentiation of Bacillus anthracis from B. cereus and for the differentiation of the Sterne vaccine strain from field isolates and the Ames strain, which was used in the recent anthrax bioterrorist attack. The present protocol thus combines the high specificity and sensitivity of real-time PCR with excellent biosafety and the low hands-on time necessary for the processing of large numbers of samples, which is extremely important during control programs involving the processing of large numbers of samples. PMID:12409358

  19. Real-Time Identification and Characterization of Asbestos and Concrete Materials with Radioactive Contamination

    SciTech Connect

    Xu, George; Zhang, Xi-Cheng

    1999-06-01

    Concrete and asbestos-containing materials were widely used in U.S. Department of Energy (DOE) building construction in the 1940s and 1950s. Over the years, many of these porous building materials have been contaminated with radioactive sources, on and below the surface. This intractable radioactive-and-hazardous- asbestos mixed-waste-stream has created a tremendous challenge to DOE decontamination and decommissioning (D&D) project managers. The current practice to identify asbestos and to characterize radioactive contamination depth profiles involve bore sampling, and is inefficient, costly, and unsafe. A three-year research project was started on 10/1/98 at Rensselaer with the following ultimate goals: (1) development of novel non-destructive methods for identifying the hazardous asbestos in real-time and in-situ, and (2) development of new algorithms and apparatus for characterizing the radioactive contamination depth profile in real-time and in-situ.

  20. Real-Time Identification and Characterization of Asbestos and Concrete Materials with Radioactive Contamination

    SciTech Connect

    Xu, George; Zhang, Xi-Cheng

    2000-06-01

    Concrete and asbestos-containing materials were widely used in U.S. Department of Energy (DOE) building construction in the 1940s and 1950s. Over the years, many of these porous building materials have been contaminated with radioactive sources, on and below the surface. This intractable radioactive-and-hazardous-asbestos mixed-waste stream has created a tremendous challenge to DOE decontamination and decommissioning (D&D) project managers. The current practice to identify asbestos and to characterize radioactive contamination depth profiles in based solely on bore sampling, which is inefficient, costly, and unsafe. A three-year research project was started 1998 at Rensselaer with the following ultimate goals: (1) development of novel non-destructive methods for identifying the hazardous asbestos in real-time and in-situ, and (2) development of new algorithms and apparatus for characterizing the radioactive contamination depth profile in real-time and in-situ.

  1. Human Fecal Source Identification: Real-Time Quantitative PCR Method Standardization

    EPA Science Inventory

    Method standardization or the formal development of a protocol that establishes uniform performance benchmarks and practices is necessary for widespread adoption of a fecal source identification approach. Standardization of a human-associated fecal identification method has been...

  2. HUMAN FECAL SOURCE IDENTIFICATION: REAL-TIME QUANTITATIVE PCR METHOD STANDARDIZATION - abstract

    EPA Science Inventory

    Method standardization or the formal development of a protocol that establishes uniform performance benchmarks and practices is necessary for widespread adoption of a fecal source identification approach. Standardization of a human-associated fecal identification method has been...

  3. HUMAN FECAL SOURCE IDENTIFICATION: REAL-TIME QUANTITATIVE PCR METHOD STANDARDIZATION - abstract

    EPA Science Inventory

    Method standardization or the formal development of a protocol that establishes uniform performance benchmarks and practices is necessary for widespread adoption of a fecal source identification approach. Standardization of a human-associated fecal identification method has been...

  4. Human Fecal Source Identification: Real-Time Quantitative PCR Method Standardization

    EPA Science Inventory

    Method standardization or the formal development of a protocol that establishes uniform performance benchmarks and practices is necessary for widespread adoption of a fecal source identification approach. Standardization of a human-associated fecal identification method has been...

  5. Real-time full-motion color Flash lidar for target detection and identification

    NASA Astrophysics Data System (ADS)

    Nelson, Roy; Coppock, Eric; Craig, Rex; Craner, Jeremy; Nicks, Dennis; von Niederhausern, Kurt

    2015-05-01

    Greatly improved understanding of areas and objects of interest can be gained when real time, full-motion Flash LiDAR is fused with inertial navigation data and multi-spectral context imagery. On its own, full-motion Flash LiDAR provides the opportunity to exploit the z dimension for improved intelligence vs. 2-D full-motion video (FMV). The intelligence value of this data is enhanced when it is combined with inertial navigation data to produce an extended, georegistered data set suitable for a variety of analysis. Further, when fused with multispectral context imagery the typical point cloud now becomes a rich 3-D scene which is intuitively obvious to the user and allows rapid cognitive analysis with little or no training. Ball Aerospace has developed and demonstrated a real-time, full-motion LIDAR system that fuses context imagery (VIS to MWIR demonstrated) and inertial navigation data in real time, and can stream these information-rich geolocated/fused 3-D scenes from an airborne platform. In addition, since the higher-resolution context camera is boresighted and frame synchronized to the LiDAR camera and the LiDAR camera is an array sensor, techniques have been developed to rapidly interpolate the LIDAR pixel values creating a point cloud that has the same resolution as the context camera, effectively creating a high definition (HD) LiDAR image. This paper presents a design overview of the Ball TotalSight™ LIDAR system along with typical results over urban and rural areas collected from both rotary and fixed-wing aircraft. We conclude with a discussion of future work.

  6. Real-time PCR assays compared to culture-based approaches for identification of aerobic bacteria in chronic wounds.

    PubMed

    Melendez, J H; Frankel, Y M; An, A T; Williams, L; Price, L B; Wang, N-Y; Lazarus, G S; Zenilman, J M

    2010-12-01

    Chronic wounds cause substantial morbidity and disability. Infection in chronic wounds is clinically defined by routine culture methods that can take several days to obtain a final result, and may not fully describe the community of organisms or biome within these wounds. Molecular diagnostic approaches offer promise for a more rapid and complete assessment. We report the development of a suite of real-time PCR assays for rapid identification of bacteria directly from tissue samples. The panel of assays targets 14 common, clinically relevant, aerobic pathogens and demonstrates a high degree of sensitivity and specificity using a panel of organisms commonly associated with chronic wound infection. Thirty-nine tissue samples from 29 chronic wounds were evaluated and the results compared with those obtained by culture. As revealed by culture and PCR, the most common organisms were methicillin-resistant Staphylococcus aureus (MRSA) followed by Streptococcus agalactiae (Group B streptococcus) and Pseudomonas aeruginosa. The sensitivities of the PCR assays were 100% and 90% when quantitative and qualitative culture results were used as the reference standard, respectively. The assays allowed the identification of bacterial DNA from ten additional organisms that were not revealed by quantitative or qualitative cultures. Under optimal conditions, the turnaround time for PCR results is as short as 4-6 h. Real-time PCR is a rapid and inexpensive approach that can be easily introduced into clinical practice for detection of organisms directly from tissue samples. Characterization of the anaerobic microflora by real-time PCR of chronic wounds is warranted.

  7. Processor core for real time background identification of HD video based on OpenCV Gaussian mixture model algorithm

    NASA Astrophysics Data System (ADS)

    Genovese, Mariangela; Napoli, Ettore

    2013-05-01

    The identification of moving objects is a fundamental step in computer vision processing chains. The development of low cost and lightweight smart cameras steadily increases the request of efficient and high performance circuits able to process high definition video in real time. The paper proposes two processor cores aimed to perform the real time background identification on High Definition (HD, 1920 1080 pixel) video streams. The implemented algorithm is the OpenCV version of the Gaussian Mixture Model (GMM), an high performance probabilistic algorithm for the segmentation of the background that is however computationally intensive and impossible to implement on general purpose CPU with the constraint of real time processing. In the proposed paper, the equations of the OpenCV GMM algorithm are optimized in such a way that a lightweight and low power implementation of the algorithm is obtained. The reported performances are also the result of the use of state of the art truncated binary multipliers and ROM compression techniques for the implementation of the non-linear functions. The first circuit has commercial FPGA devices as a target and provides speed and logic resource occupation that overcome previously proposed implementations. The second circuit is oriented to an ASIC (UMC-90nm) standard cell implementation. Both implementations are able to process more than 60 frames per second in 1080p format, a frame rate compatible with HD television.

  8. On-site identification of meat species in processed foods by a rapid real-time polymerase chain reaction system.

    PubMed

    Furutani, Shunsuke; Hagihara, Yoshihisa; Nagai, Hidenori

    2017-09-01

    Correct labeling of foods is critical for consumers who wish to avoid a specific meat species for religious or cultural reasons. Therefore, gene-based point-of-care food analysis by real-time Polymerase Chain Reaction (PCR) is expected to contribute to the quality control in the food industry. In this study, we perform rapid identification of meat species by our portable rapid real-time PCR system, following a very simple DNA extraction method. Applying these techniques, we correctly identified beef, pork, chicken, rabbit, horse, and mutton in processed foods in 20min. Our system was sensitive enough to detect the interfusion of about 0.1% chicken egg-derived DNA in a processed food sample. Our rapid real-time PCR system is expected to contribute to the quality control in food industries because it can be applied for the identification of meat species, and future applications can expand its functionality to the detection of genetically modified organisms or mutations. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. A novel application of real-time RT-LAMP for body fluid identification: using HBB detection as the model.

    PubMed

    Su, Chih-Wen; Li, Chiao-Yun; Lee, James Chun-I; Ji, Dar-Der; Li, Shu-Ying; Daniel, Barbara; Syndercombe-Court, Denise; Linacre, Adrian; Hsieh, Hsing-Mei

    2015-06-01

    We report on a novel application of real-time reverse transcription-loop-mediated isothermal amplification (real-time RT-LAMP) to identify the presence of a specific body fluid using blood as a proof-of-concept model. By comparison with recently developed methods of body fluid identification, the RT-LAMP assay is rapid and requires only one simple heating-block maintained at a single temperature, circumventing the need for dedicated equipment. RNA was extracted from different body fluids (blood, semen, saliva, menstrual blood, sweat, and urine) for use in real-time RT-LAMP reaction. The 18S rRNA locus was used as the internal control and hemoglobin beta (HBB) as the blood-specific marker. Reverse transcription and LAMP reaction were performed in the same tube using a turbidimeter for real-time monitoring the reaction products within a threshold of 60 min. HBB LAMP products were only detected in blood and not in any of the other body fluid, but products from the 18S rRNA gene were detected in all the tested body fluids as expected. The limit of detection was a minimum of 10(-5) ng total RNA for detection of both 18S rRNA and HBB. Augmenting the detection of RT-LAMP products was performed by separation of the products using gel electrophoresis and collecting the fluorescence of calcein. The data collected indicated complete concordance with the body fluid tested regardless of the method of detection used. This is the first application of real-time RT-LAMP to detect body fluid specific RNA and indicates the use of this method in forensic biology.

  10. Real-Time Parameter Estimation Method Applied to a MIMO Process and its Comparison with an Offline Identification Method

    SciTech Connect

    Kaplanoglu, Erkan; Safak, Koray K.; Varol, H. Selcuk

    2009-01-12

    An experiment based method is proposed for parameter estimation of a class of linear multivariable systems. The method was applied to a pressure-level control process. Experimental time domain input/output data was utilized in a gray-box modeling approach. Prior knowledge of the form of the system transfer function matrix elements is assumed to be known. Continuous-time system transfer function matrix parameters were estimated in real-time by the least-squares method. Simulation results of experimentally determined system transfer function matrix compare very well with the experimental results. For comparison and as an alternative to the proposed real-time estimation method, we also implemented an offline identification method using artificial neural networks and obtained fairly good results. The proposed methods can be implemented conveniently on a desktop PC equipped with a data acquisition board for parameter estimation of moderately complex linear multivariable systems.

  11. Identification of lactic acid bacteria isolated from wine using real-time PCR.

    PubMed

    Kántor, Attila; Kluz, Maciej; Puchalski, Czeslaw; Terentjeva, Margarita; Kačániová, Miroslava

    2016-01-01

    Different lactic acid bacteria strains have been shown to cause wine spoilage, including the generation of substances undesirable for the health of wine consumers. The aim of this study was to investigate the occurrence of selected species of heterofermentative lactobacilli, specifically Lactobacillus brevis, Lactobacillus hilgardii, and Lactobacillus plantarum in six different Slovak red wines following the fermentation process. In order to identify the dominant Lactobacillus strain using quantitative (real time) polymerized chain reaction (qPCR) method, pure lyophilized bacterial cultures from the Czech Collection of Microorganisms were used. Six different red wine samples following malolactic fermentation were obtained from selected wineries. After collection, the samples were subjected to a classic plate dilution method for enumeration of lactobacilli cells. Real-time PCR was performed after DNA extraction from pure bacterial strains and wine samples. We used SYBR® Green master mix reagents for measuring the fluorescence in qPCR. The number of lactobacilli ranged from 3.60 to 5.02 log CFU mL(-1). Specific lactobacilli strains were confirmed by qPCR in all wine samples. The number of lactobacilli ranged from 10(3) to 10(6) CFU mL(-1). A melting curve with different melting temperatures (T(m)) of DNA amplicons was obtained after PCR for the comparison of T(m) of control and experimental portions, revealing that the most common species in wine samples was Lactobacillus plantarum with a T(m) of 84.64°C.

  12. Rapid identification of Campylobacter jejuni from poultry carcasses and slaughtering environment samples by real-time PCR.

    PubMed

    Ivanova, Mirena; Singh, Randhir; Dharmasena, Muthu; Gong, Chao; Krastanov, Albert; Jiang, Xiuping

    2014-06-01

    The objective of this study was to develop a real-time PCR assay for rapid identification of Campylobacter jejuni and to apply the method in analyzing samples from poultry processing. A C. jejuni-specific primer set targeting a portion of the C. jejuni hippuricase gene was developed. The specificity of the newly designed primer pair was verified using 5 C. jejuni strains and 20 other bacterial strains. Sensitivity was determined to be as low as 1 genome copy per reaction. A total of 73 samples were collected at different sites along the processing line during 2 visits to a poultry slaughterhouse and were examined by direct plating onto modified charcoal cefoperazone deoxycholate agar or after enrichment in Bolton broth followed by plating on modified charcoal cefoperazone deoxycholate agar. The newly developed real-time PCR assay was used to identify the presumptive colonies as belonging to C. jejuni. A real-time PCR assay targeting 16S ribosomal RNA was also applied to determine Campylobacter spp. prevalence. Results from the real-time PCR analysis indicated considerable variability in Campylobacter contamination, with incidence rates of 72.7 and 27.6% for sampling days A and B, respectively. Campylobacter was isolated from 100% of prescalded and preeviscerated carcasses on sampling day A. In contrast, on sampling day B, the highest number of Campylobacter-positive carcasses was recovered after evisceration (60%). The chilling process significantly reduced (P < 0.05) Campylobacter population, but the percentage of positive samples on sampling day A increased to 80%. All samples collected from the processing environment, except scalding tank 3 and the prechiller and chiller tanks, were 100% positive on day A, whereas no campylobacters were isolated from machinery on sampling day B. Our results revealed the widespread of C. jejuni in poultry processing and proved that the newly developed real-time PCR assay is a simple, specific, and inexpensive method for rapid C

  13. Real-time automated failure identification in the Control Center Complex (CCC)

    NASA Technical Reports Server (NTRS)

    Kirby, Sarah; Lauritsen, Janet; Pack, Ginger; Ha, Anhhoang; Jowers, Steven; Mcnenny, Robert; Truong, The; Dell, James

    1993-01-01

    A system which will provide real-time failure management support to the Space Station Freedom program is described. The system's use of a simplified form of model based reasoning qualifies it as an advanced automation system. However, it differs from most such systems in that it was designed from the outset to meet two sets of requirements. First, it must provide a useful increment to the fault management capabilities of the Johnson Space Center (JSC) Control Center Complex (CCC) Fault Detection Management system. Second, it must satisfy CCC operational environment constraints such as cost, computer resource requirements, verification, and validation, etc. The need to meet both requirement sets presents a much greater design challenge than would have been the case had functionality been the sole design consideration. The choice of technology, discussing aspects of that choice and the process for migrating it into the control center is overviewed.

  14. Real-time PCR identification of lake whitefish Coregonus clupeaformis in the Laurentian Great Lakes.

    PubMed

    Overdyk, L M; Braid, H E; Naaum, A M; Crawford, S S; Hanner, R H

    2016-04-01

    The purpose of this study was to develop a real-time PCR assay to specifically identify lake whitefish Coregonus clupeaformis in larval fish assemblages based on a 122 bp amplicon from the mitochondrial genome. The efficiency of the reaction, as calculated from the standard curve, was 90.77% with the standard curve having an r(2) value of 0.998. Specificity of the assay provided single melt peak in a melt-curve analysis and amplification of only the target species. The assay successfully identified target DNA in as low as 0.1% proportion of a DNA mixture. This assay was designed on the portable Smart Cycler II platform and can be used in both field and laboratory settings to successfully identify C. clupeaformis.

  15. Accurate Identification of Candida parapsilosis (Sensu Lato) by Use of Mitochondrial DNA and Real-Time PCR

    PubMed Central

    Souza, Ana Carolina R.; Ferreira, Renata C.; Gonçalves, Sarah S.; Quindós, Guillermo; Eraso, Elena; Bizerra, Fernando C.; Briones, Marcelo R. S.

    2012-01-01

    Candida parapsilosis is the Candida species isolated the second most frequently from blood cultures in South America and some European countries, such as Spain. Since 2005, this species has been considered a complex of 3 closely related species: C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis. Here, we describe a real-time TaqMan-MGB PCR assay, using mitochondrial DNA (mtDNA) as the target, which readily distinguishes these 3 species. We first used comparative genomics to locate syntenic regions between these 3 mitochondrial genomes and then selected NADH5 as the target for the real-time PCR assay. Probes were designed to include a combination of different single-nucleotide polymorphisms that are able to differentiate each species within the C. parapsilosis complex. This new methodology was first tested using mtDNA and then genomic DNA from 4 reference and 5 clinical strains. For assay validation, a total of 96 clinical isolates and 4 American Type Culture Collection (ATCC) isolates previously identified by internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing were tested. Real-time PCR using genomic DNA was able to differentiate the 3 species with 100% accuracy. No amplification was observed when DNA from other species was used as the template. We observed 100% congruence with ITS rDNA sequencing identification, including for 30 strains used in blind testing. This novel method allows a quick and accurate intracomplex identification of C. parapsilosis and saves time compared with sequencing, which so far has been considered the “gold standard” for Candida yeast identification. In addition, this assay provides a useful tool for epidemiological and clinical studies of these emergent species. PMID:22535986

  16. Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry.

    PubMed

    Lesiak, Ashton D; Musah, Rabi A

    2016-10-02

    We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

  17. A multi-target real-time PCR assay for rapid identification of meningitis-associated microorganisms.

    PubMed

    Favaro, Marco; Savini, Vincenzo; Favalli, Cartesio; Fontana, Carla

    2013-01-01

    A central nervous system (CNS) infection, such as meningitis, is a serious and life-threatening condition. Bacterial meningitis can be severe and may result in brain damage, disability or even death. Rapid diagnosis of CNS infections and identification of the pathogenic microorganisms are needed to improve the patient outcome. Bacterial culture of a patient's cerebrospinal fluid (CSF) is currently considered the "gold standard" for diagnosing bacterial meningitis. From the CSF cultures researchers can assess the in vitro susceptibility of the causative microorganism to determine the best antibiotic treatment. However, many of the culture assays, such as microscopy and the latex agglutination test are not sensitive. To enhance pathogen detection in CSF samples we developed a multi-target real-time PCR assay that can rapidly identify six different microorganisms: Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus agalactiae, Listeria monocytogenes and Cryptococcus neoformans. In this study we applied this PCR analysis to 296 CSF samples from patients who were suspected of having meningitis. Of the 296 samples that were examined, 59 samples were positive according to the CSF culture and/or molecular assays. Forty-six CSF samples were positive for both the CSF culture and our real-time PCR assay, while 13 samples were positive for the real-time PCR but negative for the traditional assays. This discrepancy may have been caused by the fact that these samples were collected from 23 patients who were treated with antimicrobials before CSF sampling.

  18. Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

    PubMed Central

    Lesiak, Ashton D.; Musah, Rabi A.

    2016-01-01

    We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species. PMID:27768072

  19. Rapid and Accurate Identification by Real-Time PCR of Biotoxin-Producing Dinoflagellates from the Family Gymnodiniaceae

    PubMed Central

    Smith, Kirsty F.; de Salas, Miguel; Adamson, Janet; Rhodes, Lesley L.

    2014-01-01

    The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR) assays targeting the large subunit ribosomal RNA (LSU rRNA) gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples. PMID:24608972

  20. Detection of bacterial 16S rRNA and identification of four clinically important bacteria by real-time PCR.

    PubMed

    Clifford, Robert J; Milillo, Michael; Prestwood, Jackson; Quintero, Reyes; Zurawski, Daniel V; Kwak, Yoon I; Waterman, Paige E; Lesho, Emil P; Mc Gann, Patrick

    2012-01-01

    Within the paradigm of clinical infectious disease research, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa represent the four most clinically relevant, and hence most extensively studied bacteria. Current culture-based methods for identifying these organisms are slow and cumbersome, and there is increasing need for more rapid and accurate molecular detection methods. Using bioinformatic tools, 962,279 bacterial 16S rRNA gene sequences were aligned, and regions of homology were selected to generate a set of real-time PCR primers that target 93.6% of all bacterial 16S rRNA sequences published to date. A set of four species-specific real-time PCR primer pairs were also designed, capable of detecting less than 100 genome copies of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa. All primers were tested for specificity in vitro against 50 species of Gram-positive and -negative bacteria. Additionally, the species-specific primers were tested against a panel of 200 clinical isolates of each species, randomly selected from a large repository of clinical isolates from diverse areas and sources. A comparison of culture and real-time PCR demonstrated 100% concordance. The primers were incorporated into a rapid assay capable of positive identification from plate or broth cultures in less than 90 minutes. Furthermore, our data demonstrate that current targets, such as the uidA gene in E.coli, are not suitable as species-specific genes due to sequence variation. The assay described herein is rapid, cost-effective and accurate, and can be easily incorporated into any research laboratory capable of real-time PCR.

  1. Dynamic data-driven inversion for terascale simulations real-time identification of airborne contaminants.

    SciTech Connect

    Ghattas, Omar; Waanders, Bart Van Bloemen; Hill, Judith C.; Akcelik, Volkan; Draganescu, Andrei I.; Biros, George

    2005-05-01

    In contrast to traditional terascale simulations that have known, fixed data inputs, dynamic data-driven (DDD) applications are characterized by unknown data and informed by dynamic observations. DDD simulations give rise to inverse problems of determining unknown data from sparse observations. The main difficulty is that the optimality system is a boundary value problem in 4D space-time, even though the forward simulation is an initial value problem. We construct special-purpose parallel multigrid algorithms that exploit the spectral structure of the inverse operator. Experiments on problems of localizing airborne contaminant release from sparse observations in a regional atmospheric transport model demonstrate that 17-million-parameter inversion can be effected at a cost of just 18 forward simulations with high parallel efficiency. On 1024 Alphaserver EV68 processors, the turnaround time is just 29 minutes. Moreover, inverse problems with 135 million parameters - corresponding to 139 billion total space-time unknowns - are solved in less than 5 hours on the same number of processors. These results suggest that ultra-high resolution data-driven inversion can be carried out sufficiently rapidly for simulation-based 'real-time' hazard assessment.

  2. Feature-level signal processing for near-real-time odor identification

    NASA Astrophysics Data System (ADS)

    Roppel, Thaddeus A.; Padgett, Mary Lou; Waldemark, Joakim T. A.; Wilson, Denise M.

    1998-09-01

    Rapid detection and classification of odor is of particular interest in applications such as manufacturing of consumer items, food processing, drug and explosives detection, and battlefield situation assessment. Various detection and classification techniques are under investigation so that end users can have access to useful information from odor sensor arrays in near-real-time. Feature-level data clustering and classification techniques are proposed that are (1) parallelizable to permit efficient hardware implementation, (2) adaptable to readily incorporate new data classes, (3) capable of gracefully handling outlier data points and failed sensor conditions, and (4) can provide confidence intervals and/or a traceable decision record along with each classification to permit validation and verification. Results from using specific techniques will be presented and compared. The techniques studied include principal components analysis, automated outlier determination, radial basis functions (RBF), multi-layer perceptrons (MLP), and pulse-coupled neural networks (PCNN). The results reported here are based on data from a testbed in which a gas sensor array is exposed to odor samples on a continuous basis. We have reported previously that more detailed and faster discrimination can be obtained by using sensor transient response in addition to steady state response. As the size of the data set grows we are able to more accurately model performance of a sensor array under realistic conditions.

  3. Rapid Identification and Enumeration of Saccharomyces cerevisiae Cells in Wine by Real-Time PCR

    PubMed Central

    Martorell, P.; Querol, A.; Fernández-Espinar, M. T.

    2005-01-01

    Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated S. cerevisiae from its sibling species Saccharomyces bayanus, Saccharomyces pastorianus, and Saccharomyces paradoxus. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of S. cerevisiae directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify S. cerevisiae in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 × 105 to 3.8 CFU/ml in sweet wine and from 5 × 106 to 50 CFU/ml in red wine. PMID:16269715

  4. [Real time PCR hybridization for the rapid and specific identification of Francisella tularensis].

    PubMed

    Bielawska-Drózd, Agata; Niemcewicz, Marcin; Gaweł, Jerzy; Bartoszcze, Michał; Graniak, Grzegorz; Joniec, Justyna; Kołodziej, Marcin

    2010-01-01

    Tularemia is highly infectious and fatal zoonotic disease caused by Gram negative bacteria Francisella tularensis. The necessity to undergo medical treatment in early phase of illness in humans and possibility of making use of bacterial aerosol by terrorists in an attack create an urgent need to implement a rapid and effective method which enables to identify the agent. In our study two primers FopA F/R and hybridization probes FopA S1/S2 designed from fopA gene sequence, were tested for their potential applicability to identify F. tularensis. In this research 50 strains of F. tularensis were used and the test gave positive results. Reaction specificity was confirmed by using of non-Francisella tularensis bacterial species. The results obtained in the real-time PCR reaction with primers Tul4 F/R and hybridization probes Tul4 S1/S2, designed from tul4 gene, were comparable to the results from previous experiment with fopA - primers set. Investigation of fopA and tul4 primers and hybridization probes properties revealed characteristic Tm (melting temperature) value of the products--61 degrees C and 60 degrees C, respectively. Detection sensitivity was remarkably higher when fopA primers set was used 1 fg/microl, and for tul4 primers set, minimal detectable concentration is 10 fg/microl.

  5. Multi-Probe Real-Time PCR Identification of Common Mycobacterium Species in Blood Culture Broth

    PubMed Central

    Foongladda, Suporn; Pholwat, Suporn; Eampokalap, Boonchuay; Kiratisin, Pattarachai; Sutthent, Ruengpung

    2009-01-01

    Mycobacterium tuberculosis complex, M. avium, and M. intracellulare are the most common causes of systemic bacterial infection in AIDS patients. To identify these mycobacterial isolates in primary blood culture broths, we developed a multiple hybridization probe-based real-time PCR assay using the LightCycler system. The primers were designed to amplify a 320-bp fragment of Mycobacterium 16S rRNA genes. Reaction specificity was evaluated using PCR amplification curves along with specific melting temperatures of probes on DNA extracted from 13 Mycobacterium species. In this study, results showed 100% accuracy for the selected bacterial panel. Detection limits were 350, 600, and 650 colony-forming unit (CFU)/ml blood culture broths for M. tuberculosis complex, M. avium, and M. intracellulare, respectively (1 to 2 CFU/reaction). To evaluate clinical applicability, 341 acid-fast bacilli in blood culture broths were analyzed. In total, 327 (96%) were positively identified: 54.5% M. tuberculosis complex, 37.5% M. avium, and 3.8% M. intracellulare. Results can be available within 3 hours of receiving a broth sample, which makes this rapid and simple assay an attractive diagnostic tool for clinical use. PMID:19095775

  6. Evaluation of a co-extraction method for real-time PCR-based body fluid identification and DNA typing.

    PubMed

    Watanabe, Ken; Iwashima, Yasuki; Akutsu, Tomoko; Sekiguchi, Kazumasa; Sakurada, Koichi

    2014-01-01

    Body fluid identification and individual identification are an important series of tests in usual criminal investigations. Recent reports have demonstrated a new approach using DNA/RNA co-extraction methods in which RNA for body fluid identification and DNA for short tandem repeat (STR) typing are extracted simultaneously from the same sample. This study evaluated a standard co-extraction kit, the AllPrep® DNA/RNA Mini Kit, in order to demonstrate the availability of the co-extraction procedure for those real-time polymerase chain reaction-based body fluid identification methods that we have validated previously. We demonstrated that the use of the Allprep Kit, for which we adjusted the lysis temperature to 56°C to improve extraction efficiency, can simultaneously extract sufficient RNA and DNA for body fluid identification and STR analysis; however, a longer incubation at a high temperature slightly affected the ΔCt value of each target gene and appeared to be not as effective for DNA extraction from old stains as from 1-day-old stains. This method is promising for future forensic investigations because the use of this kit can reduce sample consumption for body fluid identification and DNA typing.

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

    PubMed Central

    2012-01-01

    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. PMID:23146128

  8. High-throughput gender identification of Accipitridae eagles with real-time PCR using TaqMan probes.

    PubMed

    Chang, H-W; Gu, D-L; Su, S-H; Chang, C-C; Cheng, C-A; Huang, H-W; Yao, C-T; Chou, T-C; Chuang, L-Y; Cheng, C-C

    2008-07-01

    The objective was to develop high-throughput gender identification of eagles. Based on BLAST and alignment analyses, the CHD-Z and CHD-W sequences of nine species of eagles were highly homologous with Spilornis cheela hoya (S. c. hoya); therefore, TaqMan probes were designed to target their CHD-ZW-common and CHD-W-specific regions. In S. c. hoya, genders were identified using TaqMan-based, real-time PCR (amplified by P2/P8 primers); this method was validated with anatomically confirmed controls (one of each gender). Both genders had high intensities of the HEX-labeled (CHD-ZW-common) probe, whereas only females had high intensity of the FAM-labeled (CHD-W-specific) probe. The sequence of the CHD-W-specific probe designed for S. c. hoya was completely homologous with the CHD-W-specific region in Circaetus gallicus, Gyps indicus, and Gyps bengalensis, and was only one nucleotide different from those of Accipiter nisus, Spizaetus nipalensis, Aquila chrysaetos, Circus spilonotus, and Milvus migrans. For the CHD-ZW-common probe, all species listed were completely conserved. Using real-time PCR software, we established auto-calling of the genders of 15 individuals of S. c. hoya. In conclusion, this method provided accurate, high-throughput gender identification for S. c. hoya, and has considerable potential for identifying the gender of several related species of eagles.

  9. CATSI EDM: a new sensor for the real-time passive stand-off detection and identification of chemicals

    NASA Astrophysics Data System (ADS)

    Thériault, Jean-Marc; Lacasse, Paul; Lavoie, Hugo; Bouffard, François; Montembeault, Yan; Farley, Vincent; Belhumeur, Louis; Lagueux, Philippe

    2010-04-01

    DRDC Valcartier recently completed the development of the CATSI EDM (Compact Atmospheric Sounding Interferometer Engineering Development Model) for the Canadian Forces (CF). It is a militarized sensor designed to meet the needs of the CF in the development of area surveillance capabilities for the detection and identification of chemical Warfare Agents (CWA) and toxic industrial chemicals (TIC). CATSI EDM is a passive infrared double-beam Fourier spectrometer system designed for real-time stand-off detection and identification of chemical vapours at distances up to 5 km. It is based on the successful passive differential detection technology. This technique known as optical subtraction, results in a target gas spectrum which is almost free of background, thus making possible detection of weak infrared emission in strong background emission. This paper summarizes the system requirements, achievements, hardware and software characteristics and test results.

  10. Real-Time Identification of Wheel Terrain Interaction Models for Enhanced Autonomous Vehicle Mobility

    DTIC Science & Technology

    2014-04-24

    address the problem of calibrating predictive models of ground vehicles in order to enable mobile robots (UGVs) to be more informed about their own...represented in vehicle frame. This term is corrected for slip by adding a V_slip term computed by a calibrated kinematic model . • The curve...Dynamics Approach to Online Vehicle Model Identification, International Symposium on Robotics Research. 29-AUG-11, . : , Neal Seegmiller,, Forrest

  11. Identification of Leptospira biflexa by real-time homogeneous detection of rapid cycle PCR product.

    PubMed

    Woo, T H; Patel, B K; Cinco, M; Smythe, L D; Norris, M A; Symonds, M L; Dohnt, M F; Piispanen, J

    1999-02-01

    Sequence analysis of 16S rRNA genes extracted from nucleic acids databases enabled the identification of a Leptospira biflexa (L. biflexa) signature sequence, against which a reverse primer designated L613, was designed. This primer, when used in conjunction with a universal bacterial specific forward primer designated Fd1, enabled the development of a LightCycler-based PCR protocol in which fluorescence emission due to binding of SYBR Green I dye to amplified products could be detected and monitored. A melting temperature (Tm), determined from the melting curve of the amplified product immediately following the termination of thermal cycling, confirmed that the product was that of L. biflexa. Agarose gel electrophoresis therefore was not necessary for identification of PCR products. The PCR protocol was very rapid, and consisted of 30 cycles with a duration of 20 s for each cycle with the monitoring of the melting curve requiring an additional 3 min. The whole protocol was completed in less than 20 min. The PCR protocol was also specific and enabled the identification of 18 strains of L. biflexa, whilst excluding 14 strains of L. interrogans and Leptonema illini. Two examples of its utility in improving work flow of a Leptospira reference laboratory are presented in this article. The use of a simple boiling method for extraction of DNA from all the members of the Leptospiraceae family DNA further simplifies the procedure and makes its use conducive to diagnostic laboratories.

  12. Real-time ligation chain reaction for DNA quantification and identification on the FO-SPR.

    PubMed

    Knez, Karel; Spasic, Dragana; Delport, Filip; Lammertyn, Jeroen

    2015-05-15

    Different assays have been developed in the past years to meet point-of-care diagnostic tests requirements for fast and sensitive quantification and identification of targets. In this paper, we developed the ligation chain reaction (LCR) assay on the Fiber Optic Surface Plasmon Resonance (FO-SPR) platform, which enabled simultaneous quantification and cycle-to-cycle identification of DNA during amplification. The newly developed assay incorporated FO-SPR DNA melting assay, previously developed by our group. This required establishment of several assay parameters, including buffer ionic strength and thermal ramping speed as these parameters both influence the ligation enzyme performance and the hybridization yield of the gold nanoparticles (Au NPs) on the FO-SPR sensor. Quantification and identification of DNA targets was achieved over a wide concentration range with a calibration curve spanning 7 orders of magnitude and LOD of 13.75 fM. Moreover, the FO-SPR LCR assay could discriminate single nucleotide polymorphism (SNPs) without any post reaction analysis, featuring thus all the essential requirements of POC tests.

  13. [Development and application of real-time PCR for identification and detection of horse meat in animal-origin products].

    PubMed

    Li, Nan; Wang, Jiahui; Shen, Qing; Han, Chunhui; Zhang, Jing; Li, Fengqin; Xu, Jin; Jiang, Tao

    2013-11-01

    To develop a real-time PCR method for identification and detection of domestic horse meat (Equus caballus) in animal-origin products. The primer and TaqMan-probe was designed and synthesized according to the EU reference laboratory and 87 bp fragments was amplified for horse ingredients. The specificity and sensitivity was tested by artificially spiked horse meat into other domestic meat, such as cattle, sheep, pork, chicken, duck and rabbit. 122 samples of cattle and sheep products were random collected in Beijing market and the detection of horse meat was carried out. The real-time PCR in this study has high specificity and sensitivity for horse meat. No cross-reaction was observed between the horse and sheep, pork, chicken, duck and rabbit meat. There was little cross reaction between horse and cattle when the CT value reach 33. 81. The method can detect 0.1% of horse meat mixed with other domestic animal-origin products. No horse meat ingredients were detected in 122 samples in this survey. There was no horse meat mixed into cattle and sheep products in Beijing marked.

  14. Identification and evaluation of new target sequences for specific detection of Bordetella pertussis by real-time PCR.

    PubMed

    Probert, William S; Ely, Janet; Schrader, Kimmi; Atwell, Jessica; Nossoff, Angela; Kwan, Stanley

    2008-10-01

    A comparative analysis of the Bordetella pertussis, B. bronchiseptica, and B. parapertussis genome assemblies permitted the identification of regions with significant sequence divergence and the design of two new real-time PCR assays, BP283 and BP485, for the specific detection of B. pertussis. The performance characteristics of these two assays were evaluated and compared to those of culture and an existing real-time PCR assay targeting the repetitive element IS481. The testing of 324 nasopharyngeal specimens indicated that, compared to culture, the BP283 assay had a sensitivity and specificity of 100 and 96.8% and the BP485 assay had a sensitivity and specificity of 92.3 and 97.1%. Notably, B. holmesii was isolated from two specimens that were positive by the IS481 assay but negative by the BP283 and BP485 assays. These two assays represent an improvement in specificity over those of PCR assays targeting only IS481 and may be duplexed or used in conjunction with existing PCR assays to improve the molecular detection of B. pertussis.

  15. Performance of an in-house real-time polymerase chain reaction for identification of Mycobacterium tuberculosis isolates in laboratory routine diagnosis from a high burden setting

    PubMed Central

    Gallo, Juliana Failde; Pinhata, Juliana Maira Watanabe; Chimara, Erica; Gonçalves, Maria Gisele; Fukasawa, Lucila Okuyama; de Oliveira, Rosangela Siqueira

    2016-01-01

    Abstract Brazil is one of the high burden countries for tuberculosis, and a rapid diagnosis is essential for effective control of the disease. In the present study, an in-house real-time polymerase chain reaction (PCR) assay targeting the mpt64 gene for identification of Mycobacterium tuberculosis complex isolates was evaluated under routine diagnosis conditions in a reference laboratory. From May 2011 to July 2012, 1,520 isolates of mycobacteria were prospectively submitted for phenotypic and/or PRA-hsp65 identification and to real-time PCR. The mpt64 real-time PCR showed 99.7% sensitivity and 96% specificity and detected 79.4% of the cases missed by phenotypic and PRA-hsp65 identification. The in-house real-time PCR assay showed high sensitivity and specificity and was successfully implemented in the routine diagnosis of tuberculosis in a reference laboratory from a high burden setting. PMID:27598243

  16. Performance of an in-house real-time polymerase chain reaction for identification of Mycobacterium tuberculosis isolates in laboratory routine diagnosis from a high burden setting.

    PubMed

    Gallo, Juliana Failde; Pinhata, Juliana Maira Watanabe; Chimara, Erica; Gonçalves, Maria Gisele; Fukasawa, Lucila Okuyama; Oliveira, Rosangela Siqueira de

    2016-09-01

    Brazil is one of the high burden countries for tuberculosis, and a rapid diagnosis is essential for effective control of the disease. In the present study, an in-house real-time polymerase chain reaction (PCR) assay targeting the mpt64 gene for identification of Mycobacterium tuberculosis complex isolates was evaluated under routine diagnosis conditions in a reference laboratory. From May 2011 to July 2012, 1,520 isolates of mycobacteria were prospectively submitted for phenotypic and/or PRA-hsp65 identification and to real-time PCR. The mpt64 real-time PCR showed 99.7% sensitivity and 96% specificity and detected 79.4% of the cases missed by phenotypic and PRA-hsp65 identification. The in-house real-time PCR assay showed high sensitivity and specificity and was successfully implemented in the routine diagnosis of tuberculosis in a reference laboratory from a high burden setting.

  17. Novel intein-containing DNA specific primers for rapid identification of Candida glabrata using Real-Time PCR assays.

    PubMed

    Kumar, R Satish; Ramesh, S

    2014-12-01

    Candida glabrata is an opportunistic human pathogen known to cause systemic and vaginal candidiasis. Rapid detection of Candida glabrata is indispensable for appropriate selection of antifungal drugs for chemotherapy. The study describes a unique intein-containing DNA fragment for specific detection of C. glabrata. The designed oligonucleotides detected C. glabrata (Ct mean: 24.75 ± 1.1 and Tm: 70.08 ± 0.23°C) in Real-Time PCR assays. The fluorescent signals were negative when the primers were tested for cross-species and cross-genera amplifications. In conclusion, our study recommends a novel primer set for developing a quick identification system which does not require laborious and time-consuming experimentations.

  18. Isolation of epithelial cells from acrylic removable dentures and gender identification by amplification of SRY gene using real time PCR.

    PubMed

    George, Renjith; Sriram, G; Saraswathi, Tr; Sivapathasundharam, B

    2010-01-01

    This study evaluates the usefulness of acrylic dentures as the source of DNA for forensic analysis. Thirty-eight samples (21 males and 17 females) were collected and stored for different time periods. The epithelial cells adhered to the dentures were retrieved and the genomic DNA was extracted. All the samples yielded sufficient amount of DNA for analysis irrespective of the storage time. Gender determination was done by amplification of the sex determining region on the Y chromosome (SRY) using real-time polymerase chain reaction with 100% accuracy, within minimal time. With this study, we conclude that saliva-stained acrylic dentures can act as a source of forensic DNA and co-amplification of SRY gene with other routine sex typing markers will give unambiguous gender identification.

  19. Identification of the five human Plasmodium species including P. knowlesi by real-time polymerase chain reaction.

    PubMed

    Oddoux, O; Debourgogne, A; Kantele, A; Kocken, C H; Jokiranta, T S; Vedy, S; Puyhardy, J M; Machouart, M

    2011-04-01

    Recently, Plasmodium knowlesi has been recognised as the fifth Plasmodium species causing malaria in humans. Hundreds of human cases infected with this originally simian Plasmodium species have been described in Asian countries and increasing numbers are reported in Europe from travellers. The growing impact of tourism and economic development in South and Southeast Asia are expected to subsequently lead to a further increase in cases both among locals and among travellers. P. knowlesi is easily misidentified in microscopy as P. malariae or P. falciparum. We developed new primers for the rapid and specific detection of this species by low-cost real-time polymerase chain reaction (PCR) and added this method to an already existing panel of primers used for the molecular identification of the other four species in one reaction. Reference laboratories should now be able to identify undisputably and rapidly P. knowlesi, as it is a potentially fatal pathogen.

  20. Plant seed species identification from chemical fingerprints: a high-throughput application of direct analysis in real time mass spectrometry.

    PubMed

    Lesiak, Ashton D; Cody, Robert B; Dane, A John; Musah, Rabi A

    2015-09-01

    Plant species identification based on the morphological features of plant parts is a well-established science in botany. However, species identification from seeds has largely been unexplored, despite the fact that the seeds contain all of the genetic information that distinguishes one plant from another. Using seeds of genus Datura plants, we show here that the mass spectrum-derived chemical fingerprints for seeds of the same species are similar. On the other hand, seeds from different species within the same genus display distinct chemical signatures, even though they may contain similar characteristic biomarkers. The intraspecies chemical signature similarities on the one hand, and interspecies fingerprint differences on the other, can be processed by multivariate statistical analysis methods to enable rapid species-level identification and differentiation. The chemical fingerprints can be acquired rapidly and in a high-throughput manner by direct analysis in real time mass spectrometry (DART-MS) analysis of the seeds in their native form, without use of a solvent extract. Importantly, knowledge of the identity of the detected molecules is not required for species level identification. However, confirmation of the presence within the seeds of various characteristic tropane and other alkaloids, including atropine, scopolamine, scopoline, tropine, tropinone, and tyramine, was accomplished by comparison of the in-source collision-induced dissociation (CID) fragmentation patterns of authentic standards, to the fragmentation patterns observed in the seeds when analyzed under similar in-source CID conditions. The advantages, applications, and implications of the chemometric processing of DART-MS derived seed chemical signatures for species level identification and differentiation are discussed.

  1. Contribution of real-time PCR to Plasmodium species identification and to clinical decisions: a nationwide study in a non-endemic setting.

    PubMed

    Grossman, T; Schwartz, E; Vainer, J; Agmon, V; Glazer, Y; Goldmann, D; Marva, E

    2017-04-01

    Treatment choice for patients with malaria in Israeli hospitals is based on microscopy and rapid diagnostic tests (RDTs). Here, we demonstrate the cumulative value of real-time polymerase chain reaction (PCR) in optimizing the treatment of malaria. Between January 2009 and December 2015, 451 samples from 357 patients were tested in our laboratory using a real-time PCR assay. Hospital laboratory results (without real-time PCR) were compared to those obtained in our laboratory. A total of 307 patients had a malaria-positive laboratory finding in the hospital. Out of those, 288 were confirmed positive and 19 negative using real-time PCR. Two negative hospital results were found to be positive by real-time PCR. More specifically, of 153 cases positive for Plasmodium falciparum by real-time PCR, only 138 (90%) had been correctly identified at the hospitals. Similarly, 66 (67%) of 99 cases positive for P. vivax, 2 (11%) of 18 cases positive for P. ovale, and 3 (30%) of 10 cases positive for P. malariae had been correctly identified. Of 10 cases of mixed infection, only one had been identified as such at the hospital. Thus, real-time PCR was required for correct identification in 81 (28%) out of 290 positive cases. In 52 (18%) of those, there was an erroneous categorization of relapsing versus non-relapsing parasites. In a nationwide study, we found that the use of real-time PCR is definitely beneficial and may change the decision regarding the choice of treatment.

  2. The use of real-time PCR and species-specific primers for the identification and monitoring of Paecilomyces lilacinus.

    PubMed

    Atkins, Simon D; Clark, Ian M; Pande, Sonal; Hirsch, Penny R; Kerry, Brian R

    2005-01-01

    The Paecilomyces lilacinus is the most widely tested fungus for the control of root-knot and cyst nematodes. The fungus has also been implicated in a number of human and animal infections, difficulties in diagnosis often result in misdiagnosis or delays in identification leading to a delay in treatment. Here, we report the development of species-specific primers for the identification of P. lilacinus based on sequence information from the ITS gene, and their use in identifying P. lilacinus isolates, including clinical isolates of the fungus. The primer set generated a single PCR fragment of 130 bp in length that was specific to P. lilacinus and was also used to detect the presence of P. lilacinus from soil, roots and nematode eggs. Real-time PCR primers and a TaqMan probe were also developed and provided quantitative data on the population size of the fungus in two field sites. PCR, bait and culture methods were combined to investigate the presence and abundance of the fungus from two field sites in the United Kingdom where potato cyst nematode populations were naturally declining, and results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.

  3. Laser-induced breakdown spectroscopy: a tool for real-time, in vitro and in vivo identification of carious teeth.

    PubMed

    Samek, Ota; Telle, Helmut H; Beddows, David CS

    2001-01-01

    BACKGROUND: Laser Induced Breakdown Spectroscopy (LIBS) can be used to measure trace element concentrations in solids, liquids and gases, with spatial resolution and absolute quantifaction being feasible, down to parts-per-million concentration levels. Some applications of LIBS do not necessarily require exact, quantitative measurements. These include applications in dentistry, which are of a more "identify-and-sort" nature - e.g. identification of teeth affected by caries. METHODS: A one-fibre light delivery / collection assembly for LIBS analysis was used, which in principle lends itself for routine in vitro / in vivo applications in a dental practice. A number of evaluation algorithms for LIBS data can be used to assess the similarity of a spectrum, measured at specific sample locations, with a training set of reference spectra. Here, the description has been restricted to one pattern recognition algorithm, namely the so-called Mahalanobis Distance method. RESULTS: The plasma created when the laser pulse ablates the sample (in vitro / in vivo), was spectrally analysed. We demonstrated that, using the Mahalanobis Distance pattern recognition algorithm, we could unambiguously determine the identity of an "unknown" tooth sample in real time. Based on single spectra obtained from the sample, the transition from caries-affected to healthy tooth material could be distinguished, with high spatial resolution. CONCLUSIONS: The combination of LIBS and pattern recognition algorithms provides a potentially useful tool for dentists for fast material identification problems, such as for example the precise control of the laser drilling / cleaning process.

  4. Rapid molecular identification of Listeria species by use of real-time PCR and high-resolution melting analysis.

    PubMed

    Jin, Dazhi; Luo, Yun; Zhang, Zheng; Fang, Weijia; Ye, Julian; Wu, Fang; Ding, Gangqiang

    2012-05-01

    Identification of Listeria species via a molecular method is critical for food safety and clinical diagnosis. In this study, an assay integrating real-time quantitative PCR (Q-PCR) with high-resolution melting (HRM) curve analysis was developed and assessed for rapid identification of six Listeria species. The ssrA gene, which encodes a transfer-messenger RNA (tmRNA) is conserved and common to all bacterial phyla, contains a variable domain in Listeria spp. Therefore, Q-PCR and a HRM profile were applied to characterize this gene. Fifty-three Listeria species and 45 non-Listeria species were detected using one primer set, with an accuracy of 100% in reference to conventional methods. There was a 93.3% correction rate to 30 artificially contaminated samples. Thus, Q-PCR with melting profiling analysis proved able to identify Listeria species accurately. Consequently, this study demonstrates that the assay we developed is a functional tool for rapidly identifying six Listeria species, and has the potential for discriminating novel species food safety and epidemiological research.

  5. Diagnosis and identification of Leishmania spp. from Giemsa-stained slides, by real-time PCR and melting curve analysis in south-west of Iran.

    PubMed

    Khademvatan, S; Neisi, N; Maraghi, S; Saki, J

    2011-12-01

    The aim of present study was describing a real-time PCR assay for the diagnosis and direct identification of Leishmania species on Giemsa-stained slides in south-west of Iran. Altogether, 102 Giemsa-stained slides were collected from different part of south-west of Iran between 2008 and 2011. All the Giemsa-stained slides were examined under light microscope. After DNA extraction, real-time PCR amplification and detection were conducted with fluorescent SYBR Green I. For identification, PCR products were analysed with melting curve analysis. One hundred and two archived slides from suspected lesion examined by microscopy and real-time PCR. The sensitivity of the real-time PCR on Giemsa-stained slid was 98% (96/102). The melting curve analysis (T(m)) were 88·3±0·2°C for L. tropica (MHOM/IR/02/Mash10), 86·5±0·2°C for L. major (MHOM/IR/75/ER) and 89·4±0·3°C for L. infantum (MCAN/IR/97/LON 49), respectively. This study is first report in use of real-time PCR for diagnosis and identification of Leishmania spp. in Iran. Up to now, in Iran, the majority of identification of Leishmania species is restriction fragment length polymorphism (PCR-RFLP) of ITS1 and kinetoplast DNA. Our data showed that Giemsa-stained slides that were stored more than 3 years, can be use for Leishmania DNA extraction and amplification by real-time PCR. Compared to conventional PCR-based methods, the real-time PCR is extremely rapid with results and more samples can be processed at one time.

  6. Real-time PCR-based identification of Borrelia burgdorferi sensu lato species in ticks collected from humans in Romania.

    PubMed

    Briciu, Violeta T; Meyer, Fabian; Sebah, Daniela; Tăţulescu, Doina F; Coroiu, Georgiana; Lupşe, Mihaela; Carstina, Dumitru; Mihalca, Andrei D; Hizo-Teufel, Cecilia; Klier, Christiane; Huber, Ingrid; Fingerle, Volker

    2014-09-01

    The aims of our study were to determine (i) which tick species bite humans in Romania and (ii) the prevalence of Borrelia (B.) burgdorferi genospecies in these ticks. All ticks collected from patients who presented to the Clinic of Infectious Diseases Cluj Napoca in spring/summer 2010 were morphologically identified by an entomologist and tested for B. burgdorferi genospecies prevalence by a real-time PCR assay targeting the hbb gene and melting curve analysis. Out of 532 ticks, 518 were Ixodes ricinus, 10 Dermacentor marginatus, and 3 Haemaphysalis spp. ticks, and one unidentified tick due to destruction. Since evaluation of the hbb PCR revealed that it was not possible to differentiate between B. spielmanii/B. valaisiana and B. garinii/B. bavariensis, sequencing of an 800-bp fragment of the ospA gene was performed in these cases. Out of 389 investigated ticks, 43 were positive by hbb PCR for B. burgdorferi sensu lato. The positive samples were 42 Ixodes ricinus (11.1% B. burgdorferi sensu lato prevalence) and the one unidentified tick. Species identification revealed the presence of mainly B. afzelii, but also of B. garinii, B. burgdorferi sensu stricto, B. valaisiana, and B. lusitaniae. In 4 samples, differentiation between B. spielmanii/B. valaisiana was impossible. Our study shows that the most relevant human pathogenic B. burgdorferi genospecies - predominantly B. afzelii - are present in ticks collected from Romanian patients.

  7. Multi-probe real-time PCR identification of four common Candida species in blood culture broth.

    PubMed

    Foongladda, Suporn; Mongkol, Nanthanida; Petlum, Pornphan; Chayakulkeeree, Methee

    2014-06-01

    We developed a single-tube real-time polymerase chain reaction (PCR) assay with multiple hybridization probes for detecting Candida albicans, C. tropicalis, C. glabrata, and C. parapsilosis. Primers were designed to amplify 18S rRNA gene of the genus Candida, and DNA probes were designed to hybridize two areas of the amplicons. The amplification curves and specific melting peaks of the probes hybridized with PCR product were used for definite species identifications. The reaction specificity was 100 % when evaluating the assay using DNA samples from 21 isolates of fungal and bacterial species. The assay was further evaluated in 129 fungal blood culture broth samples which were culture positive for fungus. Of the 129 samples, 119 were positively identified as: C. albicans (39), C. tropicalis (30), C. parapsilosis (23), C. glabrata (20), Candida spp. (5), and two samples containing mixed C. glabrata/C. albicans and C. glabrata/C. tropicalis. The five Candida spp. were identified by sequencing analysis as C. krusei, C. dubliniensis, C. aquaetextoris, and two isolates of C. athensensis. Of the ten samples which showed negative PCR results, six were Cryptococcus neoformans, and the others were Trichosporon sp., Rhodotorula sp., Fusarium sp., and Penicillium marneffei. Our findings show that the assay was highly effective in identifying the four medically important Candida species. The results can be available within 3 h after positivity of a blood culture broth sample.

  8. Accurate and rapid identification of the Burkholderia pseudomallei near-neighbour, Burkholderia ubonensis, using real-time PCR.

    PubMed

    Price, Erin P; Sarovich, Derek S; Webb, Jessica R; Ginther, Jennifer L; Mayo, Mark; Cook, James M; Seymour, Meagan L; Kaestli, Mirjam; Theobald, Vanessa; Hall, Carina M; Busch, Joseph D; Foster, Jeffrey T; Keim, Paul; Wagner, David M; Tuanyok, Apichai; Pearson, Talima; Currie, Bart J

    2013-01-01

    Burkholderia ubonensis is an environmental bacterium belonging to the Burkholderia cepacia complex (Bcc), a group of genetically related organisms that are associated with opportunistic but generally nonfatal infections in healthy individuals. In contrast, the near-neighbour species Burkholderia pseudomallei causes melioidosis, a disease that can be fatal in up to 95% of cases if left untreated. B. ubonensis is frequently misidentified as B. pseudomallei from soil samples using selective culturing on Ashdown's medium, reflecting both the shared environmental niche and morphological similarities of these species. Additionally, B. ubonensis shows potential as an important biocontrol agent in B. pseudomallei-endemic regions as certain strains possess antagonistic properties towards B. pseudomallei. Current methods for characterising B. ubonensis are laborious, time-consuming and costly, and as such this bacterium remains poorly studied. The aim of our study was to develop a rapid and inexpensive real-time PCR-based assay specific for B. ubonensis. We demonstrate that a novel B. ubonensis-specific assay, Bu550, accurately differentiates B. ubonensis from B. pseudomallei and other species that grow on selective Ashdown's agar. We anticipate that Bu550 will catalyse research on B. ubonensis by enabling rapid identification of this organism from Ashdown's-positive colonies that are not B. pseudomallei.

  9. Identification of deletion carriers in hemophilia B: quantitative real-time polymerase chain reaction or multiple ligation probe amplification.

    PubMed

    Casaña, Pilar; Haya, Saturnino; Cid, Ana R; Oltra, Silvestre; Martínez, Francisco; Cabrera, Noelia; Aznar, José A

    2009-03-01

    Gross deletions in the F9 gene are easily detected by routinely sequencing hemophilia B-affected men. Nevertheless, a carrier diagnosis proves difficult as the presence of a normal allele does not recognize the partial or complete loss of the F9 gene and may be challenging if no DNA sample from affected men is available. This work aimed to identify hemophilia carriers in 2 families in which gross deletions of the F9 gene could be expected. The indirect genetic study was not conclusive, and sequencing did not show genetic defects in family 1. A real-time polymerase chain reaction (RT-PCR) assay using SYBR Green revealed the deletion of a copy of exon 8 in 3 women, whereas the multiple ligation-dependent probe amplification (MLPA) assay showed the deletion of a copy of exons 7 and 8 in these 3 women. These studies enabled us not only to rule out a pregnant woman as a carrier but also to confirm a complete deletion of the gene in the patient from family 2 and the heterozygous state of his mother. The advantages that the MLPA method offers are the identification of a multiple exon deletion in the same assay and commonly used technology. The RT-PCR technology used involves standardizing and analyzing each exon independently.

  10. Identification and validation of reference genes for quantitative real-time polymerase chain reaction in Cimex lectularius.

    PubMed

    Mamidala, Praveen; Rajarapu, Swapna P; Jones, Susan C; Mittapalli, Omprakash

    2011-07-01

    Quantitative real-time polymerase chain reaction (qRT-PCR) has emerged as robust methodology for gene expression studies, but reference genes are crucial for accurate normalization. Commonly used reference genes are housekeeping genes that are thought to be nonregulated; however, their expression can be unstable across different experimental conditions. We report the identification and validation of suitable reference genes in the bed bug, Cimex lectularius, by using qRT-PCR. The expression stability of eight reference genes in different tissues (abdominal cuticle, midgut, Malpighian tubules, and ovary) and developmental stages (early instar nymphs, late instar nymphs, and adults) of pesticide-susceptible and pesticide-exposed C. lectularius were analyzed using geNorm, NormFinder, and BestKeeper. Overall expression analysis of the eight reference genes revealed significant variation among samples, indicating the necessity of validating suitable reference genes for accurate quantification of mRNA transcripts. Ribosomal protein (RPL18) exhibited the most stable gene expression across all the tissue and developmental-stage samples; a-tubulin revealed the least stability across all of the samples examined. Thus, we recommend RPL18 as a suitable reference gene for normalization in gene expression studies of C. lectularius.

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

    PubMed

    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.

  12. Identification of WA-type three-line hybrid rice with real-time polymerase chain reaction (PCR) method.

    PubMed

    Cheng, Y; Gao, B D; Chen, H Y; Mao, J J; Cao, A X; Zhu, J G; Zhu, S F

    2012-02-01

    A real-time fluorescent PCR (RTF-PCR) was developed to detect and quantify wild abortive (WA)-type three-line hybrid rice (Oryza sativa L.). The mitochondrial R₂₋₆₃₀ WA gene was reported to be closely related to male sterility in plants, and developed as a molecular maker to identify the cytoplasmic male sterility system of hybrid rice. First, we got the DNA sequence of R₂₋₆₃₀ WA gene in 17 rice species with traditional PCR. Then, a pair of specific primers (P₃, P₄) and TaqMan fluorescence probe (P₃₋₁₄) were designed based on the R₂₋₆₃₀ DNA sequence. The following RTF-PCR was performed on the 17 rice species finally. The results indicate that the probes used here are specific for three-line hybrid rice F₁ and male sterile lines. We can even identify a single hybrid seed using the probes, which confirmed that the probes can be applied to the identification and quantification of the WA-type three-line hybrid rice. In addition, the RFT-PCR system can be optimized when the annealing temperature is 60 °C and the Mg²⁺ concentration is 3.5 mmol/L.

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

    PubMed Central

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

    2016-01-01

    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

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

    PubMed

    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

  15. Development, validation and implementation of a quadruplex real-time PCR assay for identification of potentially toxigenic corynebacteria.

    PubMed

    De Zoysa, Aruni; Efstratiou, Androulla; Mann, Ginder; Harrison, Timothy G; Fry, Norman K

    2016-12-01

    Toxigenic corynebacteria are uncommon in the UK; however, laboratory confirmation by the national reference laboratory can inform public health action according to national guidelines. Standard phenotypic tests for identification and toxin expression of isolates can take from ≥24 to ≥48 h from receipt. To decrease the time to result, a real-time PCR (qPCR) assay was developed for confirmation of both identification of Corynebacterium diphtheriae and Corynebacterium ulcerans/Corynebacterium pseudotuberculosis and detection of the diphtheria toxin gene. Target genes were the RNA polymerase β-subunit-encoding gene (rpoB) and A-subunit of the diphtheria toxin gene (tox). Green fluorescent protein DNA (gfp) was used as an internal process control. qPCR results were obtained within 3 to 4 h after receipt of isolate. The assay was validated according to published guidelines and demonstrated high diagnostic sensitivity (100 %), high specificity (98-100 %) and positive and negative predictive values of 91 to 100 % and 100 %, respectively, compared to both block-based PCR and the Elek test, together with a greatly reduced time from isolate receipt to reporting. Limitations of the qPCR assay were the inability to distinguish between C. ulcerans and C. pseudotuberculosis and that the presence of the toxin gene as demonstrated by qPCR may not always predict toxin expression. Thus, confirmation of expression of diphtheria toxin is always sought using the phenotypic Elek test. The new qPCR assay was formally introduced as the front-line test for putative toxigenic corynebacteria to inform public health action in England and Wales on 1 April 2014.

  16. Real-Time Operating System/360

    NASA Technical Reports Server (NTRS)

    Hoffman, R. L.; Kopp, R. S.; Mueller, H. H.; Pollan, W. D.; Van Sant, B. W.; Weiler, P. W.

    1969-01-01

    RTOS has a cost savings advantage for real-time applications, such as those with random inputs requiring a flexible data routing facility, display systems simplified by a device independent interface language, and complex applications needing added storage protection and data queuing.

  17. Real-time intraoperative full-range complex FD-OCT guided cerebral blood vessel identification and brain tumor resection in neurosurgery

    NASA Astrophysics Data System (ADS)

    Zhang, Kang; Huang, Yong; Pradilla, Gustavo; Tyler, Betty; Kang, Jin U.

    2011-03-01

    This work utilized an ultra-high-speed full-range complex-conjugate-free optical coherence tomography (FD-OCT) system to perform real-time intraoperative imaging to guide two common neurosurgical procedures: the cerebral blood vessel identification and the brain tumor resection. The cerebral blood vessel identification experiment is conducted ex vivo on human cadaver specimen. Specific cerebral arteries and veins in different positions of the specimen are visualized and the spatial relations between adjacent vessels are indentified through real-time 3D visualization. The brain tumor resection experiment is conducted in vivo on 9L gliomas established in rat brains. The normal brain-tumor margin can be clearly identified in depth of the tissue from sagittal, coronal and axial slices of the intraoperatively acquired 3D data set. The real-time full-range FD-OCT guided in vivo rat flank tumor resection is also conducted.

  18. Real-Time Intraoperative Near-Infrared Fluorescence Identification of the Extrahepatic Bile Ducts using Clinically-Available Contrast Agents

    PubMed Central

    Matsui, Aya; Tanaka, Eiichi; Choi, Hak Soo; Winer, Joshua H.; Kianzad, Vida; Gioux, Sylvain; Laurence, Rita G.; Frangioni, John V.

    2009-01-01

    Background Iatrogenic bile duct injuries are serious complications with patient morbidity. We hypothesized that the invisible near-infrared (NIR) fluorescence properties of methylene blue (MB) and indocyanine green (ICG) could be exploited for real-time, intraoperative imaging of the extrahepatic bile ducts during open and laparoscopic surgeries. Methods 2.0 mg/kg of MB and 0.05 mg/kg of ICG were intravenously injected into 35-kg female Yorkshire pigs and the extrahepatic bile ducts imaged over time using either the FLARE™ image-guided surgery system (open surgery) or a custom NIR fluorescence laparoscopy system. Surgical anatomy was confirmed using x-ray cholangiography. Contrast-to-background ratio (CBR), contrast-to-liver ratio (CLR), and chemical concentrations in the cystic duct (CD) and common bile duct (CBD) were measured, and the performance of each agent quantified. Results Using NIR fluorescence of MB, the CD and CBD could be identified with good sensitivity (CBR and CLR ≥ 4), during both open and laparoscopic surgeries, from 10 to 120 min post-injection. Functional impairment of the ducts, including constriction and injury were immediately identifiable. Using NIR fluorescence of ICG, extrahepatic bile ducts did not become visible until 90 min post-injection due to strong residual liver retention, however, between 90 to 240 min, ICG provided exquisitely high sensitivity for both CD and CBD, with CBR ≥ 8 and CLR ≥ 4. Conclusions We demonstrate that two clinically available NIR fluorophores, MB fluorescing at 700 nm and ICG fluorescing at 800 nm, provide sensitive, prolonged identification of the extrahepatic bile ducts and assessment of their functional status. PMID:20117813

  19. Development of a real-time PCR assay for the identification of Gyrodactylus parasites infecting salmonids in northern Europe.

    PubMed

    Collins, Catherine M; Kerr, Rose; McIntosh, Rebecca; Snow, Mike

    2010-06-11

    Gyrodactylus salaris is a monogenean freshwater parasite that causes high mortality in wild Atlantic salmon, and a number of countries employ monitoring programmes for its presence. A TaqMan-MGB (minor groove binding) probe real-time multiplex assay targeting the internal transcribed spacer ribosomal DNA (ITS rDNA) was developed to simultaneously identify G. salaris/G. thymalli and 2 other commonly occurring Gyrodactylus species infecting salmonids in northern Europe: G. derjavinoides and G. truttae. In addition, a Gyrodactylus genus-level assay was developed to assess parasite DNA quality. The species-specific real-time PCR method correctly identified target species from a wide geographical range and from a number of salmonid hosts. It did not amplify G. lucii or G. teuchis. These species were successfully amplified using the Gyrodactylus genus real-time assay. The species-specific real-time assay proved to be significantly faster than the currently employed molecular screening method of ITS rDNA PCR amplification followed by restriction fragment length polymorphism analyses (RFLP). However, as with ITS RFLP, the real-time method did not distinguish between G. salaris and the non-pathogenic G. thymalli, its principle advantage being a significant reduction in time to achieve an initial diagnostic screen before the employment of more in-depth analyses for those specimens giving a positive G. salaris/G. thymalli real-time result.

  20. Rapid and accurate identification of Mycobacterium tuberculosis complex and common non-tuberculous mycobacteria by multiplex real-time PCR targeting different housekeeping genes.

    PubMed

    Nasr Esfahani, Bahram; Rezaei Yazdi, Hadi; Moghim, Sharareh; Ghasemian Safaei, Hajieh; Zarkesh Esfahani, Hamid

    2012-11-01

    Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T (m)) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100 % for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100 %, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.

  1. Rapid identification of Brucella isolates to the species level by real time PCR based single nucleotide polymorphism (SNP) analysis

    PubMed Central

    Gopaul, Krishna K; Koylass, Mark S; Smith, Catherine J; Whatmore, Adrian M

    2008-01-01

    Background Brucellosis, caused by members of the genus Brucella, remains one of the world's major zoonotic diseases. Six species have classically been recognised within the family Brucella largely based on a combination of classical microbiology and host specificity, although more recently additional isolations of novel Brucella have been reported from various marine mammals and voles. Classical identification to species level is based on a biotyping approach that is lengthy, requires extensive and hazardous culturing and can be difficult to interpret. Here we describe a simple and rapid approach to identification of Brucella isolates to the species level based on real-time PCR analysis of species-specific single nucleotide polymorphisms (SNPs) that were identified following a robust and extensive phylogenetic analysis of the genus. Results Seven pairs of short sequence Minor Groove Binding (MGB) probes were designed corresponding to SNPs shown to possess an allele specific for each of the six classical Brucella spp and the marine mammal Brucella. Assays were optimised to identical reaction parameters in order to give a multiple outcome assay that can differentiate all the classical species and Brucella isolated from marine mammals. The scope of the assay was confirmed by testing of over 300 isolates of Brucella, all of which typed as predicted when compared to other phenotypic and genotypic approaches. The assay is sensitive being capable of detecting and differentiating down to 15 genome equivalents. We further describe the design and testing of assays based on three additional SNPs located within the 16S rRNA gene that ensure positive discrimination of Brucella from close phylogenetic relatives on the same platform. Conclusion The multiple-outcome assay described represents a new tool for the rapid, simple and unambiguous characterisation of Brucella to the species level. Furthermore, being based on a robust phylogenetic framework, the assay provides a platform

  2. Rapid real-time PCR assay for culture and tissue identification of Geomyces destructans: the etiologic agent of bat geomycosis (white nose syndrome).

    PubMed

    Chaturvedi, Sudha; Rudd, Robert J; Davis, April; Victor, Tanya R; Li, Xiaojiang; Appler, Kim A; Rajkumar, Sunanda S; Chaturvedi, Vishnu

    2011-10-01

    Geomyces destructans is the etiologic agent of bat geomycosis, commonly referred to as white nose syndrome (WNS). This infection has caused severe morbidity and mortality in little brown bats (Myotis lucifugus) and has also spread to other bat species with significant decline in the populations. Currently, G. destructans infection is identified by culture, ITS-PCR, and histopathology. We hypothesized that a real-time PCR assay would considerably improve detection of G. destructans in bats. The 100 bp sequence of the Alpha-L-Rhamnosidase gene was validated as a target for real-time PCR. The assay sensitivity was determined from serial dilution of DNA extracted from G. destructans conidia (5 × 10(-1)-5 × 10(7)), and the specificity was tested using DNA from 30 closely and distantly related fungi and 5 common bacterial pathogens. The real-time PCR assay was highly sensitive with detection limit of two G. destructans conidia per reaction at 40 PCR cycles. The assay was also highly specific as none of the other fungal or bacterial DNA cross-reacted in the real-time PCR assay. One hundred and forty-seven bat tissue samples, suspected of infection with G. destructans, were used to compare the real-time PCR assay to other methods employed for the detection of G. destructans. Real-time PCR was highly sensitive with 80 of 147 (55%) samples testing positive for G. destructans DNA. In comparison, histopathology examination revealed 64/147 (44%) positive samples. The internal transcribed spacer (ITS)-PCR yielded positive amplicon for G. destructans from 37 tissue samples (25%). The least sensitive assay was the fungal culture with only 17 tissue samples (12%) yielding G. destructans in culture. The data suggested that the real-time PCR assay is highly promising for rapid, sensitive, and specific identification of G. destructans. Further trials and inter-laboratory comparisons of this novel assay are recommended to improve the diagnosis of bat geomycosis.

  3. A rapid, real-time quantitative polymerase chain reaction test for the identification of pathogens in bronchoalveolar lavage samples.

    PubMed

    Orlando, Alessandro; Thoma, Gregory; Slone, Denetta S; Mains, Charles W; Bar-Or, David

    2014-03-01

    Standard bacteriologic culture techniques offer results within 2 days to 3 days, precluding a focused and timely antibiotic therapy in ventilated trauma patients. Our laboratory developed a real-time quantitative polymerase chain reaction (qPCR) test that can detect 25 different bacteria and fungi and methicillin resistance and offers results within 3 hours. The objective of this study was to compare the qPCR method to standard culture techniques. This was a prospective observational cohort study at a Level I trauma center from 2009 to 2012. Adult trauma patients on ventilation, receiving at least one bronchoalveolar lavage (BAL) with culture results were eligible for inclusion. DNA was isolated from the BAL samples and analyzed in 96-well plates using qPCR. Student's t tests were used to examine differences in mean qPCR cycle counts. Sensitivities, specificities, negative predictive values, and positive predictive values were calculated for the qPCR primer sets. There were 28 BALs in the study. The qPCR method detected a total of 165 organisms, and culture methods found 54. The qPCR test had an overall sensitivity of 85%, specificity of 74%, negative predictive value of 98%, and positive predictive value of 27%. Those organisms that were only identified through qPCR had significantly less DNA than those identified through both qPCR and quantitative culture (28.8 vs. 23.3, p < 0.001). Concurrent antibiotic therapy was found to decrease the qPCR specificity in some primer sets, and methicillin resistance was only found in BAL samples that were concurrent with antibiotics. The qPCR method shows promising initial diagnostic value. Many of the organisms not identified by quantitative culture had late cycle calls, suggesting that they might have been in quantities too low to result in culture identification. Once refined, our qPCR method has the potential to identify pathogens faster and earlier than standard quantitative culture methods, allowing for targeted

  4. Two-Color Multiplex Assay for the Identification of Orthopox Viruses with Real-Time LUX-PCR

    DTIC Science & Technology

    2005-07-14

    perfringens 13124 1000 0/2 0/2 Francisella tularensis NA 1000 0/2 0/2 Haemophlius influenzae 10211 1000 0/2 0/2 Listeia monocytogenes 15313 1000 0/2 0/2...Loveless BM, Norwood D, Zwiers SH, Mucker E, Hartmann C, et al. Smallpox and pan-orthopox virus detection by real-time 3 0-minor groove binder TaqMan ...Whitehouse CA, Hartmann C, Mucker E, et al. Monkeypox virus detection in rodents using real-time 3 0-minor groove binder TaqMan assays on the Roche

  5. Detection of mecA and ermA genes and simultaneous identification of Staphylococcus aureus using triplex real-time PCR from Malaysian S. aureus strain collections.

    PubMed

    Sabet, Negar Shafiei; Subramaniam, Geetha; Navaratnam, Parasakthi; Sekaran, Shamala Devi

    2007-05-01

    A triplex real-time polymerase chain reaction (PCR) assay was used for the simultaneous detection of mecA (methicillin resistance), ermA (erythromycin resistance) and femA (Staphylococcus aureus identification) genes in a single assay. Among 93 clinical S. aureus hospital isolates, there were 48 methicillin-resistant S. aureus (MRSA) and 45 methicillin-sensitive S. aureus (MSSA) isolates. Screening the isolates using the triplex real-time PCR assay, the mecA, ermA and femA genes were detected in all MRSA isolates. The triplex real-time PCR assay was completed within 3h and is a useful genotypic method for detecting the resistance determinants as well as for the identification of S. aureus isolates. These findings will assist the clinical laboratory in identifying these resistance genes and S. aureus rapidly, thus benefiting patient therapy. This study represents a valuable source of information for researchers to study the local antibiotic resistance pattern, which can increase our knowledge of the antibiotic resistance profile, using real-time PCR technology.

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

    EPA Science Inventory

    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...

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

    EPA Science Inventory

    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...

  8. Evaluation of a new commercial real-time PCR assay for diagnosis of Pneumocystis jirovecii pneumonia and identification of dihydropteroate synthase (DHPS) mutations.

    PubMed

    Montesinos, Isabel; Delforge, Marie-Luce; Ajjaham, Farida; Brancart, Françoise; Hites, Maya; Jacobs, Frederique; Denis, Olivier

    2017-01-01

    The PneumoGenius® real-time PCR assay is a new commercial multiplex real-time PCR method, which detects the Pneumocystis mitochondrial ribosomal large subunit (mtLSU) and two dihydropteroate synthase (DHPS) point mutations. To evaluate the clinical performance of this new real-time PCR assay we tested 120 extracted DNA samples from bronchoalveolar lavage specimens. These set of extracted DNA samples had already tested positive for Pneumocystis and patients had been classified in probable and unlikely PCP in a previous study. To evaluate de accuracy of the DHPS mutant's identification, an "in house" PCR and sequencing was performed. The sensitivity and specificity of PneumoGenius® PCR in discriminating between probable and unlikely Pneumocystis pneumonia (PCP) were 70% and 82% respectively. PneumoGenius® PCR was able to genotype more samples than "in house" DHPS PCR and sequencing. The same DHPS mutations were observed by both methods in four patients: two patients with a single mutation in position 171 (Pro57Ser) and two patients with a double mutation in position 165 (Thr55Ala) and in position 171 (Pro57Ser). A low rate of P. jirovecii (4.5%) harboring DHPS mutations was found, comparable to rates observed in other European countries. The PneumoGenius® real-time PCR is a suitable real-time PCR for PCP diagnosis and detection of DHPS mutants. The added value of DHPS mutation identification can assist in understanding the role of these mutations in prophylaxis failure or treatment outcome. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. A quantitative real-time PCR assay for the identification and enumeration of Alexandrium cysts in marine sediments

    NASA Astrophysics Data System (ADS)

    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

  10. Development of TaqMan real-time polymerase chain reaction for the detection and identification of Penicillium marneffei.

    PubMed

    Pornprasert, Sakorn; Praparattanapan, Jutarat; Khamwan, Chantana; Pawichai, Sudjai; Pimsarn, Parichat; Samleerat, Tanawan; Leechanachai, Pranee; Supparatpinyo, Khunchai

    2009-11-01

    Penicillium marneffei is a dimorphic fungus, which is endemic in Southeast Asia and responsible for emerging opportunistic infections. Diagnosis of penicilliosis may be difficult when few yeast cells are present, while a gold standard diagnosis technique requires long-term culture. In order to provide a more rapid and accurate diagnosis, we developed a TaqMan real-time PCR to detect and identify P. marneffei DNA coding for 5.8S rRNA in purified yeast DNA and clinical samples. All P. marneffei DNA preparations could be detected using specific primers and TaqMan probe. The assay has a sensitivity to detect at least 10 yeast cells in seeded blood. Moreover, it can detect P. marneffei DNA in peripheral blood samples and blood-culture bottles. Therefore, the real-time PCR assay may represent a potential tool for early diagnosis of penicilliosis marneffei.

  11. Identification of stable reference genes for gene expression studies using quantitative real time PCR in buffalo oocytes and embryos.

    PubMed

    Kumar, Parveen; Yadav, Poonam; Verma, Arpana; Singh, Dheer; De, Sachinandan; Datta, Tirtha Kumar

    2012-12-01

    The present study was aimed to validate expression stability of 6 housekeeping genes (viz. YWHAZ, SDHA, GAPDH, RPS15, RPS18 and RN18S1) in the oocytes and embryos of different stages in buffalo. A modified Trizol protocol was optimized for RNA isolation from as few as five oocytes. The expression level of selected genes was studied by an optimized real time PCR using DCT method and their stability of expression was evaluated by Microsoft Excel based visual application, geNORM. The analysis revealed that the RPS15 and GAPDH were the most stable genes across different samples. Also, the geometric mean of three genes (i.e. RPS15,RPS18 and GAPDH) were found suitable for normalization of real time PCR data from buffalo oocytes⁄embryos. The information would help in more accurate interpretation of gene expression data from oocytes⁄embryos towards understanding the molecular events in these cells during development.

  12. Detection and identification of genetically modified EE-1 brinjal (Solanum melongena) by single, multiplex and SYBR® real-time PCR.

    PubMed

    Ballari, Rajashekhar V; Martin, Asha; Gowda, Lalitha R

    2013-01-01

    Brinjal is an important vegetable crop. Major crop loss of brinjal is due to insect attack. Insect-resistant EE-1 brinjal has been developed and is awaiting approval for commercial release. Consumer health concerns and implementation of international labelling legislation demand reliable analytical detection methods for genetically modified (GM) varieties. End-point and real-time polymerase chain reaction (PCR) methods were used to detect EE-1 brinjal. In end-point PCR, primer pairs specific to 35S CaMV promoter, NOS terminator and nptII gene common to other GM crops were used. Based on the revealed 3' transgene integration sequence, primers specific for the event EE-1 brinjal were designed. These primers were used for end-point single, multiplex and SYBR-based real-time PCR. End-point single PCR showed that the designed primers were highly specific to event EE-1 with a sensitivity of 20 pg of genomic DNA, corresponding to 20 copies of haploid EE-1 brinjal genomic DNA. The limits of detection and quantification for SYBR-based real-time PCR assay were 10 and 100 copies respectively. The prior development of detection methods for this important vegetable crop will facilitate compliance with any forthcoming labelling regulations. Copyright © 2012 Society of Chemical Industry.

  13. Detection and identification of Leishmania species from clinical specimens by using a real-time PCR assay and sequencing of the cytochrome B gene.

    PubMed

    Foulet, Françoise; Botterel, Françoise; Buffet, Pierre; Morizot, Gloria; Rivollet, Danièle; Deniau, Michèle; Pratlong, Francine; Costa, Jean-Marc; Bretagne, Stéphane

    2007-07-01

    Visceral and cutaneous leishmaniases are heterogenous entities. The Leishmania species that a given patient harbors usually cannot be determined clinically, and this identification is essential to prescribe the best species-specific therapeutic regimen. Our diagnosis procedure includes a real-time PCR assay targeted at the 18S rRNA gene, which detects all Leishmania species but which is not specific for a given Leishmania species. We developed a species identification based on sequencing of the cytochrome b (cyt b) gene directly from the DNA extracted from the clinical specimen. The sequences were analyzed using the Sequence Analysis/Seqscape v2.1 software (Applied Biosystems). This software is designed to automatically identify the closest sequences from a reference library after analysis of all known or unknown polymorphic positions. The library was built with the Leishmania cyt b gene sequences available in GenBank. Fifty-three consecutive real-time PCR-positive specimens were studied for species identification. The cyt b gene was amplified in the 53 specimens. Sequencing resulted in the identification of six different species with >or=99% identity with the reference sequences over 872 nucleotides. The identification was obtained in two working days and was in accordance with the multilocus enzyme electrophoresis identification when available. Real-time PCR followed by sequencing of the cyt b gene confirmed the diagnosis of leishmaniasis and rapidly determined the infecting species directly from the clinical specimen without the need for the isolation of parasites. This technique has the potential to significantly accelerate species-adapted therapeutic decisions regarding treatment of leishmaniasis.

  14. Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay.

    PubMed

    van der Graaf-van Bloois, Linda; van Bergen, Marcel A P; van der Wal, Fimme J; de Boer, Albert G; Duim, Birgitta; Schmidt, Tracy; Wagenaar, Jaap A

    2013-10-01

    Phenotypic differentiation between Campylobacter fetus (C. fetus) subspecies fetus and C. fetus subspecies venerealis is hampered by poor reliability and reproducibility of biochemical assays. AFLP (amplified fragment length polymorphism) and MLST (multilocus sequence typing) are the molecular standards for C. fetus subspecies identification, but these methods are laborious and expensive. Several PCR assays for C. fetus subspecies identification have been described, but a reliable comparison of these assays is lacking. The aim of this study was to evaluate the most practical and routinely implementable published PCR assays designed for C. fetus species and subspecies identification. The sensitivity and specificity of the assays were calculated by using an extensively characterized and diverse collection of C. fetus strains. AFLP and MLST identification were used as reference. Two PCR assays were able to identify C. fetus strains correctly at species level. The C. fetus species identification target, gene nahE, of one PCR assay was used to develop a real-time PCR assay with 100% sensitivity and 100% specificity, but the development of a subspecies venerealis specific real-time PCR (ISCfe1) failed due to sequence variation of the target insertion sequence and prevalence in other Campylobacter species. None of the published PCR assays was able to identify C. fetus strains correctly at subspecies level. Molecular analysis by AFLP or MLST is still recommended to identify C. fetus isolates at subspecies level.

  15. 5'-MGB probes allow rapid identification of methanogens and sulfate reducers in cold marine sediments by real-time PCR and melting curve analysis.

    PubMed

    Afonina, Irina; Savvichev, Alexander; Ankoudinova, Irina; Mahoney, Walt

    2009-09-01

    The analysis of microorganism communities in uncultured environmental samples requires laborious and cumbersome techniques such as denaturing gradient gel electrophoresis of amplicons generated with 16S rRNA generic primers with subsequent fragment sequencing. We have developed a simple method for genus identification of methanogen archaea and sulfate-reducing bacteria based on a real-time PCR hybridization probe melting curve analysis. The method takes advantage of a recent explosion of microorganism sequencing data conveniently packaged in the Ribosomal Database Project. Specificity of detection is based on a genus-specific real-time PCR fluorescent 5'-MGB-probe melt. As the probes are designed to have destabilizing mismatches with undesired genera, only samples with a proper melting temperature are called positive.

  16. Identification and quantitative detection of Legionella spp. in various aquatic environments by real-time PCR assay.

    PubMed

    Kao, Po-Min; Tung, Min-Che; Hsu, Bing-Mu; Chiu, Yi-Chou; She, Cheng-Yu; Shen, Shu-Min; Huang, Yu-Li; Huang, Wen-Chien

    2013-09-01

    In this study, a SYBR green quantitative real-time PCR was developed to quantify and detect the Legionella spp. in various environmental water samples. The water samples were taken from watershed, water treatment plant, and thermal spring area in Taiwan. Legionella was detected in 13.6 % (24/176), and the detection rate for river water, raw drinking water, and thermal spring water was 10, 21.4, and 16.6 %, respectively. Using real-time PCR, concentration of Legionella spp. in detected samples ranged between 9.75 × 10(4) and 3.47 × 10(5) cells/L in river water, 6.92 × 10(4) and 4.29 × 10(5) cells/L in raw drinking water, and 5.71 × 10(4) and 2.12 × 10(6) cells/L for thermal spring water samples. The identified species included Legionella pneumophila (20.8 %), Legionella jordanis (4.2 %), Legionella nautarum (4.2 %), Legionella sp. (4.2 %), and uncultured Legionella sp. (66.6 %). The presence of L. pneumophila in aquatic environments suggested a potential public health threat that must be further examined.

  17. Real-time PCR assays for detection of Brucella spp. and the identification of genotype ST27 in bottlenose dolphins (Tursiops truncatus).

    PubMed

    Wu, Qingzhong; McFee, Wayne E; Goldstein, Tracey; Tiller, Rebekah V; Schwacke, Lori

    2014-05-01

    Rapid detection of Brucella spp. in marine mammals is challenging. Microbiologic culture is used for definitive diagnosis of brucellosis, but is time consuming, has low sensitivity and can be hazardous to laboratory personnel. Serological methods can aid in diagnosis, but may not differentiate prior exposure versus current active infection and may cross-react with unrelated Gram-negative bacteria. This study reports a real-time PCR assay for the detection of Brucella spp. and application to screen clinical samples from bottlenose dolphins stranded along the coast of South Carolina, USA. The assay was found to be 100% sensitive for the Brucella strains tested, and the limit of detection was 0.27fg of genomic DNA from Brucella ceti B1/94 per PCR volume. No amplification was detected for the non-Brucella pathogens tested. Brucella DNA was detected in 31% (55/178) of clinical samples tested. These studies indicate that the real-time PCR assay is highly sensitive and specific for the detection of Brucella spp. in bottlenose dolphins. We also developed a second real-time PCR assay for rapid identification of Brucella ST27, a genotype that is associated with human zoonotic infection. Positive results were obtained for Brucella strains which had been identified as ST27 by multilocus sequence typing. No amplification was found for other Brucella strains included in this study. ST27 was identified in 33% (18/54) of Brucella spp. DNA-positive clinical samples. To our knowledge, this is the first report on the use of a real-time PCR assay for identification of Brucella genotype ST27 in marine mammals.

  18. SNP-based real-time pyrosequencing as a sensitive and specific tool for identification and differentiation of Rickettsia species in Ixodes ricinus ticks

    PubMed Central

    2012-01-01

    Background Rickettsioses are caused by pathogenic species of the genus Rickettsia and play an important role as emerging diseases. The bacteria are transmitted to mammal hosts including humans by arthropod vectors. Since detection, especially in tick vectors, is usually based on PCR with genus-specific primers to include different occurring Rickettsia species, subsequent species identification is mainly achieved by Sanger sequencing. In the present study a real-time pyrosequencing approach was established with the objective to differentiate between species occurring in German Ixodes ticks, which are R. helvetica, R. monacensis, R. massiliae, and R. felis. Tick material from a quantitative real-time PCR (qPCR) based study on Rickettsia-infections in I. ricinus allowed direct comparison of both sequencing techniques, Sanger and real-time pyrosequencing. Methods A sequence stretch of rickettsial citrate synthase (gltA) gene was identified to contain divergent single nucleotide polymorphism (SNP) sites suitable for Rickettsia species differentiation. Positive control plasmids inserting the respective target sequence of each Rickettsia species of interest were constructed for initial establishment of the real-time pyrosequencing approach using Qiagen’s PSQ 96MA Pyrosequencing System operating in a 96-well format. The approach included an initial amplification reaction followed by the actual pyrosequencing, which is traceable by pyrograms in real-time. Afterwards, real-time pyrosequencing was applied to 263 Ixodes tick samples already detected Rickettsia-positive in previous qPCR experiments. Results Establishment of real-time pyrosequencing using positive control plasmids resulted in accurate detection of all SNPs in all included Rickettsia species. The method was then applied to 263 Rickettsia-positive Ixodes ricinus samples, of which 153 (58.2%) could be identified for their species (151 R. helvetica and 2 R. monacensis) by previous custom Sanger sequencing. Real-time

  19. Development of a qualitative real-time PCR method to detect 19 targets for identification of genetically modified organisms.

    PubMed

    Peng, Cheng; Wang, Pengfei; Xu, Xiaoli; Wang, Xiaofu; Wei, Wei; Chen, Xiaoyun; Xu, Junfeng

    2016-01-01

    As the amount of commercially available genetically modified organisms (GMOs) grows recent years, the diversity of target sequences for molecular detection techniques are eagerly needed. Considered as the gold standard for GMO analysis, the real-time PCR technology was optimized to produce a high-throughput GMO screening method. With this method we can detect 19 transgenic targets. The specificity of the assays was demonstrated to be 100 % by the specific amplification of DNA derived from reference material from 20 genetically modified crops and 4 non modified crops. Furthermore, most assays showed a very sensitive detection, reaching the limit of ten copies. The 19 assays are the most frequently used genetic elements present in GM crops and theoretically enable the screening of the known GMO described in Chinese markets. Easy to use, fast and cost efficient, this method approach fits the purpose of GMO testing laboratories.

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

    PubMed

    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. Real-time PCR strategy for the identification of Trypanosoma cruzi discrete typing units directly in chronically infected human blood.

    PubMed

    Muñoz-San Martín, Catalina; Apt, Werner; Zulantay, Inés

    2017-04-01

    The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a major public health problem in Latin America. This parasite has a complex population structure comprised by six or seven major evolutionary lineages (discrete typing units or DTUs) TcI-TcVI and TcBat, some of which have apparently resulted from ancient hybridization events. Because of the existence of significant biological differences between these lineages, strain characterization methods have been essential to study T. cruzi in its different vectors and hosts. However, available methods can be laborious and costly, limited in resolution or sensitivity. In this study, a new genotyping strategy by real-time PCR to identify each of the six DTUs in clinical blood samples have been developed and evaluated. Two nuclear (SL-IR and 18S rDNA) and two mitochondrial genes (COII and ND1) were selected to develop original primers. The method was evaluated with eight genomic DNA of T. cruzi populations belonging to the six DTUs, one genomic DNA of Trypanosoma rangeli, and 53 blood samples from individuals with chronic Chagas disease. The assays had an analytical sensitivity of 1-25fg of DNA per reaction tube depending on the DTU analyzed. The selectivity of trials with 20fg/μL of genomic DNA identified each DTU, excluding non-targets DTUs in every test. The method was able to characterize 67.9% of the chronically infected clinical samples with high detection of TcII followed by TcI. With the proposed original genotyping methodology, each DTU was established with high sensitivity after a single real-time PCR assay. This novel protocol reduces carryover contamination, enables detection of each DTU independently and in the future, the quantification of each DTU in clinical blood samples.

  2. Variation in MRSA identification results from different generations of Xpert MRSA real-time PCR testing kits from nasal swabs.

    PubMed

    Rabaan, Ali A; Bazzi, Ali M

    2017-02-06

    GeneXpert MRSA kits (Cepheid) are based on a multiplex, real-time PCR method for methicillin-resistant Staphylococcus aureus (MRSA) detection, with primers to detect each SCCmec type and the chromosomal orfX-SCCmec junction. Modifications in recent kit versions were proposed to help overcome false-positive issues in earlier kit versions. The main objective of this study was to determine whether use of any version of the GeneXpert MRSA multiplex, real-time PCR kits yielded higher than expected MRSA+ results. We also estimated the level of MRSA in our healthcare facility as a proportion of total S. aureus between 2010 and 2015. We examined results from five generations of the kits used between 2008 and 2015. Results were from nasal swab samples from 16,431 patients in the Johns Hopkins Aramco Healthcare facility in Saudi Arabia. The percentage of isolates scored as MRSA+ for the original Xpert MRSA kit was 18.57%, compared to 6.93±1.12% (mean±SD) for the other four kits. The Xpert MRSA-SA Nasal kit yielded 6.48% Invalid results, compared to 0.73±0.28% for the other four kits. The succeeding Xpert MRSA-SA Nasal G3 and Xpert MRSA-SA Nasal Complete G3 kits yielded Invalid results rates of 0.29% and 1.04% respectively. Levels of MRSA-positive isolates as a percentage of total S. aureus-containing samples ranged between 19.81% and 26.74%. In conclusion, the original Xpert MRSA kit yielded higher than expected rates of MRSA+. Issues with over-estimation of MRSA+ and/or numerous Invalid results have been overcome in the most recent modified kits.

  3. Evaluation of three real-time PCR assays for differential identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria species in liquid culture media.

    PubMed

    Jung, Yu Jung; Kim, Ji-Youn; Song, Dong Joon; Koh, Won-Jung; Huh, Hee Jae; Ki, Chang-Seok; Lee, Nam Yong

    2016-06-01

    We evaluated the analytical performance of M. tuberculosis complex (MTBC)/nontuberculous mycobacteria (NTM) PCR assays for differential identification of MTBC and NTM using culture-positive liquid media. Eighty-five type strains and 100 consecutive mycobacterial liquid media cultures (MGIT 960 system) were analyzed by a conventional PCR assay (MTB-ID(®) V3) and three real-time PCR assays (AdvanSure™ TB/NTM real-time PCR, AdvanSure; GENEDIA(®) MTB/NTM Detection Kit, Genedia; Real-Q MTB & NTM kit, Real-Q). The accuracy rates for reference strains were 89.4%, 100%, 98.8%, and 98.8% for the MTB-ID V3, AdvanSure, Genedia, and Real-Q assays, respectively. Cross-reactivity in the MTB-ID V3 assay was mainly attributable to non-mycobacterium Corynebacterineae species. The diagnostic performance was determined using clinical isolates grown in liquid media, and the overall sensitivities for all PCR assays were higher than 95%. In conclusion, the three real-time PCR assays showed better performance in discriminating mycobacterium species and non-mycobacterium Corynebacterineae species than the conventional PCR assay.

  4. Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species.

    PubMed

    Zhang, Jing; Hung, Guo-Chiuan; Nagamine, Kenjiro; Li, Bingjie; Tsai, Shien; Lo, Shyh-Ching

    2016-01-01

    Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan-Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan-Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

  5. Applying a Geospatial Visualization Based on USSD Messages to Real Time Identification of Epidemiological Risk Areas in Developing Countries: A Case of Study of Paraguay.

    PubMed

    Ochoa, Silvia; Talavera, Julia; Paciello, Julio

    2015-01-01

    The identification of epidemiological risk areas is one of the major problems in public health. Information management strategies are needed to facilitate prevention and control of disease in the affected areas. This paper presents a model to optimize geographical data collection of suspected or confirmed disease occurrences using the Unstructured Supplementary Service Data (USSD) mobile technology, considering its wide adoption even in developing countries such as Paraguay. A Geographic Information System (GIS) is proposed for visualizing potential epidemiological risk areas in real time, that aims to support decision making and to implement prevention or contingency programs for public health.

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

    PubMed Central

    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. PMID:27711171

  7. Identification of suitable reference genes for quantitative real-time PCR normalization in blotched snakehead Channa maculata.

    PubMed

    Mao, H; Chen, K; Zhu, X; Luo, Q; Zhao, J; Li, W; Wu, X; Xu, H

    2017-04-07

    A systematic study was conducted to identify reliable reference genes for normalization of gene expression analysis in the blotched snakehead Channa maculata under normal physiological conditions. Firstly, the partial complementary (c)DNA of nine candidate reference genes (actb, tmem104, ube2l3, ef1α, churc1, tmem256, rpl13a, sep15 and g6pd) were cloned from C. maculata. The expression levels of these genes were then assessed in embryos of different developmental stages and various tissue types of adult fish using quantitative real-time (qrt-)PCR. RefFinder algorithm was used to evaluate the expression stability of these genes based on their cycle-threshold (Ct ) values in the qrt-PCR analysis. Results showed that there was no single best reference gene for all stages of embryos and adult tissues tested. Furthermore, it was found that, among the nine candidate genes tested, actb and tmem104 were the most stable reference genes across adult tissue types, while sep15 and tmem256 were the most stable ones across developmental stages of embryos. These stable reference genes are recommended for normalization of gene expression analysis in C. maculata.

  8. Development of a real-time PCR Assay for identification of Coccidioides immitis by use of the BD Max system.

    PubMed

    Mitchell, Marilyn; Dizon, Dominic; Libke, Robert; Peterson, Michael; Slater, David; Dhillon, Akashdeep

    2015-03-01

    Rapid real-time PCR (RT-PCR) can be performed in a community hospital setting to identify Coccidioides species using the new Becton Dickinson molecular instrument BD Max. Following sample preparation, DNA extraction and PCR were performed on the BD Max using the BD Max extraction kit ExK-DNA-1 test strip and a master mix prepared by BioGX (Birmingham, AL). Sample preparation took 2 h, and testing on the BD Max took an additional 2 h. Method sensitivity and specificity were evaluated along with the limits of detection to confirm that this convenient method would provide medically useful information. Using serial dilutions, the lower limit of detection was determined to be 1 CFU/μl. Testing with this method was validated using samples from various body sites, including bronchial alveolar lavage (BAL) fluid; sputum and lung tissue samples; and pleural and spinal fluids. Safety protocols were established, and specimen preparation processes were developed for the various types of specimens. The range for the cycle threshold (CT) indicating adequate fluorescent signal to signify a positive result was established along with the acceptable range for the internal standard. Positive controls run with each batch were prepared by spiking a pooled BAL fluid specimen with a known dilution of Coccidioides immitis organism. Our experience with testing >330 patient samples shows that clinically relevant information can be available within 4 h using an RT-PCR method on the BD Max to identify Coccidioides spp., with sensitivity equivalent to culture.

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

    PubMed

    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.

  10. Rapid identification of aminoglycoside-induced deafness gene mutations using multiplex real-time polymerase chain reaction.

    PubMed

    Huang, Shasha; Xiang, Guangxin; Kang, Dongyang; Wang, Chen; Kong, Yanling; Zhang, Xun; Liang, Shujian; Mitchelson, Keith; Xing, Wanli; Dai, Pu

    2015-07-01

    Exposure to aminoglycoside antibiotics can induce ototoxicity in genetically susceptible individuals carrying certain mitochondrial DNA (mtDNA) mutations (C1494T and A1555G), resulting in hearing loss. So, a rapid diagnostic approach is needed to accurately identify subjects carrying such gene mutations. In the present study, we describe a rapid and reliable four-color, real-time quantitative polymerase chain reaction (qPCR) assay for simultaneously detecting two mtDNA 12S rRNA gene variants, A1555G and C1494T, which are prevalent in the Han Chinese population. This multiplex assay incorporates three allele-specific TaqMan probes labeled with different fluorophores in a single reaction, providing high genotyping accuracy for clinical blood samples. Tests with C1494T, A1555G and wild-type DNA exhibited high sensitivity, specificity, reproducibility and accuracy of discriminating mutations from wild-type. This study shows that this simple and inexpensive method can be used for routine molecular diagnostics and potentially for large-scale genetic screening. Copyright © 2015. Published by Elsevier Ireland Ltd.

  11. Identification of appropriate reference genes for normalizing transcript expression by quantitative real-time PCR in Litsea cubeba.

    PubMed

    Lin, Liyuan; Han, Xiaojiao; Chen, Yicun; Wu, Qingke; Wang, Yangdong

    2013-12-01

    Quantitative real-time PCR has emerged as a highly sensitive and widely used method for detection of gene expression profiles, via which accurate detection depends on reliable normalization. Since no single control is appropriate for all experimental treatments, it is generally advocated to select suitable internal controls prior to use for normalization. This study reported the evaluation of the expression stability of twelve potential reference genes in different tissue/organs and six fruit developmental stages of Litsea cubeba in order to screen the superior internal reference genes for data normalization. Two softwares-geNorm, and NormFinder-were used to identify stability of these candidate genes. The cycle threshold difference and coefficient of variance were also calculated to evaluate the expression stability of candidate genes. F-BOX, EF1α, UBC, and TUA were selected as the most stable reference genes across 11 sample pools. F-BOX, EF1α, and EIF4α exhibited the highest expression stability in different tissue/organs and different fruit developmental stages. Besides, a combination of two stable reference genes would be sufficient for gene expression normalization in different fruit developmental stages. In addition, the relative expression profiles of DXS and DXR were evaluated by EF1α, UBC, and SAMDC. The results further validated the reliability of stable reference genes and also highlighted the importance of selecting suitable internal controls for L. cubeba. These reference genes will be of great importance for transcript normalization in future gene expression studies on L. cubeba.

  12. Development of PrimeTime-Real-Time PCR for Species Identification of Soybean Cyst Nematode (Heterodera glycines Ichinohe, 1952) in North Carolina

    PubMed Central

    Ye, Weimin

    2012-01-01

    Soybean cyst nematode (SCN) is an obligate, sedentary parasite that is a major pathogen of soybean and accounts for an estimated 1 billion dollars in production losses annually in the United States of America. This paper describes the development of a real-time PCR method for rapid, sensitive, species-specific and accurate identification of SCN alone or on mixed populations with other nematodes in North Carolina. The 83-bp DNA fragment of PrimeTime-real-time PCR was designed based on a 477-bp-SCN-SCAR marker previously proved to be SCN-specific. A total of 44 populations including cyst forming nematodes (Heterodera glycines, H. fici, H. schachtii, H. trifolii, Cactodera weissi, Globodera tabacum, Meloidodera floridensis and other unidentified cyst nematodes) and non-cyst forming nematodes (Ditylenchus dipsaci, Meloidogyne incognita and Xiphinema chambersi) were tested in this study, all SCN populations are tested positive and non-SCN populations negative. This assay for the detection and identification has been successfully applied for testing a single SCN cyst, a 2nd-stage-SCN juvenile, a single SCN egg, up to ten SCN cysts, a 10-fold dilution of a single 2nd-stage-SCN juvenile and 20-fold dilution of one SCN cyst. The assay is not SCN-race specific. It gave an accurate positive result when SCN is mixed with other cyst species. Also, nematode universal primers/probes for real-time PCR amplification as a nematode endogenous control to detect the presence of 18S ribosomal RNA (rRNA) gene were employed in this assay, so that a SCN-negative sample can be tested to exclude false negative. This method will be very useful for a broad range of research programs as well as the regulatory response and management of SCN in North Carolina and other region of the southeastern U.S.A. PMID:23481469

  13. A real-time ITS1-PCR based method in the diagnosis and species identification of Leishmania parasite from human and dog clinical samples in Turkey.

    PubMed

    Toz, Seray Ozensoy; Culha, Gulnaz; Zeyrek, Fadile Yıldız; Ertabaklar, Hatice; Alkan, M Ziya; Vardarlı, Aslı Tetik; Gunduz, Cumhur; Ozbel, Yusuf

    2013-01-01

    Human visceral leishmaniasis (VL) caused by L. infantum and cutaneous leishmaniasis (CL) caused by L. tropica and L. infantum have been reported in Turkey. L. infantum is also responsible for canine leishmaniasis (CanL) and it is widely common in the country. The main aim of the present study was to design a real-time PCR method based on the internal transcribed spacer 1 (ITS1) region in the diagnosis of all clinical forms of leishmaniasis in Mediterranean, and to identify the species directly from clinical samples. Totally, 315 clinical specimens, human/canine visceral (blood, bone marrow, lymph node) and cutaneous (lesion aspiration) samples, and 51 Turkish Leishmania isolates typed by isoenzymatic method were included in the study. For optimization, DNA samples of the 34 strains were amplified by conventional ITS1-PCR and then sequenced for designing the primers and probes, allowing the species identification. Following the validation with the isolates, the test was applied on clinical samples and melting temperatures were used for genotyping. A group of PCR products were further sequenced for confirmation and assigning the inter- and intraspecies heterogeneity. The diagnosis of leishmaniasis is successfully achieved by the new real-time PCR method, and the test identified 80.43% of human and canine VL samples as L.infantum and 6.52% as L.tropica; 52.46% of CL samples as L. infantum and 26.90% as L. tropica. In 13.04% of visceral and 20.62% of cutaneous samples, two peaks were observed. However, the higher peak was found to be concordant with the sequencing results in 96.96%, in terms of species identification. The real-time ITS1 PCR assay clearly identified the leishmanial species in 81.58% of all clinical samples. Genotypic variations of Leishmania parasites in Turkey within species and intraspecies were observed, and L. tropica is also found as causative agent of human and canine VL in Turkey.

  14. A Real-Time ITS1-PCR Based Method in the Diagnosis and Species Identification of Leishmania Parasite from Human and Dog Clinical Samples in Turkey

    PubMed Central

    Toz, Seray Ozensoy; Culha, Gulnaz; Zeyrek, Fadile Yıldız; Ertabaklar, Hatice; Alkan, M. Ziya; Vardarlı, Aslı Tetik; Gunduz, Cumhur; Ozbel, Yusuf

    2013-01-01

    Human visceral leishmaniasis (VL) caused by L. infantum and cutaneous leishmaniasis (CL) caused by L. tropica and L. infantum have been reported in Turkey. L. infantum is also responsible for canine leishmaniasis (CanL) and it is widely common in the country. The main aim of the present study was to design a real-time PCR method based on the internal transcribed spacer 1 (ITS1) region in the diagnosis of all clinical forms of leishmaniasis in Mediterranean, and to identify the species directly from clinical samples. Totally, 315 clinical specimens, human/canine visceral (blood, bone marrow, lymph node) and cutaneous (lesion aspiration) samples, and 51 Turkish Leishmania isolates typed by isoenzymatic method were included in the study. For optimization, DNA samples of the 34 strains were amplified by conventional ITS1-PCR and then sequenced for designing the primers and probes, allowing the species identification. Following the validation with the isolates, the test was applied on clinical samples and melting temperatures were used for genotyping. A group of PCR products were further sequenced for confirmation and assigning the inter- and intraspecies heterogeneity. The diagnosis of leishmaniasis is successfully achieved by the new real-time PCR method, and the test identified 80.43% of human and canine VL samples as L.infantum and 6.52% as L.tropica; 52.46% of CL samples as L. infantum and 26.90% as L. tropica. In 13.04% of visceral and 20.62% of cutaneous samples, two peaks were observed. Hovewer, the higher peak was found to be concordant with the sequencing results in 96.96%, in terms of species identification. The real-time ITS1 PCR assay clearly identified the leishmanial species in 81.58% of all clinical samples. Genotypic variations of Leishmania parasites in Turkey within species and intraspecies were observed, and L. tropica is also found as causative agent of human and canine VL in Turkey. PMID:23675543

  15. Visualizing the store-operated channel complex assembly in real time: identification of SERCA2 as a new member.

    PubMed

    Sampieri, Alicia; Zepeda, Angelica; Asanov, Alexander; Vaca, Luis

    2009-05-01

    Depletion of intracellular calcium stores leads to the activation of calcium influx via the so-called store-operated channels (SOCs). Recent evidence positions Orai proteins as the putative channels responsible for this process. The stromal interacting molecule (STIM1) has been recently identified as the calcium sensor located at the endoplasmic reticulum (ER), and responsible for communicating the deplete state of calcium stores to Orai at the plasma membrane (PM). However, recent experimental findings suggest that Orai and STIM1 are only part of a larger molecular complex required to modulate store-operated calcium entry (SOCE). In the present study we describe the assembly of the several of the components from the SOC complex in real-time, utilizing a novel imaging method. Using FRET imaging we show that under resting conditions (with calcium stores replenished) STIM1 travels continuously through the ER associated to the microtubule tracking protein, EB1. Upon depletion of the ER STIM1 dissociates from EB1 and aggregates into macromolecular complexes at the ER which includes the microsomal calcium ATPase. This association follows the assembly of Orai into macromolecular aggregates at the PM. We show that STIM1-Orai association follows a similar time course as that of Orai aggregation at the PM. During this last step of the process, calcium-selective, whole-cell inward currents developed, simultaneously. We show that this process is fully reversible. Replenishing intracellular calcium stores induces STIM1-Orai complex dissociation and shuts down inward currents. Under these conditions STIM1 re-associates to EB1, and reinitiates its travel through the ER.

  16. Identification and Validation of Reference Genes for Quantitative Real-Time PCR Normalization and Its Applications in Lycium

    PubMed Central

    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. PMID:24810586

  17. In Vivo Protein Interaction Network Identified with a Novel Real-Time Cross-Linked Peptide Identification Strategy

    PubMed Central

    Weisbrod, Chad R.; Chavez, Juan D.; Eng, Jimmy K.; Yang, Li; Zheng, Chunxiang; Bruce, James E.

    2013-01-01

    Protein interaction topologies are critical determinants of biological function. Large-scale or proteome-wide measurements of protein interaction topologies in cells currently pose an unmet challenge that could dramatically improve understanding of complex biological systems. A primary impediment includes direct protein topology and interaction measurements from living systems since interactions that lack biological significance may be introduced during cell lysis. Furthermore, many biologically relevant protein interactions will likely not survive the lysis/sample preparation and may only be measured with in vivo methods. As a step toward meeting this challenge, a new mass spectrometry method called Real-time Analysis for Cross-linked peptide Technology (ReACT) has been developed that enables assignment of cross-linked peptides “on-the-fly”. Using ReACT, 708 unique cross-linked (<5% FDR) peptide pairs were identified from cross-linked E. coli cells. These data allow assembly of the first protein interaction network that also contains topological features of every interaction, as it existed in cells during cross-linker application. Of the identified interprotein cross-linked peptide pairs, 40% are derived from known interactions and provide new topological data that can help visualize how these interactions exist in cells. Other identified cross-linked peptide pairs are from proteins known to be involved within the same complex, but yield newly discovered direct physical interactors. ReACT enables the first view of these interactions inside cells, and the results acquired with this method suggest cross-linking can play a major role in future efforts to map the interactome in cells. PMID:23413883

  18. Identification and testing of reference genes for Sesame gene expression analysis by quantitative real-time PCR.

    PubMed

    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.

  19. Real-time detection and identification of Chlamydophila species in veterinary specimens by using SYBR green-based PCR assays.

    PubMed

    Nordentoft, Steen; Kabell, Susanne; Pedersen, Karl

    2011-09-01

    Infections caused by members of the Chlamydiaceae family have long been underestimated due to the requirement of special laboratory facilities for the detection of this group of intracellular pathogens. Furthermore, new studies of this group of intracellular pathogens have revealed that host specificity of different species is not as clear as recently believed. As most members of the genus Chlamydophila have shown to be transmissible from animals to humans, sensitive and fast detection methods are required. In this study, SYBR green-based real-time assays were developed that detect all members of Chlamydiaceae and differentiate the most prevalent veterinary Chlamydophila species: Cp. psittaci, Cp. abortus, Cp. felis, and Cp. caviae. By adding bovine serum albumin to the master mixes, target DNA could be detected directly in crude lysates of enzymatically digested conjunctival or pharyngeal swabs or tissue specimens from heart, liver, and spleen without further purification. The assays were evaluated on veterinary specimens where all samples were screened using a family-specific PCR, and positive samples were further tested using species-specific PCRs. Cp. psittaci was detected in 47 birds, Cp. felis was found in 10 cats, Cp. caviae was found in one guinea pig, and Cp. abortus was detected in one sheep. The screening assay appeared more sensitive than traditional microscopical examination of stained tissue smears. By combining a fast, robust, and cost-effective method for sample preparation with a highly sensitive family-specific PCR, we were able to screen for Chlamydiaceae in veterinary specimens and confirm the species in positive samples with additional PCR assays.

  20. Quantitative Real-Time PCR technique for the identification of E.coli residual DNA in streptokinase recombinant product.

    PubMed

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

    2017-08-31

    One of the most important biopharmaceuticals worldwide is recombinant streptokinase, which is usually produced in Escherichia coli (E.coli). During the biopharmaceutical purification steps, residual host cell DNA may remain in the product, and since residual host cell DNA is considered as a contamination and risk factor, it is necessary to purify the recombinant product as much as possible. Therefore, it is necessary to control the production procedure in order 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. To do so, we designed specific primers and a probe to detect all strains of E.coli. To determine the specificity of the primers and the probe, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. Based on the results obtained from BLAST and PCR on the samples, it was confirmed that the assay detects no genomic DNA but E.coli's. Therefore, the specificity of the test 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) were prepared and tested in triplicate. Based on the results, the sensitivity of the test was determined to be 10(1) copies/µL or 35 fg/µL. Furthermore, to determine the reproducibility of the assay, Inter-assay and Intra-assay were performed and determined to be 0.86% and 1.69%, respectively. Given the results for specificity, sensitivity, precision, and the reproducibility of the test, this assay can be used as a specific and sensitive method to evaluate the contamination of recombinant streptokinase or any other recombinant product produced in E.coli.

  1. Identification and validation of reference genes for quantitative real-time PCR studies in long yellow daylily, Hemerocallis citrina Borani

    PubMed Central

    Wang, Jinyao; Kang, Xiuping; Weng, Yiqun

    2017-01-01

    Gene expression analysis using reverse transcription quantitative real-time PCR (RT-qPCR) requires the use of reference gene(s) in the target species. The long yellow daylily, Hemerocallis citrina Baroni. is rich in beneficial secondary metabolites and is considered as a functional vegetable. It is widely cultivated and consumed in East Asian countries. However, reference genes for use in RT-qPCR in H. citrina are not available. In the present study, six potential reference genes, actin (ACT), AP-4 complex subunit (AP4), tubulin (TUB), ubiquitin (UBQ), 18S and 60S ribosomal RNA, were selected and their expression stability in different developmental stages, organs and accessions was evaluated using four statistical software packages (geNorm, NormFinder, BestKeeper, and RefFinder). For commercial flower buds of different landraces, the combination of 60S, TUB, and AP4 was appropriate whereas ACT and 60S was suitable for normalization of different organs. In addition, AP4 exhibited the most stable expression in flower buds among different developmental stages. UBQ was less stable than the other reference genes under the experimental conditions except under different organs was 18S. The relative expression levels of two genes, primary-amine oxidase (HcAOC3) and tyrosine aminotransferase (HcTAT) which play important roles in alkaloid biosynthesis were also examined in different organs of the ‘Datong’ landrace, which further confirmed the results of selected reference genes. This is the first report to evaluate the stability of reference genes in the long yellow daylily that can serve as a foundation for RT-qPCR analysis of gene expression in this species. PMID:28362875

  2. A novel real-time PCR assay for the specific identification and quantification of Weissella viridescens in blood sausages.

    PubMed

    Gómez-Rojo, Erica M; Romero-Santacreu, L; Jaime, I; Rovira, J

    2015-12-23

    Weissella viridescens has been identified as one of the lactic acid bacteria (LAB) responsible for the spoilage of "morcilla de Burgos". In order to identify and quantify this bacterium in "morcilla de Burgos", a new specific PCR procedure has been developed. The primers and Taqman probe were designed on the basis of a sequence from the gene recN. To confirm the specificity of the primers, 77 strains from the genera Carnobacterium, Enterococcus, Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Vagococcus and Weissella were tested by conventional PCR. The specificity of the primers and the correct functioning of the probe was confirmed by performing real-time PCR (qPCR) with 21 W. viridescens strains and 27 strains from other LAB genera. The levels of detection and quantification for the qPCR procedure proposed herein were determined for a pure culture of W. viridescens CECT 283(T) and for "morcilla de Burgos" artificially inoculated with this species. The primers were specific for W. viridescens, with only one product of 91 bp being observed for this species. Similarly, the qPCR reactions were found to be specific, amplifying at a mean CT of 15.0±0.4 only for W. viridescens strains. The limit of detection (LOD) and quantification (LOQ) for this procedure was established in 0.082 pg for genomic DNA from W. viridescens. With regard to the artificially inoculated "morcilla", the limit of quantification was established in 80 CFU/reaction and the limit of detection in 8 CFU/reaction. Consequently, the qPCR developed herein can be considered to be a good, fast, simple and accurate tool for the specific detection and quantification of W. viridescens in meat samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. HybProbes-based real-time PCR assay for specific identification of Streptomyces scabies and Streptomyces europaeiscabiei, the potato common scab pathogens.

    PubMed

    Xu, R; Falardeau, J; Avis, T J; Tambong, J T

    2016-02-01

    The aim of this study was to develop and validate a HybProbes-based real-time PCR assay targeting the trpB gene for specific identification of Streptomyces scabies and Streptomyces europaeiscabiei. Four primer pairs and a fluorescent probe were designed and evaluated for specificity in identifying S. scabies and Streptomyces europaeiscabiei, the potato common scab pathogens. The specificity of the HybProbes-based real-time PCR assay was evaluated using 46 bacterial strains, 23 Streptomyces strains and 23 non-Streptomyces bacterial species. Specific and strong fluorescence signals were detected from all nine strains of S. scabies and Streptomyces europaeiscabiei. No fluorescence signal was detected from 14 strains of other Streptomyces species and all non-Streptomyces strains. The identification was corroborated by the melting curve analysis that was performed immediately after the amplification step. Eight of the nine S. scabies and S. europaeiscabiei strains exhibited a unique melting peak, at Tm of 69·1°C while one strain, Warba-6, had a melt peak at Tm of 65·4°C. This difference in Tm peaks could be attributed to a guanine to cytosine mutation in strain Warba-6 at the region spanning the donor HybProbe. The reported HybProbes assay provides a more specific tool for accurate identification of S. scabies and S. europaeiscabiei strains. This study reports a novel assay based on HybProbes chemistry for rapid and accurate identification of the potato common scab pathogens. Since the HybProbes chemistry requires two probes for positive identification, the assay is considered to be more specific than conventional PCR or TaqMan real-time PCR. The developed assay would be a useful tool with great potential in early diagnosis and detection of common scab pathogens of potatoes in infected plants or for surveillance of potatoes grown in soil environment. © 2015 Her Majesty the Queen in Right of Canada © 2015 The Society for Applied Microbiology.

  4. Multiplex real-time PCR for detection, identification and quantification of 'Candidatus Liberibacter solanacearum' in potato plants with zebra chip.

    PubMed

    Li, Wenbin; Abad, Jorge A; French-Monar, Ronald D; Rascoe, John; Wen, Aimin; Gudmestad, Neil C; Secor, Gary A; Lee, Ing-Ming; Duan, Yongping; Levy, Laurene

    2009-07-01

    The new Liberibacter species, 'Candidatus Liberibacter solanacearum' (Lso) recently associated with potato/tomato psyllid-transmitted diseases in tomato and capsicum in New Zealand, was found to be consistently associated with a newly emerging potato zebra chip (ZC) disease in Texas and other southwestern states in the USA. A species-specific primer LsoF was developed for both quantitative real-time PCR (qPCR) and conventional PCR (cPCR) to detect and quantify Lso in infected samples. In multiplex qPCR, a plant cytochrome oxidase (COX)-based probe-primer set was used as a positive internal control for host plants, which could be used to reliably access the DNA extraction quality and to normalize qPCR data for accurate quantification of the bacterial populations in environment samples. Neither the qPCR nor the cPCR using the primer and/or probe sets with LsoF reacted with other Liberibacter species infecting citrus or other potato pathogens. The low detection limit of the multiplex qPCR was about 20 copies of the target 16S rDNA templates per reaction for field samples. Lso was readily detected and quantified in various tissues of ZC-affected potato plants collected from fields in Texas. A thorough but uneven colonization of Lso was revealed in various tissues of potato plants. The highest Lso populations were about 3x10(8) genomes/g tissue in the root, which were 3-order higher than those in the above-ground tissues of potato plants. The Lso bacterial populations were normally distributed across the ZC-affected potato plants collected from fields in Texas, with 60% of ZC-affected potato plants harboring an average Lso population from 10(5) to 10(6) genomes/g tissue, 4% of plants hosting above 10(7) Lso genomes/g tissue, and 8% of plants holding below 10(3) Lso genomes/g tissue. The rapid, sensitive, specific and reliable multiplex qPCR showed its potential to become a powerful tool for early detection and quantification of the new Liberibacter species associated

  5. Identification of Cereulide-Producing Bacillus cereus by Nucleic Acid Chromatography and Reverse Transcription Real-Time PCR.

    PubMed

    Ueda, Shigeko; Yamaguchi, Manami; Eguchi, Kayoko; Iwase, Miki

    2016-01-01

    RNA extracts were analyzed with a nucleic acid sequence-based amplification (NASBA) - nucleic acid chromatography and a reverse transcription-quantitative PCR assay (RT-qPCR) based on the TaqMan probe for identification of cereulide-producing Bacillus cereus. All 100 emetic B. cereus strains were found to give positive results, but 50 diarrheal B. cereus strains and other bacterial species showed negative results in the NASBA-chromatography. That is, the assay could selectively identify the emetic strains among B. cereus strains. Also, the B. cereus contents of more than 10(7) cfu/ml were required for the identification of the cereulide-producing strains in this assay. In qRT-PCR assays, all 100 emetic type strains of B. cereus produced 10(2) - 10(4) copy numbers per ng of the RNA preparation, and the strains produced 10(4) copies including ones which had the high vacuolation activities of HEp-2 cells.

  6. Development of a real-time PCR assay (SYBR Green I) for rapid identification and quantification of scyphomedusae Aurelia sp.1 planulae

    NASA Astrophysics Data System (ADS)

    Wang, Jianyan; Zhen, Yu; Mi, Tiezhu; Yu, Zhigang; Wang, Guoshan

    2015-07-01

    The complicated life cycle of Aurelia spp., comprising benthic asexually-reproducing polyps and sexually-reproducing medusae, makes it hard for researchers to identify and track them, especially for early stage individuals, such as planulae. To solve this problem, we developed a real-time PCR assay (SYBR Green I) to identify planulae in both cultured and natural seawater samples. Species-specific primers targeting Aurelia sp.1 mitochondrial 16S rDNA (mt 16S rDNA) regions were designed. Using a calibration curve constructed with plasmids containing the Aurelia sp.1 mt 16S rDNA fragment and a standard curve for planulae, the absolute number of mt 16S rDNA copies per planula was determined and from that the total number of planulae per sample was estimated. For the field samples, a 100-fold dilution of the sample DNA combined with a final concentration of 0.2 μg/μL BSA in the PCR reaction mixture was used to remove real-time PCR inhibitors. Samples collected in Jiaozhou Bay from July to September 2012 were subsequently analyzed using this assay. Peak Aurelia sp.1 planula abundance occurred in July 2012 at stations near Hongdao Island and Qingdao offshore; abundances were very low in August and September. The real-time PCR assay (SYBR Green I) developed here negates the need for traditional microscopic identification, which is laborious and time-consuming, and can detect and quantify jellyfish planulae in field plankton samples rapidly and specifically.

  7. Flight Testing and Real-Time System Identification Analysis of a UH-60A Black Hawk Helicopter with an Instrumented External Sling Load

    NASA Technical Reports Server (NTRS)

    McCoy, Allen H.

    1998-01-01

    Helicopter external air transportation plays an important role in today's world. For both military and civilian helicopters, external sling load operations offer an efficient and expedient method of handling heavy, oversized cargo. With the ability to reach areas otherwise inaccessible by ground transportation, helicopter external load operations are conducted in industries such as logging, construction, and fire fighting, as well as in support of military tactical transport missions. Historically, helicopter and load combinations have been qualified through flight testing, requiring considerable time and cost. With advancements in simulation and flight test techniques there is potential to substantially reduce costs and increase the safety of helicopter sling load certification. Validated simulation tools make possible accurate prediction of operational flight characteristics before initial flight tests. Real time analysis of test data improves the safety and efficiency of the testing programs. To advance these concepts, the U.S. Army and NASA, in cooperation with the Israeli Air Force and Technion, under a Memorandum of Agreement, seek to develop and validate a numerical model of the UH-60 with sling load and demonstrate a method of near real time flight test analysis. This thesis presents results from flight tests of a U.S. Army Black Hawk helicopter with various external loads. Tests were conducted as the U.S. first phase of this MOA task. The primary load was a container express box (CONEX) which contained a compact instrumentation package. The flights covered the airspeed range from hover to 70 knots. Primary maneuvers were pitch and roll frequency sweeps, steps, and doublets. Results of the test determined the effect of the suspended load on both the aircraft's handling qualities and its control system's stability margins. Included were calculations of the stability characteristics of the load's pendular motion. Utilizing CIFER(R) software, a method for near-real

  8. Genotype-specific real-time PCR combined with high-resolution melting analysis for rapid identification of red-spotted grouper nervous necrosis virus.

    PubMed

    Toubanaki, Dimitra K; Karagouni, Evdokia

    2017-08-01

    A real-time genotype-specific polymerase chain reaction (PCR) assay combined with high-resolution melting (HRM) analysis was developed to assess the most common genotypes of nervous necrosis viruses or nodaviruses. Nodaviruses are the causal agents of viral nervous necrosis infections, which have been wreaking havoc in the aquaculture industry worldwide, with fish mortality up to 100%. The four different genotypes of nodaviruses correlate with differences in viral pathogenicity. Therefore, rational development of effective vaccines and diagnostics requires analysis of genetic variation among viruses. The aim of the present study was to develop a real-time tetra-primer genotype-specific PCR assay for genotype identification. Four primers were utilized for simultaneous amplification of nodavirus genotype-specific products in a single closed-tube PCR after a reverse-transcription reaction using RNA isolated from fish samples. For high-throughput sample analysis, SYBR Green-based real-time PCR was used in combination with HRM analysis. The assay was evaluated in terms of specificity and sensitivity. The analysis resulted in melting curves that were indicative of each genotype. The detection limit when using reference plasmids was 100 ag/µL for both genotypes, while the sensitivity of the assays when testing a complex mixture was 10 fg/µL for red-spotted grouper nervous necrosis virus (RGNNV) and 100 fg/µL for striped jack nervous necrosis virus (SJNNV). To test the capability of this method under real-world conditions, 58 samples were examined. All samples belonged to the RGNNV genotype, which was fully validated. The results were in full agreement with genotyping by reference methods. The proposed methodology provides a rapid, sensitive, specific, robust and automatable assay for nodavirus genotyping, making it a useful tool for diagnosis and screening for epidemiological studies.

  9. A centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria.

    PubMed

    Choi, Goro; Jung, Jae Hwan; Park, Byung Hyun; Oh, Seung Jun; Seo, Ji Hyun; Choi, Jong Seob; Kim, Do Hyun; Seo, Tae Seok

    2016-06-21

    In this study, we developed a centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria contaminated milk samples. The microdevice was designed to contain identical triplicate functional units and each unit has four reaction chambers, thereby making it possible to perform twelve direct-RPA reactions simultaneously. The integrated microdevice consisted of two layers: RPA reagents were injected in the top layer, while spiked milk samples with food poisoning bacteria were loaded into sample reservoirs in the bottom layer. For multiplex bacterial detection, the target gene-specific primers and probes were dried in each reaction chamber. The introduced samples and reagents could be equally aliquoted and dispensed into each reaction chamber by centrifugal force, and then the multiplex direct-RPA reaction was executed. The target genes of bacteria spiked in milk could be amplified at 39 °C without a DNA extraction step by using the direct-RPA cocktails, which were a combination of a direct PCR buffer and RPA enzymes. As the target gene amplification proceeded, the increased fluorescence signals coming from the reaction chambers were recorded in real-time at an interval of 2 min. The entire process, including the sample distribution, the direct-RPA reaction, and the real-time analysis, was accomplished with a custom-made portable genetic analyzer and a miniaturized optical detector. Monoplex, duplex, and triplex food poisoning bacteria (Salmonella enterica, Escherichia coli O157:H7, and Vibrio parahaemolyticus) detection was successfully performed with a detection sensitivity of 4 cells per 3.2 μL of milk samples within 30 min. By implementing the direct-PRA on the miniaturized centrifugal microsystem, the on-site food poisoning bacteria analysis would be feasible with high speed, sensitivity, and multiplicity.

  10. Molecular identification of Mycobacterium avium subsp. silvaticum by duplex high-resolution melt analysis and subspecies-specific real-time PCR.

    PubMed

    Rónai, Zsuzsanna; Csivincsik, Ágnes; Dán, Ádám

    2015-05-01

    Accurate identification of mycobacterial species and subspecies is essential to evaluate their significance and to perform epidemiological studies. The subspecies of Mycobacterium avium have different attributes but coincide in their zoonotic potential. Our knowledge about M. avium subsp. silvaticum is limited, since its identification is uncertain. Mycobacterium avium subsp. avium and M. avium subsp. silvaticum can be discriminated from each other based only on phenotypic characteristics, as they have almost identical genome sequences. Here we describe the development of a diagnostic method which enables the molecular identification of M. avium subsp. silvaticum and discrimination from M. avium subsp. avium based on genomic differences in a duplex high-resolution melt and M. avium subsp. silvaticum-specific mismatch real-time PCR. The developed assay was tested on reference strains and 199 field isolates, which were analyzed by phenotypic methods previously. This assay not only identified all 63 M. avium subsp. silvaticum and 138 M. avium subsp. avium strains correctly but also enabled the detection of mixed M. avium subsp. avium-M. avium subsp. silvaticum cultures. This is the first time that such a large panel of strains has been analyzed, and we also report the first isolation of M. avium subsp. silvaticum from red fox, red deer, wild boar, cattle, and badger. This assay is reliable, rapid, simple, inexpensive, and robust. It eliminates the long-existing problem of ambiguous phenotypic identification and opens up the possibility for detailed and comprehensive strain studies.

  11. Molecular Identification of Mycobacterium avium subsp. silvaticum by Duplex High-Resolution Melt Analysis and Subspecies-Specific Real-Time PCR

    PubMed Central

    Csivincsik, Ágnes; Dán, Ádám

    2015-01-01

    Accurate identification of mycobacterial species and subspecies is essential to evaluate their significance and to perform epidemiological studies. The subspecies of Mycobacterium avium have different attributes but coincide in their zoonotic potential. Our knowledge about M. avium subsp. silvaticum is limited, since its identification is uncertain. Mycobacterium avium subsp. avium and M. avium subsp. silvaticum can be discriminated from each other based only on phenotypic characteristics, as they have almost identical genome sequences. Here we describe the development of a diagnostic method which enables the molecular identification of M. avium subsp. silvaticum and discrimination from M. avium subsp. avium based on genomic differences in a duplex high-resolution melt and M. avium subsp. silvaticum-specific mismatch real-time PCR. The developed assay was tested on reference strains and 199 field isolates, which were analyzed by phenotypic methods previously. This assay not only identified all 63 M. avium subsp. silvaticum and 138 M. avium subsp. avium strains correctly but also enabled the detection of mixed M. avium subsp. avium-M. avium subsp. silvaticum cultures. This is the first time that such a large panel of strains has been analyzed, and we also report the first isolation of M. avium subsp. silvaticum from red fox, red deer, wild boar, cattle, and badger. This assay is reliable, rapid, simple, inexpensive, and robust. It eliminates the long-existing problem of ambiguous phenotypic identification and opens up the possibility for detailed and comprehensive strain studies. PMID:25740770

  12. Development and validation of real-time PCR tests for the identification of four Spodoptera species: Spodoptera eridania, Spodoptera frugiperda, Spodoptera littoralis, and Spodoptera litura (Lepidoptera: Noctuidae).

    PubMed

    Van de Vossenberg, B T L H; Van der Straten, M J

    2014-08-01

    The genus Spodoptera comprises 31 species, 4 of which are listed as quarantine pests for the European Union: Spodoptera eridania (Cramer), Spodoptera frugiperda (Smith), Spodoptera littoralis (Boisduval), and Spodoptera litura (F.). In international trade, the earlier life stages (eggs and larvae) are being intercepted at point of inspection most frequently, challenging the possibilities of morphological identification. To realize a rapid and reliable identification for all stages, we developed and validated four simplex real-time polymerase chain reaction identification tests based on the mitochondrial cytochrome b gene using dual-labeled hydrolysis probes. Method validation on dilutions of extracted DNA of the target organisms showed that low levels of template (up to 0.2-100 pg) can reliably be identified. No cross-reactivity was observed with 14 nontarget Spodoptera and 5 non-Spodoptera species in the specific Spodoptera tests. The tests showed to be repeatable, reproducible (both 100%), and robust. The new Spodoptera tests have proven to be suitable tools for routine identification of all life stages of S. eridania, S. frugiperda, S. littoralis, and S. litura.

  13. Rapid Detection and Identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in Mosquito Vectors and Blood Samples by High Resolution Melting Real-Time PCR

    PubMed Central

    Thanchomnang, Tongjit; Intapan, Pewpan M.; Tantrawatpan, Chairat; Lulitanond, Viraphong; Chungpivat, Sudchit; Taweethavonsawat, Piyanan; Kaewkong, Worasak; Sanpool, Oranuch; Janwan, Penchom; Choochote, Wej

    2013-01-01

    A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors. PMID:24516268

  14. A Simultaneous Analytical Method for Duplex Identification of Porcine and Horse in the Meat Products by EvaGreen based Real-time PCR

    PubMed Central

    2015-01-01

    A duplex real-time polymerase chain reaction (PCR) based assay for the detection of porcine and horse meat in sausages was designed by using EvaGreen fluorescent dye. Primers were selected from mitochondrial 12S rRNA and 16S rRNA genes which are powerful regions for identification of horse and porcine meat. DNA from reference samples and industrial products was successfully extracted using the GIDAGEN® Multi-Fast DNA Isolation Kit. Genomes were identified based on their specific melting peaks (Mp) which are 82.5℃ and 78℃ for horse and porcine, respectively. The assay used in this study allowed the detection of as little as 0.0001% level of horse meat and 0.001% level of porcine meat in the experimental admixtures. These findings indicate that EvaGreen based duplex real-time PCR is a potentially sensitive, reliable, rapid and accurate assay for the detection of meat species adulterated with porcine and horse meats. PMID:26761852

  15. Real-time PCR detection and quantification of elephantid DNA: species identification for highly processed samples associated with the ivory trade.

    PubMed

    Wozney, Kristyne Michelle; Wilson, Paul J

    2012-06-10

    The ivory industry is the single most serious threat to global elephant populations. A highly sensitive, species-specific real-time PCR assay has been developed to detect and quantify African elephant (Loxodonta africana), Asian elephant (Elephas maximus) and Woolly Mammoth (Mammuthus primigenius) mitochondrial DNA from highly processed samples involved in the international ivory trade. This assay is especially useful for highly processed samples where there are no distinguishing morphological features to identify the species of origin. Using species-specific Taqman(®) probes targeting a region of the mitochondrial cytochrome b gene, we developed an assay that can be used to positively identify samples containing elephant or Woolly mammoth DNA faster and more cost-effectively than traditional sequencing methods. Furthermore, this assay provides a diagnostic result based on probe hybridization that eliminates ambiguities associated with traditional DNA sequence protocols involving low template DNA. The real-time method is highly sensitive, producing accurate and reproducible results in samples with as few as 100 copies of template DNA. This protocol can be applied to the enforcement of the Convention on the International Trade of Endangered Species (CITES), when positive identification of species from illegally traded products is required by conservation officers in wildlife forensic cases. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  16. Rapid detection and identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in mosquito vectors and blood samples by high resolution melting real-time PCR.

    PubMed

    Thanchomnang, Tongjit; Intapan, Pewpan M; Tantrawatpan, Chairat; Lulitanond, Viraphong; Chungpivat, Sudchit; Taweethavonsawat, Piyanan; Kaewkong, Worasak; Sanpool, Oranuch; Janwan, Penchom; Choochote, Wej; Maleewong, Wanchai

    2013-12-01

    A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

  17. Multiplex PCR method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species.

    PubMed

    Trotta, Michele; Schönhuth, Susana; Pepe, Tiziana; Cortesi, M Luisa; Puyet, Antonio; Bautista, José M

    2005-03-23

    Mitochondrial 16S rRNA sequences from morphological validated grouper (Epinephelus aeneus, E. caninus, E. costae, and E. marginatus; Mycteroperca fusca and M. rubra), Nile perch (Lates niloticus), and wreck fish (Polyprion americanus) were used to develop an analytical system for group diagnosis based on two alternative Polymerase Chain Reaction (PCR) approaches. The first includes conventional multiplex PCR in which electrophoretic migration of different sizes of bands allowed identification of the fish species. The second approach, involving real-time PCR, produced a single amplicon from each species that showed different Tm values allowing the fish groups to be directly identified. Real-time PCR allows the quick differential diagnosis of the three groups of species and high-throughput screening of multiple samples. Neither PCR system cross-reacted with DNA samples from 41 common marketed fish species, thus conforming to standards for species validation. The use of these two PCR-based methods makes it now possible to discriminate grouper from substitute fish species.

  18. Detection, quantitation and identification of enteroviruses from surface waters and sponge tissue from the Florida Keys using real-time RT-PCR

    USGS Publications Warehouse

    Donaldson, K.A.; Griffin, Dale W.; Paul, J.H.

    2002-01-01

    A method was developed for the quantitative detection of pathogenic human enteroviruses from surface waters in the Florida Keys using Taqman (R) one-step Reverse transcription (RT)-PCR with the Model 7700 ABI Prism (R) Sequence Detection System. Viruses were directly extracted from unconcentrated grab samples of seawater, from seawater concentrated by vortex flow filtration using a 100kD filter and from sponge tissue. Total RNA was extracted from the samples, purified and concentrated using spin-column chromatography. A 192-196 base pair portion of the 5??? untranscribed region was amplified from these extracts. Enterovirus concentrations were estimated using real-time RT-PCR technology. Nine of 15 sample sites or 60% were positive for the presence of pathogenic human enteroviruses. Considering only near-shore sites, 69% were positive with viral concentrations ranging from 9.3viruses/ml to 83viruses/g of sponge tissue (uncorrected for extraction efficiency). Certain amplicons were selected for cloning and sequencing for identification. Three strains of waterborne enteroviruses were identified as Coxsackievirus A9, Coxsackievirus A16, and Poliovirus Sabin type 1. Time and cost efficiency of this one-step real-time RT-PCR methodology makes this an ideal technique to detect, quantitate and identify pathogenic enteroviruses in recreational waters. Copyright ?? 2002 Elsevier Science Ltd.

  19. A Simultaneous Analytical Method for Duplex Identification of Porcine and Horse in the Meat Products by EvaGreen based Real-time PCR.

    PubMed

    Sakalar, Ergün; Ergün, Seyma Özçirak; Akar, Emine

    2015-01-01

    A duplex real-time polymerase chain reaction (PCR) based assay for the detection of porcine and horse meat in sausages was designed by using EvaGreen fluorescent dye. Primers were selected from mitochondrial 12S rRNA and 16S rRNA genes which are powerful regions for identification of horse and porcine meat. DNA from reference samples and industrial products was successfully extracted using the GIDAGEN® Multi-Fast DNA Isolation Kit. Genomes were identified based on their specific melting peaks (Mp) which are 82.5℃ and 78℃ for horse and porcine, respectively. The assay used in this study allowed the detection of as little as 0.0001% level of horse meat and 0.001% level of porcine meat in the experimental admixtures. These findings indicate that EvaGreen based duplex real-time PCR is a potentially sensitive, reliable, rapid and accurate assay for the detection of meat species adulterated with porcine and horse meats.

  20. Real-Time PCR and Sequencing Assays for Rapid Detection and Identification of Avian Schistosomes in Environmental Samples

    PubMed Central

    Mull, Bonnie J.; Brant, Sara V.; Loker, Eric S.; Collinson, Jeremy; Secor, W. Evan; Hill, Vincent R.

    2015-01-01

    Cercarial dermatitis, also known as swimmer's itch, is an allergenic skin reaction followed by intense itching caused by schistosome cercariae penetrating human skin. Cercarial dermatitis outbreaks occur globally and are frequently associated with freshwater lakes and are occasionally associated with marine or estuarine waters where birds reside year-round or where migratory birds reside. In this study, a broadly reactive TaqMan assay targeting 18S rRNA gene (ribosomal DNA [rDNA]) sequences that was based on a genetically diverse panel of schistosome isolates representing 13 genera and 20 species (the 18S rDNA TaqMan assay) was developed. A PCR assay was also developed to amplify a 28S rDNA region for subsequent sequencing to identify schistosomes. When applied to surface water samples seeded with Schistosoma mansoni cercariae, the 18S rDNA TaqMan assay enabled detection at a level of 5 S. mansoni cercariae in 100 liters of lake water. The 18S rDNA TaqMan and 28S rDNA PCR sequencing assays were also applied to 100-liter water samples collected from lakes in Nebraska and Wisconsin where there were reported dermatitis outbreaks. Avian schistosome DNA was detected in 11 of 34 lake water samples using the TaqMan assay. Further 28S rDNA sequence analysis of positive samples confirmed the presence of avian schistosome DNA and provided a preliminary identification of the avian schistosomes in 10 of the 11 samples. These data indicate that the broadly schistosome-reactive TaqMan assay can be effective for rapid screening of large-volume water samples for detection of avian schistosomes, thereby facilitating timely response actions to mitigate or prevent dermatitis outbreaks. Additionally, samples positive by the 18S rDNA TaqMan assay can be further assayed using the 28S rDNA sequencing assay to both confirm the presence of schistosomes and contribute to their identification. PMID:25862226

  1. Real-time PCR and sequencing assays for rapid detection and identification of avian schistosomes in environmental samples.

    PubMed

    Jothikumar, Narayanan; Mull, Bonnie J; Brant, Sara V; Loker, Eric S; Collinson, Jeremy; Secor, W Evan; Hill, Vincent R

    2015-06-15

    Cercarial dermatitis, also known as swimmer's itch, is an allergenic skin reaction followed by intense itching caused by schistosome cercariae penetrating human skin. Cercarial dermatitis outbreaks occur globally and are frequently associated with freshwater lakes and are occasionally associated with marine or estuarine waters where birds reside year-round or where migratory birds reside. In this study, a broadly reactive TaqMan assay targeting 18S rRNA gene (ribosomal DNA [rDNA]) sequences that was based on a genetically diverse panel of schistosome isolates representing 13 genera and 20 species (the 18S rDNA TaqMan assay) was developed. A PCR assay was also developed to amplify a 28S rDNA region for subsequent sequencing to identify schistosomes. When applied to surface water samples seeded with Schistosoma mansoni cercariae, the 18S rDNA TaqMan assay enabled detection at a level of 5 S. mansoni cercariae in 100 liters of lake water. The 18S rDNA TaqMan and 28S rDNA PCR sequencing assays were also applied to 100-liter water samples collected from lakes in Nebraska and Wisconsin where there were reported dermatitis outbreaks. Avian schistosome DNA was detected in 11 of 34 lake water samples using the TaqMan assay. Further 28S rDNA sequence analysis of positive samples confirmed the presence of avian schistosome DNA and provided a preliminary identification of the avian schistosomes in 10 of the 11 samples. These data indicate that the broadly schistosome-reactive TaqMan assay can be effective for rapid screening of large-volume water samples for detection of avian schistosomes, thereby facilitating timely response actions to mitigate or prevent dermatitis outbreaks. Additionally, samples positive by the 18S rDNA TaqMan assay can be further assayed using the 28S rDNA sequencing assay to both confirm the presence of schistosomes and contribute to their identification.

  2. A HRM Real-Time PCR Assay for Rapid and Specific Identification of the Emerging Pest Spotted-Wing Drosophila (Drosophila suzukii)

    PubMed Central

    Dhami, Manpreet K.; Kumarasinghe, Lalith

    2014-01-01

    Spotted wing drosophila (Drosophila suzukii) is an emerging pest that began spreading in 2008 and its distribution now includes 13 countries across two continents. Countries where it is established have reported significant economic losses of fresh produce, such as cherries due to this species of fly. At larval stages, it is impossible to identify due to its striking similarities with other cosmopolitan and harmless drosophilids. Molecular methods allow identification but the current technique of DNA barcoding is time consuming. We developed and validated a rapid, highly sensitive and specific assay based on real-time PCR and high resolution melt (HRM) analysis using EvaGreen DNA intercalating dye chemistry. Performance characteristics of this qualitative assay, validation and applicability in a New Zealand quarantine framework are discussed. Application of this robust and independently validated assay across the spectrum of key food production and border protection industries will allow us to reduce the further spread of this damaging species worldwide. PMID:24927410

  3. Identification and validation of reference genes for accurate normalization of real-time quantitative PCR data in kiwifruit.

    PubMed

    Ferradás, Yolanda; Rey, Laura; Martínez, Óscar; Rey, Manuel; González, Ma Victoria

    2016-05-01

    Identification and validation of reference genes are required for the normalization of qPCR data. We studied the expression stability produced by eight primer pairs amplifying four common genes used as references for normalization. Samples representing different tissues, organs and developmental stages in kiwifruit (Actinidia chinensis var. deliciosa (A. Chev.) A. Chev.) were used. A total of 117 kiwifruit samples were divided into five sample sets (mature leaves, axillary buds, stigmatic arms, fruit flesh and seeds). All samples were also analysed as a single set. The expression stability of the candidate primer pairs was tested using three algorithms (geNorm, NormFinder and BestKeeper). The minimum number of reference genes necessary for normalization was also determined. A unique primer pair was selected for amplifying the 18S rRNA gene. The primer pair selected for amplifying the ACTIN gene was different depending on the sample set. 18S 2 and ACT 2 were the candidate primer pairs selected for normalization in the three sample sets (mature leaves, fruit flesh and stigmatic arms). 18S 2 and ACT 3 were the primer pairs selected for normalization in axillary buds. No primer pair could be selected for use as the reference for the seed sample set. The analysis of all samples in a single set did not produce the selection of any stably expressing primer pair. Considering data previously reported in the literature, we validated the selected primer pairs amplifying the FLOWERING LOCUS T gene for use in the normalization of gene expression in kiwifruit.

  4. [Dynamic detection of surface blood flow in rat heart and its application in real time identification of myocardial infarction model].

    PubMed

    Lei, Q; Chen, C; Wu, X L; Chen, W J; Yi, T; Ma, M D; He, Y; Shui, X R; Huang, S A; Chen, C; Lei, W

    2017-04-04

    Objective: To establish a method for monitoring the surface blood flow in the heart of rats, and to clarify the relationship between the degree of myocardial infarction and the blood perfusion on the surface of the heart, so as to provide a new indicator for the identification of rat myocardial infarction model. Methods: The rats were divided into control group (n=23) and model group (n=107), the rat hearts were scanned by the laser doppler perfusion imager before and after operation respectively, and the data was analyzed to acquire the rate of surface blood flow change of the heart. Myocardial infarction size of model group was detected by NBT. Model group were divided into three subgroups of mild myocardial infarction, moderate myocardial infarction and severe myocardial infarction according to the myocardial infarction size, and an analysis was made on the correlativity between rate of surface blood flow change of the heart and myocardial infarction size. Results: Myocardial infarction size was highly correlated to the rate of surface blood flow change of the heart in model group (r=0.849 6, P<0.000 1). There was no significant correlation between infarction size and heart blood flow in the mild myocardial infarction subgroup (r=-0.133 6, P>0.05), while the correlation in moderate myocardial infarction was significant (r=0.721 7, P<0.000 1), and the highest correlation was shown in severe myocardial infarction subgroup (r=0.910 2, P<0.000 1). Conclusion: The heart surface blood flow has a close relationship with the myocardial infarction size in rat, so the change of heart blood perfusion can beused as an effective reference to establish and identify rat myocardial infarction model.

  5. Multiplex real-time PCR and blood culture for identification of bloodstream pathogens in patients with suspected sepsis.

    PubMed

    Westh, H; Lisby, G; Breysse, F; Böddinghaus, B; Chomarat, M; Gant, V; Goglio, A; Raglio, A; Schuster, H; Stuber, F; Wissing, H; Hoeft, A

    2009-06-01

    Severe sepsis is increasingly a cause of death. Rapid and correct initial antimicrobial treatment reduces mortality. The aetiological agent(s) cannot always be found in blood cultures (BCs). A novel multiplex PCR test (SeptiFast (alpha version)) that allows identification of 20 bacterial and fungal species directly from blood was used, comparatively with BC, in a multicentre trial of patients with suspected bacterial or fungal sepsis. Five hundred and fifty-eight paired samples from 359 patients were evaluated. The rate of positivity was 17% for BC and 26% for SeptiFast. Ninety-six microorganisms were isolated with BC, and 186 microorganisms were identified with SeptiFast; 231 microorganisms were found by combining the two tests. Of the 96 isolates identified with BC, 22 isolates were considered to be contaminants. Of the remaining 74 non-contaminant BC isolates available for comparison with SeptiFast, 50 were identified as a species identical to the species identified with SeptiFast in the paired sample. Of the remaining 24 BC isolates for which the species, identified in the BC, could not be detected in the paired SeptiFast sample, 18 BC isolates were identified as a species included in the SeptiFast master list, and six BC isolates were identified as a species not included in the SeptiFast master list. With SeptiFast, 186 microorganisms were identified, 12 of which were considered to be contaminants. Of the 174 clinically relevant microorganisms identified with SeptiFast, 50 (29%) were detected by BC. More than half of the remaining microorganisms identified with SeptiFast (but not isolated after BC) were also found in routine cultures of other relevant samples taken from the patients. Future clinical studies should assess whether the use of SeptiFast is of significant advantage in the detection of bloodstream pathogens.

  6. Multicentric Evaluation of a New Real-Time PCR Assay for Quantification of Cryptosporidium spp. and Identification of Cryptosporidium parvum and Cryptosporidium hominis

    PubMed Central

    Chapey, E.; Dutoit, E.; Guyot, K.; Hasseine, L.; Jeddi, F.; Menotti, J.; Paraud, C.; Pomares, C.; Rabodonirina, M.; Rieux, A.; Derouin, F.

    2013-01-01

    Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidium-positive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed. PMID:23720792

  7. Real-time enzyme-digesting identification of double-strand DNA in a resonance-cantilever embedded micro-chamber.

    PubMed

    Xu, Tiegang; Yu, Haitao; Xu, Pengcheng; Xu, Wangjie; Chen, Wenqing; Chen, Chuanzhao; Li, Xinxin

    2014-03-21

    A novel direct identification of double-strand DNA is proposed by using real-time enzyme-digestion in a resonant-cantilever embedded microfluidic chip. The new gene-level detection method is expected to replace the conventional DNA-hybridization based gene-detection that suffers from not only nonspecific adsorption induced false-positives but also complicated single-strand DNA preparation and hybridization. Since a detected DNA chain features a unique cutting site for a certain restriction-enzyme, the accurately cut-off mass (representing the length of the digested segment) can be online recorded by the frequency-shift signal of the resonant micro-cantilever sensor. This enzyme-digestion technique is confirmed by experimental identification of the stx2 gene of E. coli O157:H7. The direct-PCR sample is directly analyzed by using our lab-made cantilever-embedded microfluidic-chip. The 3776 bp DNA is immobilized via biotin-streptavidin binding and the added mass is recorded by a frequency-decrease of 15.9 kHz within 10 min. Then, with EcoRV-enzyme digestion at the site of 2635 bp, the cut-off mass is real-time detected by a frequency-increase of 10.2 kHz within 6 min. The detected frequency-shift ratio of 15.9/10.2 = 64.2% is consistent with the length ratio between the cut-off fragment and the whole DNA chain (2635/3776 = 69.8%). Hence, the simple and accurate double-strand detection method is verified experimentally.

  8. Exploring the effects of driving experience on hazard awareness and risk perception via real-time hazard identification, hazard classification, and rating tasks.

    PubMed

    Borowsky, Avinoam; Oron-Gilad, Tal

    2013-10-01

    This study investigated the effects of driving experience on hazard awareness and risk perception skills. These topics have previously been investigated separately, yet a novel approach is suggested where hazard awareness and risk perception are examined concurrently. Young, newly qualified drivers, experienced drivers, and a group of commercial drivers, namely, taxi drivers performed three consecutive tasks: (1) observed 10 short movies of real-world driving situations and were asked to press a button each time they identified a hazardous situation; (2) observed one of three possible sub-sets of 8 movies (out of the 10 they have seen earlier) for the second time, and were asked to categorize them into an arbitrary number of clusters according to the similarity in their hazardous situation; and (3) observed the same sub-set for a third time and following each movie were asked to rate its level of hazardousness. The first task is considered a real-time identification task while the other two are performed using hindsight. During it participants' eye movements were recorded. Results showed that taxi drivers were more sensitive to hidden hazards than the other driver groups and that young-novices were the least sensitive. Young-novice drivers also relied heavily on materialized hazards in their categorization structure. In addition, it emerged that risk perception was derived from two major components: the likelihood of a crash and the severity of its outcome. Yet, the outcome was rarely considered under time pressure (i.e., in real-time hazard identification tasks). Using hindsight, when drivers were provided with the opportunity to rate the movies' hazardousness more freely (rating task) they considered both components. Otherwise, in the categorization task, they usually chose the severity of the crash outcome as their dominant criterion. Theoretical and practical implications are discussed. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Portable, real-time alloy identification of metallic wear debris from machinery lubrication systems: laser-induced breakdown spectroscopy versus x-ray fluorescence

    NASA Astrophysics Data System (ADS)

    Suresh, Pooja

    2014-05-01

    Alloy identification of oil-borne wear debris captured on chip detectors, filters and magnetic plugs allows the machinery maintainer to assess the health of the engine or gearbox and identify specific component damage. Today, such identification can be achieved in real time using portable, at-line laser-induced breakdown spectroscopy (LIBS) and Xray fluorescence (XRF) instruments. Both techniques can be utilized in various industries including aviation, marine, railways, heavy diesel and other industrial machinery with, however, some substantial differences in application and instrument performance. In this work, the performances of a LIBS and an XRF instrument are compared based on measurements of a wide range of typical aerospace alloys including steels, titanium, aluminum and nickel alloys. Measurement results were analyzed with a staged correlation technique specifically developed for the purposes of this study - identifying the particle alloy composition using a pre-recorded library of spectral signatures. The analysis is performed in two stages: first, the base element of the alloy is determined by correlation with the stored elemental spectra and then, the alloy is identified by matching the particle's spectral signature using parametric correlation against the stored spectra of all alloys that have the same base element. The correlation analysis has achieved highly repeatable discrimination between alloys of similar composition. Portable LIBS demonstrates higher detection accuracy and better identification of alloys comprising lighter elements as compared to that of the portable XRF system, and reveals a significant reduction in the analysis time over XRF.

  10. Real-time radiography

    SciTech Connect

    Bossi, R.H.; Oien, C.T.

    1981-02-26

    Real-time radiography is used for imaging both dynamic events and static objects. Fluorescent screens play an important role in converting radiation to light, which is then observed directly or intensified and detected. The radiographic parameters for real-time radiography are similar to conventional film radiography with special emphasis on statistics and magnification. Direct-viewing fluoroscopy uses the human eye as a detector of fluorescent screen light or the light from an intensifier. Remote-viewing systems replace the human observer with a television camera. The remote-viewing systems have many advantages over the direct-viewing conditions such as safety, image enhancement, and the capability to produce permanent records. This report reviews real-time imaging system parameters and components.

  11. Real time obscuration monitoring

    NASA Astrophysics Data System (ADS)

    Agricola, Koos

    2016-09-01

    Recently a real time particle deposition monitoring system is developed. After discussions with optical system engineers a new feature has been added. This enables the real time monitoring of obscuration of exposed optical components by counting the deposited particles and sizing the obscuration area of each particle. This way the Particle Obscuration Rate (POR) can be determined. The POR can be used to determine the risk of product contamination during exposure. The particle size distribution gives information on the type of potential particle sources. The deposition moments will indicate when these sources were present.

  12. Evaluation of a novel real-time PCR test based on the ssrA gene for the identification of group B streptococci in vaginal swabs

    PubMed Central

    2009-01-01

    Background Despite the implementation of prevention guidelines, early-onset group B streptococci (GBS) disease remains a cause of neonatal morbidity and mortality worldwide. Strategies to identify women who are at risk of transmitting GBS to their infant and the administration of intrapartum antibiotics have greatly reduced the incidence of neonatal GBS disease. However, there is a requirement for a rapid diagnostic test for GBS that can be carried out in a labour ward setting especially for women whose GBS colonisation status is unknown at the time of delivery. We report the design and evaluation of a real-time PCR test (RiboSEQ GBS test) for the identification of GBS in vaginal swabs from pregnant women. Methods The qualitative real-time PCR RiboSEQ GBS test was designed based on the bacterial ssrA gene and incorporates a competitive internal standard control. The analytical sensitivity of the test was established using crude lysate extracted from serial dilutions of overnight GBS culture using the IDI Lysis kit. Specificity studies were performed using DNA prepared from a panel of GBS strains, related streptococci and other species found in the genital tract environment. The RiboSEQ GBS test was evaluated on 159 vaginal swabs from pregnant women and compared with the GeneOhm™ StrepB Assay and culture for the identification of GBS. Results The RiboSEQ GBS test is specific and has an analytical sensitivity of 1-10 cell equivalents. The RiboSEQ GBS test was 96.4% sensitive and 95.8% specific compared to "gold standard" culture for the identification of GBS in vaginal swabs from pregnant women. In this study, the RiboSEQ GBS test performed slightly better than the commercial BD GeneOhm™ StrepB Assay which gave a sensitivity of 94.6% and a specificity of 89.6% compared to culture. Conclusion The RiboSEQ GBS test is a valuable method for the rapid, sensitive and specific detection of GBS in pregnant women. This study also validates the ssrA gene as a suitable and

  13. Device-independent quantum private query

    NASA Astrophysics Data System (ADS)

    Maitra, Arpita; Paul, Goutam; Roy, Sarbani

    2017-04-01

    In quantum private query (QPQ), a client obtains values corresponding to his or her query only, and nothing else from the server, and the server does not get any information about the queries. V. Giovannetti et al. [Phys. Rev. Lett. 100, 230502 (2008)], 10.1103/PhysRevLett.100.230502 gave the first QPQ protocol and since then quite a few variants and extensions have been proposed. However, none of the existing protocols are device independent; i.e., all of them assume implicitly that the entangled states supplied to the client and the server are of a certain form. In this work, we exploit the idea of a local CHSH game and connect it with the scheme of Y. G. Yang et al. [Quantum Info. Process. 13, 805 (2014)], 10.1007/s11128-013-0692-8 to present the concept of a device-independent QPQ protocol.

  14. Development of a SYBR green I real-time PCR assay for specific identification of the fish pathogen Aeromonas salmonicida subspecies salmonicida.

    PubMed

    Fernández-Álvarez, Clara; González, Santiago F; Santos, Ysabel

    2016-12-01

    A SYBR Green I real-time polymerase chain reaction protocol for specific detection of the fish pathogen Aeromonas salmonicida subsp. salmonicida was developed and validated for rapid diagnosis of typical furunculosis. The sequence of the aopO gene of A. salmonicida subsp. salmonicida, which encodes for a serine/threonine protein kinase linked to virulence, was chosen for primer design. The selected primers amplified a 119-bp internal fragment of the aopO gene. The specificity test proved that 100 % (40/40) of the A. salmonicida subsp. salmonicida strains tested showed a positive amplification with subspecies-specific melting temperatures (Tm) of 80.75 ± 0.35 °C. Atypical A. salmonicida subspecies and other non-related bacterial fish pathogens did not amplify or showed unspecific melting profiles, except for one strain of A. salmonicida subsp. achromogenes and one strain of A. salmonicida subsp. smithia. The detection sensitivity was 21 fg of purified bacterial DNA per reaction, corresponding to 1-2 bacterial cells and 6-60 bacteria per reaction for seeded kidney and blood. The assay was highly reproducible with low variation coefficient values for intra-run and inter-run assays. The assay also allowed the specific detection of A. salmonicida subsp. salmonicida in tissues of fish naturally and experimentally infected. No amplification was detected when tissues from healthy fish or fish affected by other diseases were tested. The SYBR Green real-time PCR and melt curve analysis developed in this study is a rapid and accurate method for the specific identification of A. salmonicida subsp. salmonicida isolates and its detection on tissues of fish affected by furunculosis.

  15. Real-time monitoring for detection of retained surgical sponges and team motion in the surgical operation room using radio-frequency-identification (RFID) technology: a preclinical evaluation.

    PubMed

    Kranzfelder, Michael; Zywitza, Dorit; Jell, Thomas; Schneider, Armin; Gillen, Sonja; Friess, Helmut; Feussner, Hubertus

    2012-06-15

    Technical progress in the surgical operating room (OR) increases constantly, facilitating the development of intelligent OR systems functioning as "safety backup" in the background of surgery. Precondition is comprehensive data retrieval to identify imminent risky situations and inaugurate adequate security mechanisms. Radio-frequency-identification (RFID) technology may have the potential to meet these demands. We set up a pilot study investigating feasibility and appliance reliability of a stationary RFID system for real-time surgical sponge monitoring (passive tagged sponges, position monitoring: mayo-stand/abdominal situs/waste bucket) and OR team tracking (active transponders, position monitoring: right/left side of OR table). In vitro: 20/20 sponges (100%) were detected on the mayo-stand and within the OR-phantom, however, real-time detection accuracy declined to 7/20 (33%) when the tags were moved simultaneously. All retained sponges were detected correctly. In vivo (animal): 7-10/10 sterilized sponges (70%-100%) were detected correctly within the abdominal cavity. OR-team: detection accuracy within the OR (surveillance antenna) and on both sides of the OR table (sector antenna) was 100%. Mean detection time for position change (left to right side and contrariwise) was 30-60 s. No transponder failure was noted. This is the first combined RFID system that has been developed for stationary use in the surgical OR. Preclinical evaluation revealed a reliable sponge tracking and correct detection of retained textiles (passive RFID) but also demonstrated feasibility of comprehensive data acquisition of team motion (active RFID). However, detection accuracy needs to be further improved before implementation into the surgical OR. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Complementary use of MALDI-TOF MS and real-time PCR-melt curve analysis for rapid identification of methicillin-resistant staphylococci and VRE.

    PubMed

    Chan, Wai-Sing; Chan, Tsz-Ming; Lai, Tsz-Wan; Chan, Jasper Fuk-Woo; Lai, Raymond Wai-Man; Lai, Christopher Koon-Chi; Tang, Bone Siu-Fai

    2015-02-01

    To develop a rapid method for routine screening of methicillin-resistant staphylococci and VRE for clinical isolates and positive blood cultures. Our method consisted of two parts: MALDI-TOF MS was used for identification of staphylococci and enterococci, followed by antibiotic resistance detection by real-time PCR-melt curve analysis without DNA extraction. The latter part included a triplex reaction for staphylococcal culture isolates (mecA, mecALGA251 and Panton-Valentine leucocidin genes), dual PCR of mecA/mecALGA251 and nuc genes for staphylococcal blood cultures, and a duplex reaction for enterococci (vanA and vanB genes). A total of 124 clinical isolates and 56 positive blood cultures were tested. MALDI-TOF MS was performed using Microflex LT (Bruker Daltonik, Bremen, Germany) and Rotor-Gene Q (Qiagen, Hilden, Germany) was used for real-time PCR-melt curve analysis. The total assay time was <2.5 h. The results revealed 100% concordance with antibiotic susceptibility testing or other reference methods for all culture isolates and enterococcal blood cultures. The percentage of concordance for staphylococcal blood cultures was 97.5%. The method described herein was fast, economical, reliable and capable of detecting mecALGA251, vanB1 and vanB2 genotypes, which are not included in most commercial assays. Large-scale screening is required to further test the performance of this protocol, especially for genotypes that are infrequently encountered. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Development and testing of real-time PCR assays for determining fecal loading and source identification (cattle, human, etc.) in surface water and groundwater

    NASA Astrophysics Data System (ADS)

    McKay, L. D.; Layton, A.; Gentry, R.

    2004-12-01

    A multi-disciplinary group of researchers at the University of Tennessee is developing and testing a series of microbial assay methods based on real-time PCR to detect fecal bacterial concentrations and host sources in water samples. Real-time PCR is an enumeration technique based on the unique and conserved nucleic acid sequences present in all organisms. The first research task was development of an assay (AllBac) to detect total amount of Bacteroides, which represents up to 30 percent of fecal mass. Subsequent assays were developed to detect Bacteroides from cattle (BoBac) and humans (HuBac) using 16sRNA genes based on DNA sequences in the national GenBank, as well as sequences from local fecal samples. The assays potentially have significant advantages over conventional bacterial source tracking methods because: 1. unlike traditional enumeration methods, they do not require bacterial cultivation; 2. there are no known non-fecal sources of Bacteroides; 3. the assays are quantitative with results for total concentration and for each species expressed in mg/l; and 4. they show little regional variation within host species, meaning that they do not require development of extensive local gene libraries. The AllBac and BoBac assays have been used in a study of fecal contamination in a small rural watershed (Stock Creek) near Knoxville, TN, and have proven useful in identification of areas where cattle represent a significant fecal input and in development of BMPs. It is expected that these types of assays (and future assays for birds, hogs, etc.) could have broad applications in monitoring fecal impacts from Animal Feeding Operations, as well as from wildlife and human sources.

  18. DNA barcoding, species-specific PCR and real-time PCR techniques for the identification of six Tribolium pests of stored products

    PubMed Central

    Zhang, Tao; Wang, Yi-Jiao; Guo, Wei; Luo, Dan; Wu, Yi; Kučerová, Zuzana; Stejskal, Václav; Opit, George; Cao, Yang; Li, Fu-Jun; Li, Zhi-Hong

    2016-01-01

    Flour beetles of the genus Tribolium Macleay (Coleoptera: Tenebrionidae) are important stored product pests in China and worldwide. They are often found or are intercepted in grain depots, flour mills, and entry-exit ports, etc. Traditionally, Tribolium species are identified according to the morphological characteristics of the adult. However, it is almost impossible to rapidly identify adult fragments and non-adult stages based on external morphological characteristics. Molecular techniques for the rapid and accurate identification of Tribolium species are required, particularly for pest monitoring and the quarantine of stored products pests. Here, we establish DNA barcoding, species-specific PCR, and real-time PCR techniques for the identification of six stored-product pest Tribolium species including T. castaneum, T. confusum, T. destructor, T. madens, T. freemani and T. brevicornis. We detected the mitochondrial DNA cytochrome oxidase subunit I (COI) barcodes for Tribolium from 18 geographic populations and 101 individuals, built a Tribolium DNA barcode library, and designed species-specific primers and TaqMan probes for the above six Tribolium species. The three techniques were applied to identify Tribolium collected from stored samples and samples captured from quarantine ports. The results demonstrated that three techniques were all able to identify the six species of Tribolium both rapidly and accurately. PMID:27352804

  19. A real-time PCR assay for the rapid identification of the autoimmune disease-associated allele HLA-DQB1*0602

    PubMed Central

    Gersuk, Vivian H.; Nepom, Gerald T.

    2011-01-01

    Many autoimmune diseases share a genetic association with the presence or absence of HLA-DQB1*0602, including type I diabetes, multiple sclerosis, and narcolepsy. High resolution HLA typing to determine the presence of this allele is cumbersome and expensive by currently available techniques. We present a real-time PCR assay for the identification of HLA-DQB1*0602, using sequence-specific primers and probes, that provides rapid and sensitive identification of this allele, involves minimal hands-on time, and provides a major cost savings compared to existing methods. The assay allows the simultaneous determination of both the presence and the number of copies of this allele. Since there is no post-PCR handling, the risk of contamination is avoided. We have validated the assay using 44 blinded and 32 unblinded samples, previously typed by standard techniques, which were identified with 100% accuracy, sensitivity, and specificity. Further, using a narcolepsy cohort of 734 subjects, we demonstrated the robustness of the assay to analyze DNA isolated from buccal swabs, demonstrating the applicability of this assay as an alternative approach to traditional HLA typing methods. PMID:19317743

  20. Direct analysis in real time - high resolution mass spectrometry (DART-HRMS): a high throughput strategy for identification and quantification of anabolic steroid esters.

    PubMed

    Doué, Mickael; Dervilly-Pinel, Gaud; Pouponneau, Karinne; Monteau, Fabrice; Le Bizec, Bruno

    2015-07-01

    High throughput screening is essential for doping, forensic, and food safety laboratories. While hyphenated chromatography-mass spectrometry (MS) remains the approach of choice, recent ambient MS techniques, such as direct analysis in real time (DART), offer more rapid and more versatile strategies and thus gain in popularity. In this study, the potential of DART hyphenated with Orbitrap-MS for fast identification and quantification of 21 anabolic steroid esters has been evaluated. Direct analysis in high resolution scan mode allowed steroid esters screening by accurate mass measurement (Resolution = 60 000 and mass error < 3 ppm). Steroid esters identification was further supported by collision-induced dissociation (CID) experiments through the generation of two additional ions. Moreover, the use of labelled internal standards allowed quantitative data to be recovered based on isotopic dilution approach. Linearity (R(2)  > 0.99), dynamic range (from 1 to 1000 ng mL(-1) ), bias (<10%), sensitivity (1 ng mL(-1) ), repeatability and reproducibility (RSD < 20%) were evaluated as similar to those obtained with hyphenated chromatography-mass spectrometry techniques. This innovative high throughput approach was successfully applied for the characterization of oily commercial preparations, and thus fits the needs of the competent authorities in the fight against forbidden or counterfeited substances.

  1. Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms.

    PubMed

    Cottenet, Geoffrey; Blancpain, Carine; Sonnard, Véronique; Chuah, Poh Fong

    2013-08-01

    Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers' perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories.

  2. DNA barcoding, species-specific PCR and real-time PCR techniques for the identification of six Tribolium pests of stored products.

    PubMed

    Zhang, Tao; Wang, Yi-Jiao; Guo, Wei; Luo, Dan; Wu, Yi; Kučerová, Zuzana; Stejskal, Václav; Opit, George; Cao, Yang; Li, Fu-Jun; Li, Zhi-Hong

    2016-06-29

    Flour beetles of the genus Tribolium Macleay (Coleoptera: Tenebrionidae) are important stored product pests in China and worldwide. They are often found or are intercepted in grain depots, flour mills, and entry-exit ports, etc. Traditionally, Tribolium species are identified according to the morphological characteristics of the adult. However, it is almost impossible to rapidly identify adult fragments and non-adult stages based on external morphological characteristics. Molecular techniques for the rapid and accurate identification of Tribolium species are required, particularly for pest monitoring and the quarantine of stored products pests. Here, we establish DNA barcoding, species-specific PCR, and real-time PCR techniques for the identification of six stored-product pest Tribolium species including T. castaneum, T. confusum, T. destructor, T. madens, T. freemani and T. brevicornis. We detected the mitochondrial DNA cytochrome oxidase subunit I (COI) barcodes for Tribolium from 18 geographic populations and 101 individuals, built a Tribolium DNA barcode library, and designed species-specific primers and TaqMan probes for the above six Tribolium species. The three techniques were applied to identify Tribolium collected from stored samples and samples captured from quarantine ports. The results demonstrated that three techniques were all able to identify the six species of Tribolium both rapidly and accurately.

  3. Real-Time Identification of Smoldering and Flaming Combustion Phases in Forest Using a Wireless Sensor Network-Based Multi-Sensor System and Artificial Neural Network.

    PubMed

    Yan, Xiaofei; Cheng, Hong; Zhao, Yandong; Yu, Wenhua; Huang, Huan; Zheng, Xiaoliang

    2016-08-04

    Diverse sensing techniques have been developed and combined with machine learning method for forest fire detection, but none of them referred to identifying smoldering and flaming combustion phases. This study attempts to real-time identify different combustion phases using a developed wireless sensor network (WSN)-based multi-sensor system and artificial neural network (ANN). Sensors (CO, CO₂, smoke, air temperature and relative humidity) were integrated into one node of WSN. An experiment was conducted using burning materials from residual of forest to test responses of each node under no, smoldering-dominated and flaming-dominated combustion conditions. The results showed that the five sensors have reasonable responses to artificial forest fire. To reduce cost of the nodes, smoke, CO₂ and temperature sensors were chiefly selected through correlation analysis. For achieving higher identification rate, an ANN model was built and trained with inputs of four sensor groups: smoke; smoke and CO₂; smoke and temperature; smoke, CO₂ and temperature. The model test results showed that multi-sensor input yielded higher predicting accuracy (≥82.5%) than single-sensor input (50.9%-92.5%). Based on these, it is possible to reduce the cost with a relatively high fire identification rate and potential application of the system can be tested in future under real forest condition.

  4. Real-Time Identification of Smoldering and Flaming Combustion Phases in Forest Using a Wireless Sensor Network-Based Multi-Sensor System and Artificial Neural Network

    PubMed Central

    Yan, Xiaofei; Cheng, Hong; Zhao, Yandong; Yu, Wenhua; Huang, Huan; Zheng, Xiaoliang

    2016-01-01

    Diverse sensing techniques have been developed and combined with machine learning method for forest fire detection, but none of them referred to identifying smoldering and flaming combustion phases. This study attempts to real-time identify different combustion phases using a developed wireless sensor network (WSN)-based multi-sensor system and artificial neural network (ANN). Sensors (CO, CO2, smoke, air temperature and relative humidity) were integrated into one node of WSN. An experiment was conducted using burning materials from residual of forest to test responses of each node under no, smoldering-dominated and flaming-dominated combustion conditions. The results showed that the five sensors have reasonable responses to artificial forest fire. To reduce cost of the nodes, smoke, CO2 and temperature sensors were chiefly selected through correlation analysis. For achieving higher identification rate, an ANN model was built and trained with inputs of four sensor groups: smoke; smoke and CO2; smoke and temperature; smoke, CO2 and temperature. The model test results showed that multi-sensor input yielded higher predicting accuracy (≥82.5%) than single-sensor input (50.9%–92.5%). Based on these, it is possible to reduce the cost with a relatively high fire identification rate and potential application of the system can be tested in future under real forest condition. PMID:27527175

  5. Device-independent certification of entangled measurements.

    PubMed

    Rabelo, Rafael; Ho, Melvyn; Cavalcanti, Daniel; Brunner, Nicolas; Scarani, Valerio

    2011-07-29

    We present a device-independent protocol to test if a given black-box measurement device is entangled, that is, has entangled eigenstates. Our scheme involves three parties and is inspired by entanglement swapping; the test uses the Clauser-Horne-Shimony-Holt Bell inequality, checked between each pair of parties. In the case where all particles are qubits, we characterize quantitatively the deviation of the measurement device from a perfect Bell-state measurement.

  6. Pragmatic Approach to Device-Independent Color

    NASA Technical Reports Server (NTRS)

    Brandt, R. D.; Capraro, K. S.

    1995-01-01

    JPL has been producing images of planetary bodies for over 30 years. The results of an effort to implement device-independent color on three types of devices are described. The goal is to produce near the same eye-brain response when the observer views the image produced by each device under the correct lighting conditions. The procedure used to calibrate and obtain each device profile is described.

  7. Pragmatic Approach to Device-Independent Color

    NASA Technical Reports Server (NTRS)

    Brandt, R. D.; Capraro, K. S.

    1995-01-01

    JPL has been producing images of planetary bodies for over 30 years. The results of an effort to implement device-independent color on three types of devices are described. The goal is to produce near the same eye-brain response when the observer views the image produced by each device under the correct lighting conditions. The procedure used to calibrate and obtain each device profile is described.

  8. Device-independent quantum key distribution

    NASA Astrophysics Data System (ADS)

    Hänggi, Esther

    2010-12-01

    In this thesis, we study two approaches to achieve device-independent quantum key distribution: in the first approach, the adversary can distribute any system to the honest parties that cannot be used to communicate between the three of them, i.e., it must be non-signalling. In the second approach, we limit the adversary to strategies which can be implemented using quantum physics. For both approaches, we show how device-independent quantum key distribution can be achieved when imposing an additional condition. In the non-signalling case this additional requirement is that communication is impossible between all pairwise subsystems of the honest parties, while, in the quantum case, we demand that measurements on different subsystems must commute. We give a generic security proof for device-independent quantum key distribution in these cases and apply it to an existing quantum key distribution protocol, thus proving its security even in this setting. We also show that, without any additional such restriction there always exists a successful joint attack by a non-signalling adversary.

  9. design of multiplexed detection assays for identification of avian influenza a virus subtypes pathogenic to humans by SmartCycler real-time reverse transcription-PCR.

    PubMed

    Wang, Wei; Ren, Peijun; Mardi, Sek; Hou, Lili; Tsai, Cheguo; Chan, Kwok Hung; Cheng, Peter; Sheng, Jun; Buchy, Philippe; Sun, Bing; Toyoda, Tetsuya; Lim, Wilina; Peiris, J S Malik; Zhou, Paul; Deubel, Vincent

    2009-01-01

    Influenza A virus (IAV) epidemics are the result of human-to-human or poultry-to-human transmission. Tracking seasonal outbreaks of IAV and other avian influenza virus (AIV) subtypes that can infect humans, aquatic and migratory birds, poultry, and pigs is essential for epidemiological surveillance and outbreak alerts. In this study, we performed four real-time reverse transcription-PCR (rRT-PCR) assays for identification of the IAV M and hemagglutinin (HA) genes from six known AIVs infecting pigs, birds, and humans. IAV M1 gene-positive samples tested by single-step rRT-PCR and a fluorogenic Sybr green I detection system were further processed for H5 subtype identification by using two-primer-set multiplex and Sybr green I rRT-PCR assays. H5 subtype-negative samples were then tested with either a TaqMan assay for subtypes H1 and H3 or a TaqMan assay for subtypes H2, H7, and H9 and a beacon multiplex rRT-PCR identification assay. The four-tube strategy was able to detect 10 RNA copies of the HA genes of subtypes H1, H2, H3, H5, and H7 and 100 RNA copies of the HA gene of subtype H9. At least six H5 clades of H5N1 viruses isolated in Southeast Asia and China were detected by that test. Using rRT-PCR assays for the M1 and HA genes in 202 nasopharyngeal swab specimens from children with acute respiratory infections, we identified a total of 39 samples positive for the IAV M1 gene and subtypes H1 and H3. When performed with a portable SmartCycler instrument, the assays offer an efficient, flexible, and reliable platform for investigations of IAV and AIV in remote hospitals and in the field.

  10. rpoB gene: a target for identification of LAB cocci by PCR-DGGE and melting curves analyses in real time PCR.

    PubMed

    Renouf, Vincent; Claisse, Olivier; Lonvaud-Funel, Aline

    2006-10-01

    Lactic acid bacteria (LAB) are essential in the quality of many fermented beverages like beer, cider and wine. In the two later cases, they convert malic acid into lactic acid during the malolactic fermentation. After fermentation, microbial stabilization is needed to prevent the development of spoilage bacteria species. Among them, cocci lead to different alterations: Pediococcus sp., and some strains of Leuconostoc mesenteroides and Oenococcus oeni can produce exopolysaccharides which modify wine viscosity and lead to ropiness. They also can produce acetic acid, biogenic amine, ethyl carbamate and volatile phenols. Therefore detection and identification are crucial. Results of phenotypic tests and DNA-DNA probes are not accurate enough. 16S RNA gene which is currently used for bacterial species identification presents intraspecies heterogeneity. The rpoB gene is an alternative to this limitation. However previous PCR targeting partial sequence of rpoB gene could not delimit cocci species. Therefore we compared the rpoB gene sequence of the six main cocci species found in fermented beverages: P. damnosus, P. dextrinicus, P. parvulus, P. pentosaceus, L. mesenteroides and O. oeni. The most discriminating partial sequence of the rpoB gene was chosen for designing primers. By PCR-DGGE the reliability of these primers was verified. It was controlled in a mixture of several cocci and other lactic acid bacteria (Lactobacillus sp.). Then we adapted the primers and the PCR conditions in order to achieve the identification of cocci species by real time PCR program including the fluorescent dye SYBR Green I, which gives faster results. PCR melt curves were established and a specific T(m) was attributed to each species.

  11. Real-Time PCR

    NASA Astrophysics Data System (ADS)

    Evrard, A.; Boulle, N.; Lutfalla, G. S.

    Over the past few years there has been a considerable development of DNA amplification by polymerase chain reaction (PCR), and real-time PCR has now superseded conventional PCR techniques in many areas, e.g., the quantification of nucleic acids and genotyping. This new approach is based on the detection and quantification of a fluorescent signal proportional to the amount of amplicons generated by PCR. Real-time detection is achieved by coupling a thermocycler with a fluorimeter. This chapter discusses the general principles of quantitative real-time PCR, the different steps involved in implementing the technique, and some examples of applications in medicine. The polymerase chain reaction (PCR) provides a way of obtaining a large number of copies of a double-stranded DNA fragment of known sequence. This DNA amplification technique, developed in 1985 by K. Mullis (Cetus Corporation), saw a spectacular development over the space of a few years, revolutionising the methods used up to then in molecular biology. Indeed, PCR has many applications, such as the detection of small amounts of DNA, cloning, and quantitative analysis (assaying), each of which will be discussed further below.

  12. Real-time cosmology

    NASA Astrophysics Data System (ADS)

    Quercellini, Claudia; Amendola, Luca; Balbi, Amedeo; Cabella, Paolo; Quartin, Miguel

    2012-12-01

    In recent years, improved astrometric and spectroscopic techniques have opened the possibility of measuring the temporal change of radial and transverse position of sources in the sky over relatively short time intervals. This has made at least conceivable to establish a novel research domain, which we dub “real-time cosmology”. We review for the first time most of the work already done in this field, analysing the theoretical framework as well as some foreseeable observational strategies and their capability to constrain models. We first focus on real-time measurements of the overall redshift drift and angular separation shift in distant sources, which allows the observer to trace the background cosmic expansion and large scale anisotropy, respectively. We then examine the possibility of employing the same kind of observations to probe peculiar and proper accelerations in clustered systems, and therefore their gravitational potential. The last two sections are devoted to the future change of the cosmic microwave background on “short” time scales, as well as to the temporal shift of the temperature anisotropy power spectrum and maps. We conclude revisiting in this context the usefulness of upcoming experiments (like CODEX and Gaia) for real-time observations.

  13. Duplex Real-time PCR assay and SYBR green I melting curve analysis for molecular identification of HPV genotypes 16, 18, 31, 35, 51 and 66.

    PubMed

    Tsakogiannis, D; Papacharalampous, M; Toska, E; Kyriakopoulou, Z; Dimitriou, T G; Ruether, I G A; Komiotis, D; Markoulatos, P

    2015-02-01

    Long-term infection with high-risk HPV genotypes is the leading cause of cervical cancer. In the present study a Duplex Real-time PCR assay was developed in order to identify HPV types 16, 18, 31, 35, 51 and 66 in three reactions, through SYBR green I melting curve analysis. The method utilizes type-specific primer sets that allowed the amplification of highly conserved regions of L1 gene. Reconstitution experiments were conducted by using HPV DNA plasmids in order to determine the sensitivity of the assay. The newly designed assay has a limit of detection of 10 copies per reaction. The most prevalent HPV genotype in single and in multiple HPV infections was HPV16 followed by HPV18, HPV51, HPV31, HPV35 and HPV66. The proposed method is a simple, specific, sensitive and cost-effective assay that can be easily incorporated in small and medium size laboratories for the rapid identification of the most clinically important HPV genotypes.

  14. Optimising the diagnostic strategy for onychomycosis from sample collection to FUNGAL identification evaluation of a diagnostic kit for real-time PCR.

    PubMed

    Petinataud, Dimitri; Berger, Sibel; Ferdynus, Cyril; Debourgogne, Anne; Contet-Audonneau, Nelly; Machouart, Marie

    2016-05-01

    Onychomycosis is a common nail disorder mainly due to dermatophytes for which the conventional diagnosis requires direct microscopic observation and culture of a biological sample. Nevertheless, antifungal treatments are commonly prescribed without a mycological examination having been performed, partly because of the slow growth of dermatophytes. Therefore, molecular biology has been applied to this pathology, to support a quick and accurate distinction between onychomycosis and other nail damage. Commercial kits are now available from several companies for improving traditional microbiological diagnosis. In this paper, we present the first evaluation of the real-time PCR kit marketed by Bio Evolution for the diagnosis of dermatophytosis. Secondly, we compare the efficacy of the kit on optimal and non-optimal samples. This study was conducted on 180 nails samples, processed by conventional methods and retrospectively analysed using this kit. According to our results, this molecular kit has shown high specificity and sensitivity in detecting dermatophytes, regardless of sample quality. On the other hand, and as expected, optimal samples allowed the identification of a higher number of dermatophytes by conventional mycological diagnosis, compared to non-optimal samples. Finally, we have suggested several strategies for the practical use of such a kit in a medical laboratory for quick pathogen detection.

  15. Real-time PCR Tests in Dutch Exotic Mosquito Surveys; Implementation of Aedes aegypti and Aedes albopictus Identification Tests, and the Development of Tests for the Identification of Aedes atropalpus and Aedes japonicus japonicus (Diptera: Culicidae).

    PubMed

    van de Vossenberg, B T L H; Ibáñez-Justicia, A; Metz-Verschure, E; van Veen, E J; Bruil-Dieters, M L; Scholte, E J

    2015-05-01

    Since 2009, The Netherlands Food and Consumer Product Safety Authority carries out surveys focusing on, amongst others, the presence of invasive mosquito species (IMS). Special attention is given to exotic container-breeding Aedes species Aedes aegypti (L.), Aedes albopictus (Skuse), Aedes atropalpus (Coquillett), and Aedes japonicus japonicus (Theobald). This study describes the implementation of real-time PCR tests described by Hill et al. (2008) for the identification of Ae. aegypti and Ae. albopictus, and the development of two novel real-time PCR tests for the identification of Ae. atropalpus and Ae. j. japonicus. Initial test showed that optimization of elements of the Ae. aegypti and Ae. albopictus tests was needed. Method validation tests were performed to determine if the implemented and newly developed tests are fit for routine diagnostics. Performance criteria of analytical sensitivity, analytical specificity, selectivity, repeatability, and reproducibility were determined. In addition, experiments were performed to determine the influence of environmental conditions on the usability of DNA extracted from mosquito specimens trapped in BG-Sentinel traps. The real-time PCR tests were demonstrated to be sensitive, specific, repeatable, reproducible, and are less prone to false negative results compared to partial cytochrome c oxidase I gene sequencing owing to the DNA fragmentation caused by environmental influences.

  16. Specific Real-Time PCR for Simultaneous Detection and Identification of Legionella pneumophila Serogroup 1 in Water and Clinical Samples▿ †

    PubMed Central

    Mérault, N.; Rusniok, C.; Jarraud, S.; Gomez-Valero, L.; Cazalet, C.; Marin, M.; Brachet, E.; Aegerter, P.; Gaillard, J. L.; Etienne, J.; Herrmann, J. L.; Lawrence, C.; Buchrieser, C.

    2011-01-01

    Legionella pneumophila, a bacterium that replicates within aquatic amoebae and persists in the environment as a free-living microbe, is the causative agent of Legionnaires' disease. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease, and within the 15 serogroups (Sg), L. pneumophila Sg1 causes more than 84% of Legionnaires' disease worldwide. Thus, rapid and specific identification of L. pneumophila Sg1 is of the utmost importance for evaluation of the contamination of collective water systems and the risk posed. Previously we had shown that about 20 kb of the 33-kb locus carrying the genes coding for the proteins involved in lipopolysaccharide biosynthesis (LPS gene cluster) by L. pneumophila was highly specific for Sg1 strains and that three genes (lpp0831, wzm, and wzt) may serve as genetic markers. Here we report the sequencing and comparative analyses of this specific region of the LPS gene cluster in L. pneumophila Sg6, -10, -12, -13, and -14. Indeed, the wzm and wzt genes were present only in the Sg1 LPS gene cluster, which showed a very specific gene content with respect to the other five serogroups investigated. Based on this observation, we designed primers and developed a classical and a real-time PCR method for the detection and simultaneous identification of L. pneumophila Sg1 in clinical and environmental isolates. Evaluation of the selected primers with 454 Legionella and 38 non-Legionella strains demonstrated 100% specificity. Sensitivity, specificity, and predictive values were further evaluated with 209 DNA extracts from water samples of hospital water supply systems and with 96 respiratory specimens. The results showed that the newly developed quantitative Sg1-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk L. pneumophila Sg1 in water and clinical samples. PMID:21193672

  17. Development and validation of a rotor-gene real-time PCR assay for detection, identification, and quantification of Chlamydia trachomatis in a single reaction.

    PubMed

    Jalal, Hamid; Stephen, Hannah; Curran, Martin D; Burton, Janet; Bradley, Michelle; Carne, Christopher

    2006-01-01

    A multitarget real-time PCR (MRT-PCR) for detection of Chlamydia trachomatis DNA was developed and validated. There were three targets for amplification in a single reaction: the cryptic plasmid (CP), the major outer membrane protein (MOMP) gene, and an internal control. The assay had the following characteristics: (i) detection and confirmation of the presence of C. trachomatis DNA in a single reaction, (ii) detection of all genovars of C. trachomatis without any cross-reactivity with pathogenic bacteria or commensal organisms of the oropharynx and genital tract, (iii) a 95% probability of detection with three copies of MOMP and one copy of CP per reaction mixture, (iv) identification of the inhibition of amplification, (v) a quantitative dynamic range of 25 to 250,000 genome copies per reaction mixture, (vi) high intra- and interassay reproducibilities, and (vii) correct identification of all samples in the validation panel. There were 146 COBAS Amplicor PCR (Amplicor PCR)-positive samples and 122 Amplicor PCR-negative samples in the panel. MRT-PCR detected CP DNA alone in 6 (4%) Amplicor PCR-positive samples and both CP and MOMP DNAs in 140 (96%) of 146 Amplicor PCR-positive samples. The quantity of MOMP DNA in 95 (68%) of 140 samples was within the dynamic range of the assay. The median C. trachomatis load in these samples was 321 genome copies per reaction mixture (range, 26 to 40,137 genome copies per reaction mixture). Due to the inclusion of two different C. trachomatis-specific targets, the assay confirmed 259 (97%) of 268 results in a single reaction. This assay could be used in the qualitative format for the routine detection of C. trachomatis and in the quantitative format for study of the pathogenesis of C. trachomatis-associated diseases. The assay demonstrated the potential to eliminate the need for confirmatory testing in almost all samples, thus reducing the turnaround time and the workload.

  18. Molecular diagnosis of canine visceral leishmaniasis: identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR.

    PubMed

    Quaresma, Patrícia Flávia; Murta, Silvane Maria Fonseca; Ferreira, Eduardo de Castro; da Rocha-Lima, Ana Cristina Vianna Mariano; Xavier, Ana Amélia Prates; Gontijo, Célia Maria Ferreira

    2009-09-01

    The efficacies of polymerase chain reaction (PCR) procedures for the diagnosis of canine visceral leishmaniasis (CVL), and of PCR-restriction fragment length polymorphism (RFLP) analysis for the identification of Leishmania species, have been assessed. Quantitative real-time PCR employing a SYBR Green dye-based system was standardised for the quantification of Leishmania kDNA minicircles. Skin, peripheral blood and bone marrow samples collected from 217 dogs, asymptomatic or symptomatic for CVL, were analysed. The PCR method, which was based on the amplification of a 120 bp kDNA fragment conserved across Leishmania species, was able to detect the presence in clinical samples of protozoan parasite DNA in amounts as low as 0.1 fg. Bone marrow and skin samples proved to be more suitable than peripheral blood for the detection of Leishmania by PCR and presented positive indices of 84.9% and 80.2%, respectively. PCR-RFLP analysis indicated that 192 of the PCR-positive dogs were infected with Leishmania infantum chagasi, whilst L. braziliensis was identified in two other animals. Quantitative PCR revealed that bone marrow samples from dogs presenting positive conventional tests contained a higher number of copies of Leishmania kDNA than peripheral blood, although no significant differences were detected between symptomatic and asymptomatic dogs in terms of parasite load. This study demonstrates that PCR can be used for the detection of Leishmania in clinical samples derived from naturally infected dogs, and that PCR-RFLP represents a rapid and sensitive tool for the identification of Leishmania species. Additionally, qPCR is effective in quantifying Leishmania DNA load in clinical samples.

  19. Molecular Detection and Species-Specific Identification of Medically Important Aspergillus Species by Real-Time PCR in Experimental Invasive Pulmonary Aspergillosis ▿

    PubMed Central

    Walsh, Thomas J.; Wissel, Mark C.; Grantham, Kevin J.; Petraitiene, Ruta; Petraitis, Vidmantas; Kasai, Miki; Francesconi, Andrea; Cotton, Margaret P.; Hughes, Johanna E.; Greene, Lora; Bacher, John D.; Manna, Pradip; Salomoni, Martin; Kleiboeker, Steven B.; Reddy, Sushruth K.

    2011-01-01

    Diagnosis of invasive pulmonary aspergillosis (IPA) remains a major challenge to clinical microbiology laboratories. We developed rapid and sensitive quantitative PCR (qPCR) assays for genus- and species-specific identification of Aspergillus infections by use of TaqMan technology. In order to validate these assays and understand their potential diagnostic utility, we then performed a blinded study of bronchoalveolar lavage (BAL) fluid specimens from well-characterized models of IPA with the four medically important species. A set of real-time qPCR primers and probes was developed by utilizing unique ITS1 regions for genus- and species-specific detection of the four most common medically important Aspergillus species (Aspergillus fumigatus, A. flavus, A. niger, and A. terreus). Pan-Aspergillus and species-specific qPCRs with BAL fluid were more sensitive than culture for detection of IPA caused by A. fumigatus in untreated (P < 0.0007) and treated (P ≤ 0.008) animals, respectively. For infections caused by A. terreus and A. niger, culture and PCR amplification from BAL fluid yielded similar sensitivities for untreated and treated animals. Pan-Aspergillus PCR was more sensitive than culture for detection of A. flavus in treated animals (P = 0.002). BAL fluid pan-Aspergillus and species-specific PCRs were comparable in sensitivity to BAL fluid galactomannan (GM) assay. The copy numbers from the qPCR assays correlated with quantitative cultures to determine the pulmonary residual fungal burdens in lung tissue. Pan-Aspergillus and species-specific qPCR assays may improve the rapid and accurate identification of IPA in immunocompromised patients. PMID:21976757

  20. Measurement-device-independent quantum digital signatures

    NASA Astrophysics Data System (ADS)

    Puthoor, Ittoop Vergheese; Amiri, Ryan; Wallden, Petros; Curty, Marcos; Andersson, Erika

    2016-08-01

    Digital signatures play an important role in software distribution, modern communication, and financial transactions, where it is important to detect forgery and tampering. Signatures are a cryptographic technique for validating the authenticity and integrity of messages, software, or digital documents. The security of currently used classical schemes relies on computational assumptions. Quantum digital signatures (QDS), on the other hand, provide information-theoretic security based on the laws of quantum physics. Recent work on QDS Amiri et al., Phys. Rev. A 93, 032325 (2016);, 10.1103/PhysRevA.93.032325 Yin, Fu, and Zeng-Bing, Phys. Rev. A 93, 032316 (2016), 10.1103/PhysRevA.93.032316 shows that such schemes do not require trusted quantum channels and are unconditionally secure against general coherent attacks. However, in practical QDS, just as in quantum key distribution (QKD), the detectors can be subjected to side-channel attacks, which can make the actual implementations insecure. Motivated by the idea of measurement-device-independent quantum key distribution (MDI-QKD), we present a measurement-device-independent QDS (MDI-QDS) scheme, which is secure against all detector side-channel attacks. Based on the rapid development of practical MDI-QKD, our MDI-QDS protocol could also be experimentally implemented, since it requires a similar experimental setup.

  1. Fully device-independent quantum key distribution

    NASA Astrophysics Data System (ADS)

    Vidick, Thomas

    2013-03-01

    The laws of quantum mechanics allow unconditionally secure key distribution protocols. Nevertheless, security proofs of traditional quantum key distribution (QKD) protocols rely on a crucial assumption, the trustworthiness of the quantum devices used in the protocol. In device-independent QKD, even this last assumption is relaxed: the devices used in the protocol may have been adversarially prepared, and there is no a priori guarantee that they perform according to specification. Proving security in this setting had been a central open problem in quantum cryptography. We give the first device-independent proof of security of a protocol for quantum key distribution that guarantees the extraction of a linear amount of key even when the devices are subject to a constant rate of noise. Our only assumptions are that the laboratories in which each party holds his or her own device are spatially isolated, and that both devices, as well as the eavesdropper, are bound by the laws of quantum mechanics. All previous proofs of security relied either on the use of many independent pairs of devices, or on the absence of noise.

  2. Documentation for DIGLIB: Device Independent Graphics Library

    SciTech Connect

    Brand, H.R.

    1984-09-01

    DIGLIB (Device Independent Graphics LIBrary) is a collection of FORTRAN callable subroutines designed with the following goals: (1) easily usable by the casual graphics programmer for 2D plotting; (2) device independent (as much as possible); (3) small and reasonably fast; (4) device drivers are as simple as possible, and therefore easy to write, and device drivers may be written in FORTRAN when desired; (5) maintainable; (6) compatible with RT-11, RSX-11M, IAS, and VMS; and (7) compatible with MACRO, FORTRAN, F4P, FORTRAN-77, and OMSI PASCAL. DIGLIB comes very close to meeting all of the above design goals. Maintainability has been enhanced by using the INCLUDE feature of FORTRAN-IV-PLUS. Compatibility with RT-11 has been maintained by writing a FORTRAN-IV-PLUS to FORTRAN-IV conversion program. Compatibility with OMSI PASCAL has been maintained by avoiding any FORTRAN I/O. Drivers are available for a variety of devices under RSX-11M, TSX, RT-11 and VMS. These include Tektronix 4000 series terminals, Retro-Graphics enhancements to several terminal types, HP 26xx terminals, and plotters.

  3. Real-Time Revolution?

    PubMed

    Berlin, Joey

    2016-03-01

    Austin Regional Clinic (ARC) physicians and officials know patient feedback is important, but getting patients to provide it can be a challenge. A pilot program of a new, real-time feedback system provided ARC patients a high-tech convenience previous attempts lacked and produced participation numbers dwarfing those past efforts. ARC's initial results with the system, in which patients answer five to seven questions on a computer tablet and can leave free-text comments, were so successful the clinic is already planning to expand it to all of its locations by the end of June.

  4. Real time Faraday spectrometer

    DOEpatents

    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.

  5. Molecular characterization of Swiss Ceratopogonidae (Diptera) and evaluation of real-time PCR assays for the identification of Culicoides biting midges.

    PubMed

    Wenk, Claudia E; Kaufmann, Christian; Schaffner, Francis; Mathis, Alexander

    2012-03-23

    Biting midges of the genus Culicoides (Diptera, Ceratopogonidae) are vectors of several viruses of veterinary relevance, and they can cause insect bite hypersensitivity. As the morphological identification of these tiny insects is a difficult task in many cases, alternative approaches are expedient. With the aim to develop real-time PCRs, we determined partial mitochondrial cytochrome oxidase I gene (mt COI) sequences from 380 Culicoides midges representing three regions of Switzerland, namely the Alps, Midland north of the Alps (Atlantic climate), and South of the Alps (Mediterranean climate). The same region was also sequenced from non-biting midges of the genera Atrichopogon, Brachypogon, Dasyhelea, Forcipomyia and Serromyia. A total of 21 Culicoides species were identified by morphology. Sequence variability (haplotypes) was observed in all species. For each of C. grisescens and C. obsoletus, a novel cryptic species was identified. Whereas all individuals of C. grisescens and of the cryptic C. obsoletus species (O2) originated only from Alpine sites, the known C. obsoletus (O1) species was found in all three regions. Further, a sister taxon to C. pulicaris was identified based on the mt COI sequences and named Culicoides sp. Alignments of available mtCOI sequences from Ceratopogonidae (GenBank, this study) were used to design real-time PCR primers and probes to distinguish C. chiopterus, C. deltus, C. dewulfi, C. grisescens (including the cryptic species), C. imicola, C. lupicaris, C. obsoletus O1, C. obsoletus O2, C. pulicaris, C. scoticus and Culicoides sp. Specificities of primers and probes was tested with cloned targets representing 1 to 4 haplotypes of 18 Culicoides spp. and 1 haplotype each from 4 other Ceratopogonidae. No cross-reactivity was observed when plasmid template representing 5 × 10(6) gene copies was tested, but it was evident (Ct values ≤ 30) in few instances when plasmid template representing 5 × 10(9) gene copies was utilized, the

  6. Identification and Validation of Reference Genes for Quantification of Target Gene Expression with Quantitative Real-time PCR for Tall Fescue under Four Abiotic Stresses

    PubMed Central

    Hu, Baoyun; Tan, Zhiqun; Huang, Bingru

    2015-01-01

    Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species. PMID:25786207

  7. Evaluation of hand hygiene compliance and associated factors with a radio-frequency-identification-based real-time continuous automated monitoring system.

    PubMed

    Dufour, J-C; Reynier, P; Boudjema, S; Soto Aladro, A; Giorgi, R; Brouqui, P

    2017-04-01

    Hand hygiene is a major means for preventing healthcare-associated infections. One critical point in understanding poor compliance is the lack of relevant markers used to monitor practices systematically. This study analysed hand hygiene compliance and associated factors with a radio-frequency-identification-based real-time continuous automated monitoring system in an infectious disease ward with 17 single bedrooms. Healthcare workers (HCWs) were tracked while performing routine care over 171 days. A multi-level multi-variate logistics model was used for data analysis. The main outcome measures were hand disinfection before entering the bedroom (outside use) and before entering the patient care zone, defined as the zone surrounding the patient's bed (inside/bedside use). Variables analysed included HCWs' characteristics and behaviour, patients, room layouts, path chains and duration of HCWs' paths. In total, 4629 paths with initial hand hygiene opportunities when entering the patient care zone were selected, of which 763 (16.5%), 285 (6.1%) and 3581 (77.4%) were associated with outside use, inside/bedside use and no use, respectively. Hand hygiene is caregiver-dependent. The shorter the duration of the HCW's path, the worse the bedside hand hygiene. Bedside hand hygiene is improved when one or two extra HCWs are present in the room. Hand hygiene compliance at the bedside, as analysed using the continuous monitoring system, depended upon the HCW's occupation and personal behaviour, number of HCWs, time spent in the room and (potentially) dispenser location. Meal tray distribution was a possible factor in the case of failure to disinfect hands. Copyright © 2017 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

  8. Real-time RT-PCR for detection, identification and absolute quantification of viral haemorrhagic septicaemia virus using different types of standards.

    PubMed

    Lopez-Vazquez, C; Bandín, I; Dopazo, C P

    2015-05-21

    In the present study, 2 systems of real-time RT-PCR-one based on SYBR Green and the other on TaqMan-were designed to detect strains from any genotype of viral haemorrhagic septicaemia virus (VHSV), with high sensitivity and repeatability/reproducibility. In addition, the method was optimized for quantitative purposes (qRT-PCR), and standard curves with different types of reference templates were constructed and compared. Specificity was tested against 26 isolates from 4 genotypes. The sensitivity of the procedures was first tested against cell culture isolation, obtaining a limit of detection (LD) of 100 TCID50 ml-1 (100-fold below the LD using cell culture), at a threshold cycle value (Ct) of 36. Sensitivity was also evaluated using RNA from crude (LD = 1 fg; 160 genome copies) and purified virus (100 ag; 16 copies), plasmid DNA (2 copies) and RNA transcript (15 copies). No differences between both chemistries were observed in sensitivity and dynamic range. To evaluate repeatability and reproducibility, all experiments were performed in triplicate and on 3 different days, by workers with different levels of experience, obtaining Ct values with coefficients of variation always <5. This fact, together with the high efficiency and R2 values of the standard curves, encouraged us to analyse the reliability of the method for viral quantification. The results not only demonstrated that the procedure can be used for detection, identification and quantification of this virus, but also demonstrated a clear correlation between the regression lines obtained with different standards, which will help scientists to compare sensitivity results between different studies.

  9. Development of a novel DNA extraction method for identification and quantification of Mycobacterium avium subsp. paratuberculosis from tissue samples by real-time PCR.

    PubMed

    Park, Kun Taek; Allen, Andrew J; Davis, William C

    2014-04-01

    Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease in ruminants and possibly associated with human Crohn's disease. One impediment in furthering our understanding of this potential association has been the lack of an accurate method for detection of Map in affected tissues. Real time polymerase chain reaction (RT-PCR) methods have been reported to have different sensitivities in detection of Map. This is in part attributable to the difficulties of extracting Map DNA and removing PCR inhibitors from the clinical specimens. The maximum efficiency of RT-PCR can only be achieved by using high quality DNA samples. In this study, we present a novel pre-treatment method which significantly increases Map DNA recovery and decreases PCR inhibitors (p<0.05). When the pre-treatment method was combined with the DNeasy Blood and Tissue kit (Qiagen), PCR inhibition was not detected in any of three different RT-PCR methods tested in this study. The results obtained with the IS900 probe showed an excellent Kappa value (0.849) and a high correlation coefficient r (0.940) compared to the results of culture method. When used to examine unknown field samples (n=15), more positive tissues were identified with DNA extracts prepared with pre-treatment method than without (5 vs 3). This improved Map DNA extraction method from tissue samples will make RT-PCR a more powerful tool for a wide range of applications for Map identification and quantification. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Identification and validation of reference genes for quantification of target gene expression with quantitative real-time PCR for tall fescue under four abiotic stresses.

    PubMed

    Yang, Zhimin; Chen, Yu; Hu, Baoyun; Tan, Zhiqun; Huang, Bingru

    2015-01-01

    Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

  11. Computerized patient identification for the EMBRACA clinical trial using real-time data from the PRAEGNANT network for metastatic breast cancer patients.

    PubMed

    Hein, Alexander; Gass, Paul; Walter, Christina Barbara; Taran, Florin-Andrei; Hartkopf, Andreas; Overkamp, Friedrich; Kolberg, Hans-Christian; Hadji, Peyman; Tesch, Hans; Ettl, Johannes; Wuerstlein, Rachel; Lounsbury, Debra; Lux, Michael P; Lüftner, Diana; Wallwiener, Markus; Müller, Volkmar; Belleville, Erik; Janni, Wolfgang; Fehm, Tanja N; Wallwiener, Diethelm; Ganslandt, Thomas; Ruebner, Matthias; Beckmann, Matthias W; Schneeweiss, Andreas; Fasching, Peter A; Brucker, Sara Y

    2016-07-01

    As breast cancer is a diverse disease, clinical trials are becoming increasingly diversified and are consequently being conducted in very small subgroups of patients, making study recruitment increasingly difficult. The aim of this study was to assess the use of data from a remote data entry system that serves a large national registry for metastatic breast cancer. The PRAEGNANT network is a real-time registry with an integrated biomaterials bank that was designed as a scientific study and as a means of identifying patients who are eligible for clinical trials, based on clinical and molecular information. Here, we report on the automated use of the clinical data documented to identify patients for a clinical trial (EMBRACA) for patients with metastatic breast cancer. The patients' charts were assessed by two independent physicians involved in the clinical trial and also by a computer program that tested patients for eligibility using a structured query language script. In all, 326 patients from two study sites in the PRAEGNANT network were included in the analysis. Using expert assessment, 120 of the 326 patients (37 %) appeared to be eligible for inclusion in the EMBRACA study; with the computer algorithm assessment, a total of 129 appeared to be eligible. The sensitivity of the computer algorithm was 0.87 and its specificity was 0.88. Using computer-based identification of patients for clinical trials appears feasible. With the instrument's high specificity, its application in a large cohort of patients appears to be feasible, and the workload for reassessing the patients is limited.

  12. Isolation and Identification of Campylobacter spp. from Poultry and Poultry By-Products in Tunisia by Conventional Culture Method and Multiplex Real-Time PCR.

    PubMed

    Jribi, Hela; Sellami, Hanen; Mariam, Siala; Smaoui, Salma; Ghorbel, Asma; Hachicha, Salma; Benejat, Lucie; Messadi-Akrout, Feriel; Mégraud, Francis; Gdoura, Radhouane

    2017-10-01

    Thermophilic Campylobacter spp. are one of the primary causes of bacterial human diarrhea. The consumption of poultry meats, by-products, or both is suspected to be a major cause of human campylobacteriosis. The aims of this study were to determine the prevalence of thermophilic Campylobacter spp. in fresh poultry meat and poultry by-products by conventional culture methods and to confirm Campylobacter jejuni and Campylobacter coli isolates by using the multiplex PCR assay. Two hundred fifty fresh poultry samples were collected from a variety of supermarkets and slaughterhouses located in Sfax, Tunisia, including chicken (n =149) and turkey (n =101). The samples were analyzed using conventional microbiological examinations according to the 2006 International Organization for Standardization method (ISO 10272-1) for Campylobacter spp. Concurrently, a real-time PCR was used for identification of C. jejuni and C. coli . Of the 250 samples of poultry meat and poultry by-products, 25.6% (n = 64) were contaminated with Campylobacter spp. The highest prevalence of Campylobacter spp. was found in chicken meat (26.8%) followed by turkey meat (23.7%). Among the different products, poultry breasts showed the highest contamination (36.6%) followed by poultry by-products (30%), poultry wings (28%) and poultry legs (26%) showed the lowest contamination, and no contamination was found on neck skin. Of the 64 thermophilic Campylobacter isolates, C. jejuni (59.7%) was the most frequently isolated species and 10.9% of the isolates were identified as C. coli . All of the 64 Campylobacter isolates identified by the conventional culture methods were further confirmed by PCR. The seasonal peak of Campylobacter spp. contamination was in the warm seasons (spring and summer). The study concluded that high proportions of poultry meat and poultry by-products marketed in Tunisia are contaminated by Campylobacter spp. Furthermore, to ensure food safety, poultry meats must be properly cooked

  13. Rapid identification of additives in poly(vinyl chloride) lid gaskets by direct analysis in real time ionisation and single-quadrupole mass spectrometry.

    PubMed

    Rothenbacher, Thorsten; Schwack, Wolfgang

    2010-01-01

    Gaskets for lids of glass jars usually consist of poly(vinyl chloride) (PVC) containing plasticisers and additional additives, which may migrate into packed foodstuffs. To conform to legal regulations, any such migration has to be determined analytically, which is a big challenge due to the huge chemical variety of additives in use. Therefore, a rapid screening method by means of direct analysis in real time mass spectrometry (DART-MS), using a single-quadrupole mass spectrometer, was developed. On introducing a plastisol sample into the DART interface, protonated molecules and ammonium adducts were obtained as the typical ionisation products of any additives present, and cleavages of ester bonds as typical fragmentation processes. Generally, additives present in the 1% range could be directly and easily identified if ion suppressive effects deriving from specific molecules did not occur. These effects could be avoided by analysing toluene extracts of plastisol samples, and this also improved the sensitivity. Using this method, it was possible to identify phthalates, fatty acid amides, tributyl O-acetylcitrate, dibutyl sebacate, bis(2-ethylhexyl) adipate, 1,2-diisononyl 1,2-cyclohexanedicarboxylate, and even more complex additives like acetylated mono- and diacylglycerides, epoxidised soybean oil, and polyadipates, with a limit of detection of < or = 1% in PVC plastisols. Only in the case of epoxidised linseed oil were levels of > or = 5% required for identification. The detection of azodicarbonamide, used as a foaming agent within the manufacturing process, was possible in principle, but was not highly reproducible due to the very low concentrations in plastisols.

  14. Device-independent tests of quantum channels

    NASA Astrophysics Data System (ADS)

    Dall'Arno, Michele; Brandsen, Sarah; Buscemi, Francesco

    2017-03-01

    We develop a device-independent framework for testing quantum channels. That is, we falsify a hypothesis about a quantum channel based only on an observed set of input-output correlations. Formally, the problem consists of characterizing the set of input-output correlations compatible with any arbitrary given quantum channel. For binary (i.e. two input symbols, two output symbols) correlations, we show that extremal correlations are always achieved by orthogonal encodings and measurements, irrespective of whether or not the channel preserves commutativity. We further provide a full, closed-form characterization of the sets of binary correlations in the case of: (i) any dihedrally covariant qubit channel (such as any Pauli and amplitude-damping channels) and (ii) any universally-covariant commutativity-preserving channel in an arbitrary dimension (such as any erasure, depolarizing, universal cloning and universal transposition channels).

  15. Measurement-device-independent quantum cryptography

    SciTech Connect

    Xu, Feihu; Curty, Marcos; Qi, Bing; Lo, Hoi-Kwong

    2014-12-18

    In theory, quantum key distribution (QKD) provides information-theoretic security based on the laws of physics. Owing to the imperfections of real-life implementations, however, there is a big gap between the theory and practice of QKD, which has been recently exploited by several quantum hacking activities. To fill this gap, a novel approach, called measurement-device-independent QKD (mdiQKD), has been proposed. In addition, it can remove all side-channels from the measurement unit, arguably the most vulnerable part in QKD systems, thus offering a clear avenue toward secure QKD realisations. In this study, we review the latest developments in the framework of mdiQKD, together with its assumptions, strengths, and weaknesses.

  16. Measurement-device-independent quantum cryptography

    DOE PAGES

    Xu, Feihu; Curty, Marcos; Qi, Bing; ...

    2014-12-18

    In theory, quantum key distribution (QKD) provides information-theoretic security based on the laws of physics. Owing to the imperfections of real-life implementations, however, there is a big gap between the theory and practice of QKD, which has been recently exploited by several quantum hacking activities. To fill this gap, a novel approach, called measurement-device-independent QKD (mdiQKD), has been proposed. In addition, it can remove all side-channels from the measurement unit, arguably the most vulnerable part in QKD systems, thus offering a clear avenue toward secure QKD realisations. In this study, we review the latest developments in the framework of mdiQKD,more » together with its assumptions, strengths, and weaknesses.« less

  17. Detector-device-independent quantum key distribution

    SciTech Connect

    Lim, Charles Ci Wen; Korzh, Boris; Martin, Anthony; Bussières, Félix; Thew, Rob; Zbinden, Hugo

    2014-12-01

    Recently, a quantum key distribution (QKD) scheme based on entanglement swapping, called measurement-device-independent QKD (mdiQKD), was proposed to bypass all measurement side-channel attacks. While mdiQKD is conceptually elegant and offers a supreme level of security, the experimental complexity is challenging for practical systems. For instance, it requires interference between two widely separated independent single-photon sources, and the secret key rates are dependent on detecting two photons—one from each source. Here, we demonstrate a proof-of-principle experiment of a QKD scheme that removes the need for a two-photon system and instead uses the idea of a two-qubit single-photon to significantly simplify the implementation and improve the efficiency of mdiQKD in several aspects.

  18. Real time automated inspection

    DOEpatents

    Fant, K.M.; Fundakowski, R.A.; Levitt, T.S.; Overland, J.E.; Suresh, B.R.; Ulrich, F.W.

    1985-05-21

    A method and apparatus are described relating to the real time automatic detection and classification of characteristic type surface imperfections occurring on the surfaces of material of interest such as moving hot metal slabs produced by a continuous steel caster. A data camera transversely scans continuous lines of such a surface to sense light intensities of scanned pixels and generates corresponding voltage values. The voltage values are converted to corresponding digital values to form a digital image of the surface which is subsequently processed to form an edge-enhanced image having scan lines characterized by intervals corresponding to the edges of the image. The edge-enhanced image is thresholded to segment out the edges and objects formed by the edges by interval matching and bin tracking. Features of the objects are derived and such features are utilized to classify the objects into characteristic type surface imperfections. 43 figs.

  19. Real time automated inspection

    DOEpatents

    Fant, Karl M.; Fundakowski, Richard A.; Levitt, Tod S.; Overland, John E.; Suresh, Bindinganavle R.; Ulrich, Franz W.

    1985-01-01

    A method and apparatus relating to the real time automatic detection and classification of characteristic type surface imperfections occurring on the surfaces of material of interest such as moving hot metal slabs produced by a continuous steel caster. A data camera transversely scans continuous lines of such a surface to sense light intensities of scanned pixels and generates corresponding voltage values. The voltage values are converted to corresponding digital values to form a digital image of the surface which is subsequently processed to form an edge-enhanced image having scan lines characterized by intervals corresponding to the edges of the image. The edge-enhanced image is thresholded to segment out the edges and objects formed by the edges are segmented out by interval matching and bin tracking. Features of the objects are derived and such features are utilized to classify the objects into characteristic type surface imperfections.

  20. Experimental measurement-device-independent quantum random-number generation

    NASA Astrophysics Data System (ADS)

    Nie, You-Qi; Guan, Jian-Yu; Zhou, Hongyi; Zhang, Qiang; Ma, Xiongfeng; Zhang, Jun; Pan, Jian-Wei

    2016-12-01

    The randomness from a quantum random-number generator (QRNG) relies on the accurate characterization of its devices. However, device imperfections and inaccurate characterizations can result in wrong entropy estimation and bias in practice, which highly affects the genuine randomness generation and may even induce the disappearance of quantum randomness in an extreme case. Here we experimentally demonstrate a measurement-device-independent (MDI) QRNG based on time-bin encoding to achieve certified quantum randomness even when the measurement devices are uncharacterized and untrusted. The MDI-QRNG is randomly switched between the regular randomness generation mode and a test mode, in which four quantum states are randomly prepared to perform measurement tomography in real time. With a clock rate of 25 MHz, the MDI-QRNG generates a final random bit rate of 5.7 kbps. Such implementation with an all-fiber setup provides an approach to construct a fully integrated MDI-QRNG with trusted but error-prone devices in practice.

  1. Identification of Common Bacterial Pathogens Causing Meningitis in Culture-Negative Cerebrospinal Fluid Samples Using Real-Time Polymerase Chain Reaction

    PubMed Central

    2016-01-01

    Background. Meningitis is a serious communicable disease with high morbidity and mortality rates. It is an endemic disease in Egypt caused mainly by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. In some settings, bacterial meningitis is documented depending mainly on positive cerebrospinal fluid (CSF) culture results or CSF positive latex agglutination test, missing the important role of prior antimicrobial intake which can yield negative culture and latex agglutination test results. This study aimed to utilize molecular technology in order to diagnose bacterial meningitis in culture-negative CSF samples. Materials and Methods. Forty culture-negative CSF samples from suspected cases of bacterial meningitis were examined by real-time polymerase chain reaction (real-time PCR) for the presence of lytA, bexA, and ctrA genes specific for Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis, respectively. Results. Positive real-time PCR results for Streptococcus pneumoniae were detected in 36 (90%) of culture-negative CSF samples while no positive results for Haemophilus influenzae or Neisseria meningitidis were detected. Four (10%) samples were negative by real-time PCR for all tested organisms. Conclusion. The use of molecular techniques as real-time PCR can provide a valuable addition to the proportion of diagnosed cases of bacterial meningitis especially in settings with high rates of culture-negative results. PMID:27563310

  2. Real time polarimetric dehazing.

    PubMed

    Mudge, Jason; Virgen, Miguel

    2013-03-20

    Remote sensing is a rich topic due to its utility in gathering detailed accurate information from locations that are not economically feasible traveling destinations or are physically inaccessible. However, poor visibility over long path lengths is problematic for a variety of reasons. Haze induced by light scatter is one cause for poor visibility and is the focus of this article. Image haze comes about as a result of light scattering off particles and into the imaging path causing a haziness to appear on the image. Image processing using polarimetric information of light scatter can be used to mitigate image haze. An imaging polarimeter which provides the Stokes values in real time combined with a "dehazing" algorithm can automate image haze removal for instant applications. Example uses are to improve visual display providing on-the-spot detection or imbedding in an active control loop to improve viewing and tracking while on a moving platform. In addition, removing haze in this manner allows the trade space for a system operational waveband to be opened up to bands which are object matched and not necessarily restricted by scatter effects.

  3. Measurement-Device-Independent Quantum Cryptography

    NASA Astrophysics Data System (ADS)

    Tang, Zhiyuan

    Quantum key distribution (QKD) enables two legitimate parties to share a secret key even in the presence of an eavesdropper. The unconditional security of QKD is based on the fundamental laws of quantum physics. Original security proofs of QKD are based on a few assumptions, e.g., perfect single photon sources and perfect single-photon detectors. However, practical implementations of QKD systems do not fully comply with such assumptions due to technical limitations. The gap between theory and implementations leads to security loopholes in most QKD systems, and several attacks have been launched on sophisticated QKD systems. Particularly, the detectors have been found to be the most vulnerable part of QKD. Much effort has been put to build side-channel-free QKD systems. Solutions such as security patches and device-independent QKD have been proposed. However, the former are normally ad-hoc, and cannot close unidentified loopholes. The latter, while having the advantages of removing all assumptions on devices, is impractical to implement today. Measurement-device-independent QKD (MDI-QKD) turns out to be a promising solution to the security problem of QKD. In MDI-QKD, all security loopholes, including those yet-to-be discovered, have been removed from the detectors, the most critical part in QKD. In this thesis, we investigate issues related to the practical implementation and security of MDI-QKD. We first present a demonstration of polarization-encoding MDI-QKD. Taking finite key effect into account, we achieve a secret key rate of 0.005 bit per second (bps) over 10 km spooled telecom fiber, and a 1600-bit key is distributed. This work, together with other demonstrations, shows the practicality of MDI-QKD. Next we investigate a critical assumption of MDI-QKD: perfect state preparation. We apply the loss-tolerant QKD protocol and adapt it to MDI-QKD to quantify information leakage due to imperfect state preparation. We then present an experimental demonstration of

  4. [DNA extraction and identification of Trichophyton rubrum by real-time polymerase chain reaction from direct nail scraping specimens of patients with onycomycosis].

    PubMed

    Berk, Elife; Kuştimur, Semra; Kalkancı, Ayşe; Oztaş, O Murat

    2011-01-01

    Trichophyton rubrum is the most frequently encountered dermatophyte species causing onichomycosis. The routine diagnosis of dermatophytes depends on the direct microscopic examination (DME) and culture methods, however due to the phenotypic identification problems related to those agents, the molecular methods come into question. The aim of this study was to evaluate the diagnostic performance of real-time polymerase chain reaction (RT-PCR) for the identification of T.rubrum by comparing to DME and culture methods, from nail samples of patients with the complaints of onychomycosis. A total of 90 patients of whom 58 were male who were admitted to the dermatology outpatients clinics of our hospital with the complaints of color/shape changes in the nails and thickening of the nail, were included in the study, together with the 20 healthy volunteer subjects as controls. The nail scraping samples obtained from the patients and controls were examined with direct microscopy using 15% potassium hydroxide, dimethyl sulphoxide and chlorazole black mixture and cultivated onto Sabouraud dextrose agar with and without cycloheximide. For DNA isolation, after the disruption of nail samples with a steel tool, phenol-chloroform-isoamyl alcohol purification method were used. The amplification and demonstration of the T.rubrum DNA have been performed by using specific primers and probes following TaqMan protocol of RT-PCR (LightCycler-Roche, USA) method. Seventy-two of the patients yielded positive and 18 yielded negative results with DME. Growth of molds was detected in the cultures of 20 (27.8%) of the 72 DME positive patients and all of the isolates were identified as T.rubrum. No fungal growth was seen in the samples of 18 patients who were DME negative. In DME positive group, 67 (93%) patients were found to be positive in RT-PCR, while 8 (44.4%) patients were RT-PCR positive in DME negative group. All of the culture positive samples (n= 20) were also found positive in RT

  5. Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.).

    PubMed

    Galeano, Esteban; Vasconcelos, Tarcísio Sales; Ramiro, Daniel Alves; De Martin, Valentina de Fátima; Carrer, Helaine

    2014-07-22

    Teak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak. Identification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes. This study proposes a first broad

  6. Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.)

    PubMed Central

    2014-01-01

    Background Teak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak. Results Identification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes. Conclusion This study

  7. Completely device-independent quantum key distribution

    NASA Astrophysics Data System (ADS)

    Aguilar, Edgar A.; Ramanathan, Ravishankar; Kofler, Johannes; Pawłowski, Marcin

    2016-08-01

    Quantum key distribution (QKD) is a provably secure way for two distant parties to establish a common secret key, which then can be used in a classical cryptographic scheme. Using quantum entanglement, one can reduce the necessary assumptions that the parties have to make about their devices, giving rise to device-independent QKD (DIQKD). However, in all existing protocols to date the parties need to have an initial (at least partially) random seed as a resource. In this work, we show that this requirement can be dropped. Using recent advances in the fields of randomness amplification and randomness expansion, we demonstrate that it is sufficient for the message the parties want to communicate to be (partially) unknown to the adversaries—an assumption without which any type of cryptography would be pointless to begin with. One party can use her secret message to locally generate a secret sequence of bits, which can then be openly used by herself and the other party in a DIQKD protocol. Hence our work reduces the requirements needed to perform secure DIQKD and establish safe communication.

  8. Comparison of DNA Microarray, Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of Fusarium spp. Obtained from Patients with Hematologic Malignancies.

    PubMed

    de Souza, Marcela; Matsuzawa, Tetsuhiro; Sakai, Kanae; Muraosa, Yasunori; Lyra, Luzia; Busso-Lopes, Ariane Fidelis; Levin, Anna Sara Shafferman; Schreiber, Angélica Zaninelli; Mikami, Yuzuru; Gonoi, Tohoru; Kamei, Katsuhiko; Moretti, Maria Luiza; Trabasso, Plínio

    2017-03-21

    The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.

  9. Rapid Differentiation and Identification of Potential Severe Strains of Citrus tristeza Virus by Real-Time Reverse Transcription Polymerase Chain Reaction Assays

    USDA-ARS?s Scientific Manuscript database

    A multiplex Taqman®-based real-time reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to detect all strains of Citrus tristeza virus (CTV) and to identify potentially severe strains of the virus. A CTV TaqMan probe (CTV-CY5) based on the coat protein (CP) gene sequences...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  11. Novel identification and differentiation of Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae suitable for both conventional and real-time PCR systems.

    PubMed

    Hinić, V; Brodard, I; Thomann, A; Cvetnić, Z; Makaya, P V; Frey, J; Abril, C

    2008-10-01

    We describe the development of a novel PCR assay for the rapid detection of members of the Brucella genus, and the differentiation between six recognized Brucella species. The assay has proven to be highly specific with the additional advantage of being suitable for use with both conventional and real-time PCR.

  12. Analysis of real-time vibration data

    USGS Publications Warehouse

    Safak, E.

    2005-01-01

    In recent years, a few structures have been instrumented to provide continuous vibration data in real time, recording not only large-amplitude motions generated by extreme loads, but also small-amplitude motions generated by ambient loads. The main objective in continuous recording is to track any changes in structural characteristics, and to detect damage after an extreme event, such as an earthquake or explosion. The Fourier-based spectral analysis methods have been the primary tool to analyze vibration data from structures. In general, such methods do not work well for real-time data, because real-time data are mainly composed of ambient vibrations with very low amplitudes and signal-to-noise ratios. The long duration, linearity, and the stationarity of ambient data, however, allow us to utilize statistical signal processing tools, which can compensate for the adverse effects of low amplitudes and high noise. The analysis of real-time data requires tools and techniques that can be applied in real-time; i.e., data are processed and analyzed while being acquired. This paper presents some of the basic tools and techniques for processing and analyzing real-time vibration data. The topics discussed include utilization of running time windows, tracking mean and mean-square values, filtering, system identification, and damage detection.

  13. Rapid identification and quantification of Campylobacter coli and Campylobacter jejuni by real-time PCR in pure cultures and in complex samples

    PubMed Central

    2011-01-01

    Background Campylobacter spp., especially Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli), are recognized as the leading human foodborne pathogens in developed countries. Livestock animals carrying Campylobacter pose an important risk for human contamination. Pigs are known to be frequently colonized with Campylobacter, especially C. coli, and to excrete high numbers of this pathogen in their faeces. Molecular tools, notably real-time PCR, provide an effective, rapid, and sensitive alternative to culture-based methods for the detection of C. coli and C. jejuni in various substrates. In order to serve as a diagnostic tool supporting Campylobacter epidemiology, we developed a quantitative real-time PCR method for species-specific detection and quantification of C. coli and C. jejuni directly in faecal, feed, and environmental samples. Results With a sensitivity of 10 genome copies and a linear range of seven to eight orders of magnitude, the C. coli and C. jejuni real-time PCR assays allowed a precise quantification of purified DNA from C. coli and C. jejuni. The assays were highly specific and showed a 6-log-linear dynamic range of quantification with a quantitative detection limit of approximately 2.5 × 102 CFU/g of faeces, 1.3 × 102 CFU/g of feed, and 1.0 × 103 CFU/m2 for the environmental samples. Compared to the results obtained by culture, both C. coli and C. jejuni real-time PCR assays exhibited a specificity of 96.2% with a kappa of 0.94 and 0.89 respectively. For faecal samples of experimentally infected pigs, the coefficients of correlation between the C. coli or C. jejuni real-time PCR assay and culture enumeration were R2 = 0.90 and R2 = 0.93 respectively. Conclusion The C. coli and C. jejuni real-time quantitative PCR assays developed in this study provide a method capable of directly detecting and quantifying C. coli and C. jejuni in faeces, feed, and environmental samples. These assays represent a new diagnostic tool for studying

  14. Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data

    PubMed Central

    2010-01-01

    Background Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes. Results By the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1α5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhβTUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development. Conclusion We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references for normalization of gene

  15. Identification and quantification of genetically modified Moonshade carnation lines using conventional and TaqMan real-time polymerase chain reaction methods.

    PubMed

    Li, Peng; Jia, Junwei; Bai, Lan; Pan, Aihu; Tang, Xueming

    2013-07-01

    Genetically modified carnation (Dianthus caryophyllus L.) Moonshade was approved for planting and commercialization in several countries from 2004. Developing methods for analyzing Moonshade is necessary for implementing genetically modified organism labeling regulations. In this study, the 5'-transgene integration sequence was isolated using thermal asymmetric interlaced (TAIL)-PCR. Based upon the 5'-transgene integration sequence, conventional and TaqMan real-time PCR assays were established. The relative limit of detection for the conventional PCR assay was 0.05 % for Moonshade using 100 ng total carnation genomic DNA, corresponding to approximately 79 copies of the carnation haploid genome, and the limits of detection and quantification of the TaqMan real-time PCR assay were estimated to be 51 and 254 copies of haploid carnation genomic DNA, respectively. These results are useful for identifying and quantifying Moonshade and its derivatives.

  16. A multiplex real-time PCR assay for identification of Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii in samples from AIDS patients with opportunistic pneumonia.

    PubMed

    Gago, Sara; Esteban, Cristina; Valero, Clara; Zaragoza, Oscar; Puig de la Bellacasa, Jorge; Buitrago, María José

    2014-04-01

    A molecular diagnostic technique based on real-time PCR was developed for the simultaneous detection of three of the most frequent causative agents of fungal opportunistic pneumonia in AIDS patients: Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii. This technique was tested in cultured strains and in clinical samples from HIV-positive patients. The methodology used involved species-specific molecular beacon probes targeted to the internal transcribed spacer regions of the rDNA. An internal control was also included in each assay. The multiplex real-time PCR assay was tested in 24 clinical strains and 43 clinical samples from AIDS patients with proven fungal infection. The technique developed showed high reproducibility (r(2) of >0.98) and specificity (100%). For H. capsulatum and Cryptococcus spp., the detection limits of the method were 20 and 2 fg of genomic DNA/20 μl reaction mixture, respectively, while for P. jirovecii the detection limit was 2.92 log10 copies/20 μl reaction mixture. The sensitivity in vitro was 100% for clinical strains and 90.7% for clinical samples. The assay was positive for 92.5% of the patients. For one of the patients with proven histoplasmosis, P. jirovecii was also detected in a bronchoalveolar lavage sample. No PCR inhibition was detected. This multiplex real-time PCR technique is fast, sensitive, and specific and may have clinical applications.

  17. Megahertz-Rate Semi-Device-Independent Quantum Random Number Generators Based on Unambiguous State Discrimination

    NASA Astrophysics Data System (ADS)

    Brask, Jonatan Bohr; Martin, Anthony; Esposito, William; Houlmann, Raphael; Bowles, Joseph; Zbinden, Hugo; Brunner, Nicolas

    2017-05-01

    An approach to quantum random number generation based on unambiguous quantum state discrimination is developed. We consider a prepare-and-measure protocol, where two nonorthogonal quantum states can be prepared, and a measurement device aims at unambiguously discriminating between them. Because the states are nonorthogonal, this necessarily leads to a minimal rate of inconclusive events whose occurrence must be genuinely random and which provide the randomness source that we exploit. Our protocol is semi-device-independent in the sense that the output entropy can be lower bounded based on experimental data and a few general assumptions about the setup alone. It is also practically relevant, which we demonstrate by realizing a simple optical implementation, achieving rates of 16.5 Mbits /s . Combining ease of implementation, a high rate, and a real-time entropy estimation, our protocol represents a promising approach intermediate between fully device-independent protocols and commercial quantum random number generators.

  18. Identification and quantification of three genetically modified insect resistant cotton lines using conventional and TaqMan real-time polymerase chain reaction methods.

    PubMed

    Yang, Litao; Pan, Aihu; Zhang, Kewei; Guo, Jinchao; Yin, Changsong; Chen, Jianxiu; Huang, Cheng; Zhang, Dabing

    2005-08-10

    As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM

  19. Identification of cattle carrying alleles associated with resistance and susceptibility to the Bovine Leukemia Virus progression by real-time PCR.

    PubMed

    Forletti, A; Juliarena, M A; Ceriani, C; Amadio, A F; Esteban, E; Gutiérrez, S E

    2013-12-01

    Previous studies have shown a significant association between polymorphisms of the BoLA DRB3 gene and Bovine Leukemia Virus (BLV) infection profile. The presence of allele *1501 has been associated with high proviral load in peripheral blood while allele *0902 has been associated with low proviral load. The purpose of this study was to develop allele-specific real-time PCRs to identify cattle carrying alleles associated with resistance (BoLA DRB3*0902) or susceptibility (BoLA DRB3*1501) to the BLV progression. Specific primers were designed and differential amplification was carried out by real-time PCR and monitored by SYBR® Green dye in DNA samples from peripheral blood. Conditions were also adjusted for traditional PCR amplification (end point amplification). These methods are rapid, simple and suitable for high throughput screening, and could aid in marker-assisted selection of BLV-resistant and susceptible cattle. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Rapid differentiation and identification of potential severe strains of Citrus tristeza virus by real-time reverse transcription-polymerase chain reaction assays.

    PubMed

    Yokomi, R K; Saponari, M; Sieburth, P J

    2010-04-01

    A multiplex Taqman-based real-time reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to identify potential severe strains of Citrus tristeza virus (CTV) and separate genotypes that react with the monoclonal antibody MCA13. Three strain-specific probes were developed using intergene sequences between the major and minor coat protein genes (CPi) in a multiplex reaction. Probe CPi-VT3 was designed for VT and T3 genotypes; probe CPi-T36 for T36 genotypes; and probe CPi-T36-NS to identify isolates in an outgroup clade of T36-like genotypes mild in California. Total nucleic acids extracted by chromatography on silica particles, sodium dodecyl sulfate-potassium acetate, and CTV virion immunocapture all yielded high quality templates for real-time PCR detection of CTV. These assays successfully differentiated CTV isolates from California, Florida, and a large panel of CTV isolates from an international collection maintained in Beltsville, MD. The utility of the assay was validated using field isolates collected in California and Florida.

  1. Simultaneous identification of pork and poultry origins in pet foods by a quick multiplex real-time PCR assay using EvaGreen florescence dye.

    PubMed

    Safdar, M; Abasıyanık, M F

    2013-12-01

    EvaGreen multiplex real-time polymerase chain reaction (EMRT-PCR) was designed for an assay that can join the advantages of multiplex PCR and real-time PCR to recognize animal genes more quickly in pet foods. EMRT-PCR based on melting temperatures discrimination by using EvaGreen fluorescence dye was developed for the analysis of pork and poultry in pet food. The method combines the use of poultry- and pork-specific primers that amplify small fragments of 12S rRNA and mitochondrial DNA genes. Appropriate mixtures of poultry and pork meat in reference samples were used to develop the assay. Gene yields of poultry and pork were represented in two melting peaks generated simultaneously at temperatures of 80.5 and 87.2 °C, respectively. Based upon the assay results, it has been concluded that EMRT-PCR assay might be an efficient tool for the verification of species origin in pet foods.

  2. Use of tissue culture techniques for producing virus-free plant in garlic and their identification through real-time PCR.

    PubMed

    Taşkın, Hatıra; Baktemur, Gökhan; Kurul, Mehmet; Büyükalaca, Saadet

    2013-01-01

    This study was performed for comparison of meristem culture technique with shoot tip culture technique for obtaining virus-free plant, comparison of micropropagation success of two different nutrient media, and determination of effectiveness of real-time PCR assay for the detection of viruses. Two different garlic species (Allium sativum and Allium tuncelianum) and two different nutrient media were used in this experiment. Results showed that Medium 2 was more successful compared to Medium 1 for both A. tuncelianum and A. sativum (Kastamonu garlic clone). In vitro plants obtained via meristem and shoot tip cultures were tested for determination of onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV) through real-time PCR assay. In garlic plants propagated via meristem culture, we could not detect any virus. OYDV and LYSV viruses were detected in plants obtained via shoot tip culture. OYDV virus was observed in amount of 80% and 73% of tested plants for A. tuncelianum and A. sativum, respectively. LYSV virus was found in amount of 67% of tested plants of A. tuncelianum and in amount of 87% of tested plants of A. sativum in this study.

  3. Use of Tissue Culture Techniques for Producing Virus-Free Plant in Garlic and Their Identification through Real-Time PCR

    PubMed Central

    Taşkın, Hatıra; Baktemur, Gökhan; Kurul, Mehmet; Büyükalaca, Saadet

    2013-01-01

    This study was performed for comparison of meristem culture technique with shoot tip culture technique for obtaining virus-free plant, comparison of micropropagation success of two different nutrient media, and determination of effectiveness of real-time PCR assay for the detection of viruses. Two different garlic species (Allium sativum and Allium tuncelianum) and two different nutrient media were used in this experiment. Results showed that Medium 2 was more successful compared to Medium 1 for both A. tuncelianum and A. sativum (Kastamonu garlic clone). In vitro plants obtained via meristem and shoot tip cultures were tested for determination of onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV) through real-time PCR assay. In garlic plants propagated via meristem culture, we could not detect any virus. OYDV and LYSV viruses were detected in plants obtained via shoot tip culture. OYDV virus was observed in amount of 80% and 73% of tested plants for A. tuncelianum and A. sativum, respectively. LYSV virus was found in amount of 67% of tested plants of A. tuncelianum and in amount of 87% of tested plants of A. sativum in this study. PMID:23935432

  4. REAL-TIME FEEDBACK FOR IMPROVING COMPLIANCE TO HAND SANITIZATION AMONG HEALTHCARE WORKERS IN AN OPEN LAYOUT ICU USING RADIOFREQUENCY IDENTIFICATION

    PubMed Central

    Waghmare, Abijeet; Ekstrand, Maria; Raj, Tony; Selvam, Sumithra; Sreerama, Sai Madhukar; Sampath, Sriram

    2015-01-01

    Objective To increase hand sanitizer usage among healthcare workers by developing and implementing a low-cost intervention using RFID and wireless mesh networks to provide real-time alarms for increasing hand hygiene compliance during opportune moments in an open layout Intensive Care Unit (ICU). Method A wireless, RFID based system was developed and deployed in the ICU. The ICU beds were divded into an intervention arm (n=10) and a control arm (n=14). Passive RFID tags were issued to the doctors, nurses and support staff of the ICU. Long range RFID readers were positioned strategically. Sensors were placed beneath the hand sanitizers to record sanitizer usage. The system would alert the HCWs by flashing a light if an opportune moment for hand sanitization was detected. Results A significant increase in hand sanitizer use was noted in the intervention arm. Usage was highest during the early part of the workday and decreased as the day progressed. Hand wash events per person hour was highest among the ancilliary staff followed by the doctors and nurses. Conclusion Real-time feedback has potential to increase hand hygiene compliance among HCWs. The system demonstrates the possibility of automating compliance monitoring in an ICU with an open layout. PMID:25957165

  5. Real-time feedback for improving compliance to hand sanitization among healthcare workers in an open layout ICU using radiofrequency identification.

    PubMed

    Radhakrishna, Kedar; Waghmare, Abijeet; Ekstrand, Maria; Raj, Tony; Selvam, Sumithra; Sreerama, Sai Madhukar; Sampath, Sriram

    2015-06-01

    The aim of this study is to increase hand sanitizer usage among healthcare workers by developing and implementing a low-cost intervention using RFID and wireless mesh networks to provide real-time alarms for increasing hand hygiene compliance during opportune moments in an open layout Intensive Care Unit (ICU). A wireless, RFID based system was developed and implemented in the ICU. The ICU beds were divded into an intervention arm (n = 10) and a control arm (n = 14). Passive RFID tags were issued to the doctors, nurses and support staff of the ICU. Long range RFID readers were positioned strategically. Sensors were placed beneath the hand sanitizers to record sanitizer usage. The system would alert the HCWs by flashing a light if an opportune moment for hand sanitization was detected. A significant increase in hand sanitizer use was noted in the intervention arm. Usage was highest during the early part of the workday and decreased as the day progressed. Hand wash events per person hour was highest among the ancilliary staff followed by the doctors and nurses. Real-time feedback has potential to increase hand hygiene compliance among HCWs. The system demonstrates the possibility of automating compliance monitoring in an ICU with an open layout.

  6. Real-time Identification and Control of Satellite Signal Impairments Solution and Application of the Stratonovich Equation Part 1. Theoretical Development

    NASA Technical Reports Server (NTRS)

    Manning, Robert M.

    2016-01-01

    As satellite communications systems become both more complex and reliant with respect to their operating environment, it has become imperative to be able to identify, during real-time operation, the onset of one or more impairments to the quality of overall communications system integrity. One of the most important aspects to monitor of a satellite link operating within the Earth's atmosphere is the signal fading due to the occurrence of rain and/or phase scintillations. This, of course, must be done in the presence of the associated measurement uncertainty or potentially faulty measurement equipment such as in the Advanced Communication Technology Satellite (ACTS) experiment. In the present work, an approach originally suggested in 1991, and apparently still considered iconoclastic, will be significantly developed and applied to the satellite communications link on which the deleterious composite signal fade is the result of one or many component fade mechanisms. Through the measurement (with the attendant uncertainty or 'error' in the measurement) of such a composite fading satellite signal, it is desired to extract the level of each of the individual fading mechanisms so they can be appropriately mitigated before they impact the overall performance of the communications network. Rather than employing simple-minded deterministic filtering to the real-time fading, the present approach is built around all the models and/or descriptions used to describe the individual fade components, including their dynamic evolution. The latter is usually given by a first-order Langevin equation. This circumstance allows the description of the associated temporal transition probability densities of each of the component processes. By using this description, along with the real-time measurements of the composite fade (along with the measurement errors), one can obtain statistical estimates of the levels of each of the component fading mechanisms as well as their predicted values

  7. Real-time polymerase chain reaction-based identification of bacteria in milk samples from bovine clinical mastitis with no growth in conventional culturing.

    PubMed

    Taponen, S; Salmikivi, L; Simojoki, H; Koskinen, M T; Pyörälä, S

    2009-06-01

    In more than 30% of milk samples from clinical and subclinical bovine mastitis, bacteria fail to grow even after 48 h of conventional culture. The "no-growth" samples are problematic for mastitis laboratories, veterinarians, and dairy producers. This study provides the first investigation of the bacteriological etiology of such samples, using a real-time PCR-based commercial reagent kit. The assay targets the DNA of the 11 most common bacterial species or groups in mastitis and the staphylococcal blaZ gene (responsible for penicillin resistance) and can identify and quantify bacterial cells even if dead or growth-inhibited. A study was made of 79 mastitic milk samples with no-growth bacteria in conventional culture, originating from cows with clinical mastitis. Of the 79 samples, 34 (43%) were positive for 1 (32 samples) or 2 (2 samples) of the target bacteria. The positive findings included 11 Staphylococcus spp. (staphylococci other than Staphylococcus aureus), 10 Streptococcus uberis, 2 Streptococcus dysgalactiae, 6 Corynebacterium bovis, 3 Staph. aureus, 1 Escherichia coli, 1 Enterococcus, and 1 Arcanobacterium pyogenes. The positive samples contained as many as 10(3) to 10(7) bacterial genome copies per milliliter of milk. This study demonstrates that in nearly half of the clinical mastitis cases in which conventional culture failed to detect bacteria, mastitis pathogens were still present, often in substantial quantities. The clearly elevated N-acetyl-beta-d-glucosaminidase activity values of the milk samples, together with clinical signs of the infected cows and quarters, confirmed the diagnosis of clinical mastitis and indicated that real-time, PCR-based bacterial findings are able to reveal bacteriological etiology. We conclude that all common mastitis bacteria can occur in large quantities in clinical mastitis samples that exhibit no growth in conventional culture, and that the real-time PCR assay is a useful tool for bacteriological diagnosis of such

  8. Identification of human herpesvirus 6 variants A and B by primer-specific real-time PCR may help to revisit their respective role in pathology.

    PubMed

    Boutolleau, David; Duros, Caroline; Bonnafous, Pascale; Caïola, Delphine; Karras, Alexandre; Castro, Nathalie De; Ouachée, Marie; Narcy, Philippe; Gueudin, Marie; Agut, Henri; Gautheret-Dejean, Agnès

    2006-03-01

    Human herpesvirus 6 (HHV-6) isolates are classified into two variants, termed HHV-6A and HHV-6B, on the basis of distinct genetic, antigenic and biological characteristics, but the specific pathogenicity of each variant remains poorly understood. To design a rapid, sensitive and specific real-time variant-specific PCR (VS-PCR) method to differentiate both variants in biological specimens. The VS-PCR was adapted from a real-time PCR assay, based on TaqMan technology, previously developed for the genome quantitation of both HHV-6 variants [Gautheret-Dejean A, Manichanh C, Thien-Ah-Koon F, Fillet AM, Mangeney N, Vidaud M, et al. Development of a real-time polymerase chain reaction assay for the diagnosis of human herpesvirus-6 infection and application to bone marrow transplant patients. J Virol Meth 2002;100:27-35], a consensual reverse primer (Taq2) being changed into two variant-specific primers named H6A and H6B. This method was applied to a large set of biological specimens obtained in different pathological contexts. The sensitivity threshold was about 10 copies/well for HHV-6A-specific PCR (PCR-A) and 1 copy/well for HHV-6B-specific PCR (PCR-B). Both assays showed a linear dynamic range from 10 to 100,000 copies of HHV-6 DNA. Regarding the specificity and the capacity of discrimination of each assay, one variant could be detected and identified in the presence of more than 1000 times higher concentrations of the other variant in virus mixtures. The comparison of the results obtained with this VS-PCR with those previously obtained with a classic PCR method allowed us to validate our new technique on a wide panel of biological samples, including numerous patients with severe HHV-6-related symptoms. The high prevalence of HHV-6B was confirmed in healthy individuals and immunocompromised patients. HHV-6A was identified in distinct samples from several patients exhibiting neurological disorders. We developed a new VS-PCR assay, able to differentiate HHV-6A and HHV-6

  9. Real-Time PCR for the Detection and Quantification of Geodermatophilaceae from Stone Samples and Identification of New Members of the Genus Blastococcus†

    PubMed Central

    Salazar, Oscar; Valverde, Aranzazu; Genilloud, Olga

    2006-01-01

    Real-time PCR (RT-PCR) technology was used for the specific detection and quantification of members of the family Geodermatophilaceae in stone samples. Differences in the nucleotide sequences of the 16S rRNA gene region were used to design a pair of family-specific primers that were used to detect and quantify by RT-PCR DNA from members of this family in stone samples from different geographical origins in Spain. These primers were applied later to identify by PCR-specific amplification new members of the family Geodermatophilaceae isolated from the same stone samples. The diversity and taxonomic position of the wild-type strains identified from ribosomal sequence analysis suggest the presence of a new lineage within the genus Blastococcus. PMID:16391063

  10. Genome Analysis and Development of a Multiplex TaqMan Real-Time PCR for Specific Identification and Detection of Clavibacter michiganensis subsp. nebraskensis.

    PubMed

    Tambong, James T; Xu, Renlin; Daayf, Fouad; Brière, Stephan; Bilodeau, Guillaume J; Tropiano, Raymond; Hartke, Allison; Reid, Lana M; Cott, Morgan; Cote, Tammy; Agarkova, Irina

    2016-12-01

    The reemergence of the Goss's bacterial wilt and blight disease in corn in the United States and Canada has prompted investigative research to better understand the genome organization. In this study, we generated a draft genome sequence of Clavibacter michiganensis subsp. nebraskensis strain DOAB 395 and performed genome and proteome analysis of C. michiganensis subsp. nebraskensis strains isolated in 2014 (DOAB 397 and DOAB 395) compared with the type strain, NCPPB 2581 (isolated over 40 years ago). The proteomes of strains DOAB 395 and DOAB 397 exhibited a 99.2% homology but had 92.1 and 91.8% homology, respectively, with strain NCPPB 2581. The majority (99.9%) of the protein sequences had a 99.6 to 100% homology between C. michiganensis subsp. nebraskensis strains DOAB 395 and DOAB 397, with only four protein sequences (0.1%) exhibiting a similarity <70%. In contrast, 3.0% of the protein sequences of strain DOAB 395 or DOAB 397 showed low homologies (<70%) with the type strain NCPPB 2581. The genome data were exploited for the development of a multiplex TaqMan real-time polymerase chain reaction (PCR) tool for rapid detection of C. michiganensis subsp. nebraskensis. The specificity of the assay was validated using 122 strains of Clavibacter and non-Clavibacter spp. A blind test and naturally infected leaf samples were used to confirm specificity. The sensitivity (0.1 to 1.0 pg) compared favorably with previously reported real-time PCR assays. This tool should fill the current gap for a reliable diagnostic technique.

  11. Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.

    PubMed

    Cura, Carolina I; Duffy, Tomas; Lucero, Raúl H; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J; Valencia Ayala, Edward; Kjos, Sonia A; Santalla, José; Mahaney, Susan M; Cayo, Nelly M; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S; Acosta Viana, Karla Y; Brutus, Laurent; Ocampo, Susana B; Aznar, Christine; Cuba Cuba, Cesar A; Gürtler, Ricardo E; Ramsey, Janine M; Ribeiro, Isabela; VandeBerg, John L; Yadon, Zaida E; Osuna, Antonio; Schijman, Alejandro G

    2015-05-01

    Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.

  12. Real time gamma-ray signature identifier

    DOEpatents

    Rowland, Mark [Alamo, CA; Gosnell, Tom B [Moraga, CA; Ham, Cheryl [Livermore, CA; Perkins, Dwight [Livermore, CA; Wong, James [Dublin, CA

    2012-05-15

    A real time gamma-ray signature/source identification method and system using principal components analysis (PCA) for transforming and substantially reducing one or more comprehensive spectral libraries of nuclear materials types and configurations into a corresponding concise representation/signature(s) representing and indexing each individual predetermined spectrum in principal component (PC) space, wherein an unknown gamma-ray signature may be compared against the representative signature to find a match or at least characterize the unknown signature from among all the entries in the library with a single regression or simple projection into the PC space, so as to substantially reduce processing time and computing resources and enable real-time characterization and/or identification.

  13. Real-time software receiver

    NASA Technical Reports Server (NTRS)

    Ledvina, Brent M. (Inventor); Psiaki, Mark L. (Inventor); Powell, Steven P. (Inventor); Kintner, Jr., Paul M. (Inventor)

    2006-01-01

    A real-time software receiver that executes on a general purpose processor. The software receiver includes data acquisition and correlator modules that perform, in place of hardware correlation, baseband mixing and PRN code correlation using bit-wise parallelism.

  14. Real-time software receiver

    NASA Technical Reports Server (NTRS)

    Ledvina, Brent M. (Inventor); Psiaki, Mark L. (Inventor); Powell, Steven P. (Inventor); Kintner, Jr., Paul M. (Inventor)

    2007-01-01

    A real-time software receiver that executes on a general purpose processor. The software receiver includes data acquisition and correlator modules that perform, in place of hardware correlation, baseband mixing and PRN code correlation using bit-wise parallelism.

  15. Real Time Conference 2014 Overview

    NASA Astrophysics Data System (ADS)

    Nomachi, Masaharu

    2015-06-01

    This article presents an overview of the 19th Real Time Conference held last May 26-30, 2014, at the Nara Prefectural New Public Hall, Nara, Japan, organized by the Research Center for Nuclear Physics of the Osaka University. The program included many invited talks and oral sessions offering an extensive overview on the following topics: real-time system architectures, intelligent signal processing, fast data transfer links and networks, trigger systems, data acquisition, processing-farms, control, monitoring and test systems, emerging real-time technologies, new standards, real-time safety and security, and some feedback on experiences. In parallel to the oral and poster presentations, industrial exhibits by companies, workshops and short courses also ran through the week.

  16. Real Time Data System (RTDS)

    NASA Technical Reports Server (NTRS)

    Muratore, John F.

    1991-01-01

    Lessons learned from operational real time expert systems are examined. The basic system architecture is discussed. An expert system is any software that performs tasks to a standard that would normally require a human expert. An expert system implies knowledge contained in data rather than code. And an expert system implies the use of heuristics as well as algorithms. The 15 top lessons learned by the operation of a real time data system are presented.

  17. Real-time vision systems

    SciTech Connect

    Johnson, R.; Hernandez, J.E.; Lu, Shin-yee

    1994-11-15

    Many industrial and defence applications require an ability to make instantaneous decisions based on sensor input of a time varying process. Such systems are referred to as `real-time systems` because they process and act on data as it occurs in time. When a vision sensor is used in a real-time system, the processing demands can be quite substantial, with typical data rates of 10-20 million samples per second. A real-time Machine Vision Laboratory (MVL) was established in FY94 to extend our years of experience in developing computer vision algorithms to include the development and implementation of real-time vision systems. The laboratory is equipped with a variety of hardware components, including Datacube image acquisition and processing boards, a Sun workstation, and several different types of CCD cameras, including monochrome and color area cameras and analog and digital line-scan cameras. The equipment is reconfigurable for prototyping different applications. This facility has been used to support several programs at LLNL, including O Division`s Peacemaker and Deadeye Projects as well as the CRADA with the U.S. Textile Industry, CAFE (Computer Aided Fabric Inspection). To date, we have successfully demonstrated several real-time applications: bullet tracking, stereo tracking and ranging, and web inspection. This work has been documented in the ongoing development of a real-time software library.

  18. Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

    PubMed Central

    Cura, Carolina I.; Duffy, Tomas; Lucero, Raúl H.; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J.; Valencia Ayala, Edward; Kjos, Sonia A.; Santalla, José; Mahaney, Susan M.; Cayo, Nelly M.; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S.; Acosta Viana, Karla Y.; Brutus, Laurent; Ocampo, Susana B.; Aznar, Christine; Cuba Cuba, Cesar A.; Gürtler, Ricardo E.; Ramsey, Janine M.; Ribeiro, Isabela; VandeBerg, John L.; Yadon, Zaida E.; Osuna, Antonio; Schijman, Alejandro G.

    2015-01-01

    Background Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). Methods/Principal Findings The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Conclusions/Significance Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. PMID:25993316

  19. Identification and validation of biomarkers of IgV(H) mutation status in chronic lymphocytic leukemia using microfluidics quantitative real-time polymerase chain reaction technology.

    PubMed

    Abruzzo, Lynne V; Barron, Lynn L; Anderson, Keith; Newman, Rachel J; Wierda, William G; O'brien, Susan; Ferrajoli, Alessandra; Luthra, Madan; Talwalkar, Sameer; Luthra, Rajyalakshmi; Jones, Dan; Keating, Michael J; Coombes, Kevin R

    2007-09-01

    To develop a model incorporating relevant prognostic biomarkers for untreated chronic lymphocytic leukemia patients, we re-analyzed the raw data from four published gene expression profiling studies. We selected 88 candidate biomarkers linked to immunoglobulin heavy-chain variable region gene (IgV(H)) mutation status and produced a reliable and reproducible microfluidics quantitative real-time polymerase chain reaction array. We applied this array to a training set of 29 purified samples from previously untreated patients. In an unsupervised analysis, the samples clustered into two groups. Using a cutoff point of 2% homology to the germline IgV(H) sequence, one group contained all 14 IgV(H)-unmutated samples; the other contained all 15 mutated samples. We confirmed the differential expression of 37 of the candidate biomarkers using two-sample t-tests. Next, we constructed 16 different models to predict IgV(H) mutation status and evaluated their performance on an independent test set of 20 new samples. Nine models correctly classified 11 of 11 IgV(H)-mutated cases and eight of nine IgV(H)-unmutated cases, with some models using three to seven genes. Thus, we can classify cases with 95% accuracy based on the expression of as few as three genes.

  20. Development of a real time PCR Taqman assay based on the TPI gene for simultaneous identification of Clostridium chauvoei and Clostridium septicum.

    PubMed

    Garofolo, G; Galante, D; Serrecchia, L; Buonavoglia, D; Fasanella, A

    2011-02-01

    In the present study, a Taqman allelic discrimination assay based on three SNPs of the TPI gene is described. It was used as a differential diagnostic tool to detect blackleg and malignant edema. Sudden deaths of grazing ruminants, such as cattle, sheep and goats, which show clinical signs related to hyperacute infective processes, encouraged the development of a rapid and precise diagnostic molecular method. Specific primers and probes for Clostridium septicum and Clostridium chauvoei were designed on the basis of the TPI gene sequence. The multiplex PCR was tested on the DNA of a total of 57 strains, including 24 Clostridium chauvoei, 20 Clostridium septicum, 1 Bacillus anthracis and 12 other Clostridium spp. The DNA samples from Clostridium chauvoei and Clostridium septicum strains were amplified. Amplification of other DNA samples was not observed, with the exception of Clostridium tertium, which showed a weak positive signal. To avoid misdiagnosis, a confirmatory assay based on a Sybr green real time PCR was proposed. The authors confirmed the efficacy and the specificity of the test used in this study, which proved to be a useful tool for the diagnosis of clostridiosis that are often diagnosed using only traditional tools.

  1. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA.

    PubMed

    Purcell, Maureen K; Pearman-Gillman, Schuyler; Thompson, Rachel L; Gregg, Jacob L; Hart, Lucas M; Winton, James R; Emmenegger, Eveline J; Hershberger, Paul K

    2016-07-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  2. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

    USGS Publications Warehouse

    Purcell, Maureen K.; Pearman-Gillman, Schuyler; Thompson, Rachel L.; Gregg, Jacob L.; Hart, Lucas M.; Winton, James R.; Emmenegger, Eveline J.; Hershberger, Paul K.

    2016-01-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  3. Real-time neutron source localization and identification with a hand-held, volumetrically-sensitive, moderating-type neutron spectrometer

    NASA Astrophysics Data System (ADS)

    Hoshor, C. B.; Myers, E. R.; Oakes, T. M.; Young, S. M.; Currie, J. E.; Scott, P. R.; Miller, W. H.; Bellinger, S. L.; McGregor, D. S.; Caruso, A. N.

    2017-09-01

    Measuring source-dependent properties of free neutrons over a large neutron energy range, with hand-portable instrumentation, continues to push the frontier of neutron detection instrumentation design and analysis techniques. Building on prior work - C.B. Hoshor, et al., A portable and wide energy range semiconductor-based neutron spectrometer, Nucl. Instrum. Methods Phys. Res. A 803 (2015) 68-81 - which focused on demonstrating one-dimensional-based energy-dependent neutron measurement and analysis with a new class of solid-state moderating-type spectrometer, this work introduces two ;core; algorithmic methodologies that expand the analysis of neutron thermalization measurements to three spatial dimensions to determine the location and identity of neutron radiation sources in real time. Two extensions of these core methodologies are then proposed to further improve both the accuracy and reliability of source location and identity determinations with this new class of hand-held instrumentation. In 432 preliminary simulation tests, these method extensions are shown to decrease the average source location error by 64% and provide correct identity determinations in all test cases.

  4. Visualisation and identification of peak exposure events in aluminium smelter pot rooms using hydrogen fluoride and aerosol real-time portable spectrometers.

    PubMed

    Skaugset, Nils Petter; Berlinger, Balázs; Radziuk, Bernhard; Tørring, Håvard; Synnes, Ole; Thomassen, Yngvar

    2014-05-01

    A recently developed novel portable real-time hydrogen fluoride spectrometer was used with an aerosol PM10 spectrometer under a PIMEX telemetric measurement strategy to visualize and identify simultaneous occupational air peak exposure events to hydrogen fluoride and PM10 aerosol sub-fractions in aluminium smelter pot rooms using Søderberg or Prebake anode technologies. The hydrogen fluoride and the aerosol concentration data measured during different work operations are plotted and evaluated applying the synchronised videos and air concentrations measured by the spectrometers. The main point-emission sources of HF and PM10 were identified and assessed. The major finding in the study was that the main source of PM10 and HF was partly open cells in a Søderberg pot room, whereas in a Prebake pot room, the point emissions of the two contaminants were associated with hot bath residues and hot replaced anodes. In order to prevent the simultaneous exposure to HF and PM10 among pot room workers it is important to prevent workers from being close to these point-sources under unfavourable ventilation. Storage of hot residues outside electrolytic cells without any point source ventilation should not occur.

  5. Identification and Evaluation of Suitable Reference Genes for Gene Expression Studies in the Whitefly Bemisia tabaci (Asia I) by Reverse Transcription Quantitative Real-Time PCR

    PubMed Central

    Collins, Carl; Patel, Mitulkumar V.; Colvin, John; Bailey, David; Seal, Susan

    2014-01-01

    This study presents a reliable method for performing reverse transcription quantitative real-time PCR (RT-qPCR) to measure gene expression in the whitefly Bemisia tabaci (Asia I) (Gennadius) (Hemiptera: Aleyrodidae), utilising suitable reference genes for data normalisation. We identified orthologs of commonly used reference genes (actin (ACT), cyclophilin 1 (CYP1), elongation factor 1α (EF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13A), and α-tubulin (TUB1A)), measured the levels of their transcripts by RT-qPCR during development and in response to thermal stress, and evaluated their suitability as endogenous controls using geNorm, BestKeeper, and NormFinder programs. Overall, TUB1A, RPL13A, and CYP1 were the most stable reference genes during B. tabaci development, and TUB1A, GAPDH, and RPL13A were the most stable reference genes in the context of thermal stress. An analysis of the effects of reference gene choice on the transcript profile of a developmentally-regulated gene encoding vitellogenin demonstrated the importance of selecting the correct endogenous controls for RT-qPCR studies. We propose the use of TUB1A, RPL13A, and CYP1 as endogenous controls for transcript profiling studies of B. tabaci development, whereas the combination of TUB1A, GAPDH, and RPL13A should be employed for studies into thermal stress. The data presented here will assist future transcript profiling studies in whiteflies. PMID:25373210

  6. Simultaneous detection and identification of STI pathogens by multiplex Real-Time PCR in genital tract specimens in a selected area of Apulia, a region of Southern Italy.

    PubMed

    Del Prete, Raffaele; Ronga, Luigi; Lestingi, Mirella; Addati, Grazia; Angelotti, Umberto Filippo; Di Carlo, Domenico; Miragliotta, Giuseppe

    2017-08-01

    Genital tract infections are globally a major cause of morbidity in sexually active individuals. The aim of this study was to investigate the prevalence and associations of co-infections of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma hominis (MH), Mycoplasma genitalium, Ureaplasma urealyticum (UU) and Ureaplasma parvum (UP) in specimens collected from female (SF) and male (SM) patients. 1575 samples from 1575 individuals from the geographical area around Bari, Apulia region in Southern Italy, were collected and analyzed by a multiplex Real-Time PCR (mRT-PCR) (Anyplex(TM) II STI-7, Seegene, Inc., Seoul, Korea) assay. 455/1575 (28.89%) samples resulted positive for at least one of the targets named above. Statistically significant differences in prevalence of the pathogens between SF and SM were not detected except for UP (24.92% in SF vs 8.91% in SM). Prevalence of co-infections was 6.84 and 3.96% in SF and SM, respectively. Moreover, MH presence in SF, but not in SM, was associated with UU and UP. Our data suggest different patterns of infections between females and male and the importance of an increased vigilance of sexually transmitted pathogens to reduce the burden on general population and the sequelae or the complications on reproductive organs.

  7. Identification of sexually dimorphic gene expression in brain tissue of the fish Leporinus macrocephalus through mRNA differential display and real time PCR analyses.

    PubMed

    Alves-Costa, Fernanda A; Wasko, A P

    2010-03-01

    Differentially expressed genes in males and females of vertebrate species generally have been investigated in gonads and, to a lesser extent, in other tissues. Therefore, we attempted to identify sexually dimorphic gene expression in the brains of adult males and females of Leporinus macrocephalus, a gonochoristic fish species that presents a ZZ/ZW sex determination system, throughout a comparative analysis using differential display reverse transcriptase-PCR and real-time PCR. Four cDNA fragments were characterized, representing candidate genes with differential expression between the samples. Two of these fragments presented no significant identity with previously reported gene sequences. The other two fragments, isolated from male specimens, were associated to the gene that codes for the protein APBA2 (amyloid beta (A4) precursor protein-binding, family A, member 2) and to the Rab 37 gene, a member of the Ras oncogene family. The overexpression of these genes has been associated to a greater production of the beta-amyloid protein which, in turns, is the major factor that leads to Alzheimer's disease, and to the development of brain-tumors, respectively. Quantitative RT-PCR analyses revealed a higher Apba2 gene expression in males, thus validating the previous data on differential display. L. macrocephalus may represent an interesting animal model to the understanding of the function of several vertebrate genes, including those involved in neurodegenerative and cancer diseases.

  8. Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells.

    PubMed

    Li, Tianyi; Diao, Hongying; Zhao, Lei; Xing, Yue; Zhang, Jichang; Liu, Ning; Yan, Youyou; Tian, Xin; Sun, Wei; Liu, Bin

    2017-04-05

    Oxidative stress can induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis. Although quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in gene expression studies in oxidative stress injuries, using carefully validated reference genes has not received sufficient attention in related studies. The objective of this study, therefore, was to select a set of stably expressed reference genes for use in qRT-PCR normalization in oxidative stress injuries in human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H2O2). Using geNorm analysis, we found that five stably expressed reference genes were sufficient for normalization in qRT-PCR analysis in HUVECs treated with H2O2. Genes with the most stable expression according to geNorm were U6, TFRC, RPLP0, GAPDH, and ACTB, and according to NormFinder were ALAS1, TFRC, U6, GAPDH, and ACTB. Taken together, our study demonstrated that the expression stability of reference genes may differ according to the statistical program used. U6, TFRC, RPLP0, GAPDH, and ACTB was the optimal set of reference genes for studies on gene expression performed by qRT-PCR assays in HUVECs under oxidative stress study.

  9. Identification of Valid Housekeeping Genes for Real-Time Quantitative PCR Analysis of Collapsed Lung Tissues of Neonatal Somatic Cell Nuclear Transfer-Derived Cattle.

    PubMed

    Liu, Yan; Zhang, Yunhai; Jiang, Qiuling; Rao, Man; Sheng, Zheya; Zhang, Yu; Du, Weihua; Hao, Haisheng; Zhao, Xueming; Xu, Zhe; Liu, Jianning; Zhu, Huabin

    2015-10-01

    Cloned calves produced by somatic cell nuclear transfer frequently suffer alveolar collapse as newborns. To study the underlying pathophysiological mechanisms responsible for this phenomenon, the expression profiles of numerous genes involved in lung development need to be investigated. Quantitative real-time PCR is commonly adopted in gene expression analysis. However, selection of an appropriate reference gene for normalization is critical for obtaining reliable and accurate results. Seven housekeeping genes-β-glucuronidase (GUSB), phosphoglycerate kinase 1 (PGK1), β-2-microglobolin (B2M), peptidylprolyl isomerase A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA-box binding protein (TBP), and 5.8S ribosomal RNA (5.8S rRNA)-were selected and evaluated as candidates. Their gene expression levels in the collapsed lungs of deceased neonate cloned calves and normal lung derived from normal calves were assessed. The ranking of gene expression stability was estimated by the geNorm, NormFinder, and BestKeeper programs. 5.8S rRNA and PPIA were determined to be the most stable reference genes by geNorm and BestKeeper, whereas the combination of GAPDH and TBP was suggested as reference genes by NormFinder. Taking these results into account, we conclude that 5.8S rRNA and PPIA could be the most reliable reference genes for studying the genes involved in alveolar collapse. Moreover, 5.8S rRNA could be represented as a uniform reference gene in similar cases.

  10. Identification of Streptococcus pneumoniae lytA, plyA and psaA genes in pleural fluid by multiplex real-time PCR.

    PubMed

    Sanz, Juan Carlos; Ríos, Esther; Rodríguez-Avial, Iciar; Ramos, Belén; Marín, Mercedes; Cercenado, Emilia

    2017-08-14

    The aim was to evaluate the utility of a multiplex real-time PCR to detect Streptococcus pneumoniae lytA, plyA and psaA genes in pleural fluid (PF). A collection of 81 PF samples was used. Sixty were considered positive for S. pneumoniae according to previous results (54 by an in-house lytA gene PCR and eight by universal rRNA PCR). The sensitivity for detection of the lytA, plyA and psaA genes by multiplex PCR was 100% (60/60), 98.3% (59/60) and 91.7% (55/60), respectively. The detection of all three genes was negative in 21 samples formerly confirmed as negative for S. pneumoniae (100% specificity) by the other procedures (9 by in-house lytA PCR and 12 by rRNA PCR). The use of this multiplex PCR may be a useful option to identify S. pneumoniae directly in PF samples. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  11. Identification of suitable normalizing genes for quantitative real-time RT-PCR analysis of gene expression in fetal mouse gonads.

    PubMed

    Svingen, T; Spiller, C M; Kashimada, K; Harley, V R; Koopman, P

    2009-01-01

    In biological research, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays are commonly employed to study mRNA abundance in cells and tissues. This type of assay usually relies on assessing transcript abundance relative to constitutively expressed endogenous reference genes. Therefore, it is important that the reference genes themselves are stably expressed in the cells or tissues analyzed, independent of factors such as age, sex, disease or experimental manipulations. Since no gene is expressed at the same level in all cells at all times, suitable reference genes must be identified for the specific cellular system or tissue being investigated. Here, we sought to identify stably expressed endogenous reference genes during embryonic gonad development in the mouse. We measured the transcript abundance of 10 frequently employed normalizing genes, of which 4 were stably expressed in fetal gonads from 11.5 to 14.5 dpc irrespective of sex. Based on our analysis, we suggest that Rn18s, Rps29, Tbp and Sdha are suitable reference genes for qRT-PCR expression studies during early gonad differentiation in the mouse.

  12. Comparison of real-time PCR assays for detection of pathogenic Leptospira spp. in blood and identification of variations in target sequences.

    PubMed

    Bourhy, Pascale; Bremont, Sylvie; Zinini, Farida; Giry, Claude; Picardeau, Mathieu

    2011-06-01

    Leptospirosis is considered an underdiagnosed disease. Although several PCR-based methods are currently in use, there is little information on their comparability. In this study, four quantitative real-time PCR (qPCR) assays (SYBR green and TaqMan chemistries) targeting the secY, lfb1, and lipL32 genes were evaluated as diagnostic assays. In our hands, these assays can detect between 10(2) and 10(3) bacteria/ml of pure culture, whole-blood, plasma, and serum samples. In three independent experiments, we found a slightly higher sensitivity of the PCR assays in plasma than in whole blood and serum. We also evaluated the specificity of the PCR assays on reference Leptospira strains, including newly described Leptospira species, and clinical isolates. No amplification was detected for DNA obtained from saprophytic or intermediate Leptospira species. However, among the pathogens, we identified sequence polymorphisms in target genes that result in primer and probe mismatches and affect qPCR assay performance. In conclusion, most of these assays are sensitive and specific tools for routine diagnosis of leptospirosis. However, it is important to continually evaluate and, if necessary, modify the primers and/or probes used to ensure effective detection of the circulating Leptospira isolates.

  13. Assessing direct analysis in real-time-mass spectrometry (DART-MS) for the rapid identification of additives in food packaging.

    PubMed

    Ackerman, L K; Noonan, G O; Begley, T H

    2009-12-01

    The ambient ionization technique direct analysis in real time (DART) was characterized and evaluated for the screening of food packaging for the presence of packaging additives using a benchtop mass spectrometer (MS). Approximate optimum conditions were determined for 13 common food-packaging additives, including plasticizers, anti-oxidants, colorants, grease-proofers, and ultraviolet light stabilizers. Method sensitivity and linearity were evaluated using solutions and characterized polymer samples. Additionally, the response of a model additive (di-ethyl-hexyl-phthalate) was examined across a range of sample positions, DART, and MS conditions (temperature, voltage and helium flow). Under optimal conditions, molecular ion (M+H+) was the major ion for most additives. Additive responses were highly sensitive to sample and DART source orientation, as well as to DART flow rates, temperatures, and MS inlet voltages, respectively. DART-MS response was neither consistently linear nor quantitative in this setting, and sensitivity varied by additive. All additives studied were rapidly identified in multiple food-packaging materials by DART-MS/MS, suggesting this technique can be used to screen food packaging rapidly. However, method sensitivity and quantitation requires further study and improvement.

  14. Identification of mRNA for endocannabinoid biosynthetic enzymes within hippocampal pyramidal cells and CA1 stratum radiatum interneuron subtypes using quantitative real-time polymerase chain reaction.

    PubMed

    Merrill, C B; McNeil, M; Williamson, R C; Poole, B R; Nelson, B; Sudweeks, S; Edwards, J G

    2012-08-30

    The hippocampus is required for short-term memory and contains both excitatory pyramidal cells and inhibitory interneurons. These cells exhibit various forms of synaptic plasticity, the mechanism underlying learning and memory. More recently, endocannabinoids were identified to be involved in synaptic plasticity. Our goal was to describe the distribution of endocannabinoid biosynthetic enzymes within CA1 stratum radiatum interneurons and CA3/CA1 pyramidal cells. We extracted mRNA from single interneurons and pyramidal cells and used real-time quantitative polymerase chain reaction (RT-PCR) to detect the presence of 12-lipoxygenase, N-acyl-phosphatidylethanolamine-specific phospholipase D, diacylglycerol lipase α, and type I metabotropic glutamate receptors, all known to be involved in endocannabinoid production and plasticity. We observed that the expression of endocannabinoid biosynthetic enzyme mRNA does occur within interneurons and that it is coexpressed with type I metabotropic glutamate receptors, suggesting interneurons have the potential to produce endocannabinoids. We also identified that CA3 and CA1 pyramidal cells express endocannabinoid biosynthetic enzyme mRNA. Our data provide the first molecular biological evidence for putative endocannabinoid production in interneurons, suggesting their potential ability to regulate endocannabinoid-mediated processes, such as synaptic plasticity.

  15. Sequential real-time PCR assays applied to identification of genomic signatures in formalin-fixed paraffin-embedded tissues: a case report about brucella-induced osteomyelitis.

    PubMed

    Zhang, Binxue; Wear, Douglas J; Stojadinovic, Alexander; Izadjoo, Mina

    2013-01-01

    Brucellosis is a zoonotic infection transmitted from animals to human by ingestion of infected food products, direct contact with an infected animal, or inhalation of aerosols. Brucella infection-induced osteomyelitis may present only with nonspecific clinical and radiographic findings, mild elevations in serum inflammatory markers, as well as nonspecific histological changes. We studied a case of an Iraqi war veteran with multifocal vertebral body and left iliac bone lesions on radio nucleotide scans and magnetic resonance imaging, clinically suspected osteomyelitis possibly because of Brucella. Although histomorphological findings were nonspecific, consisting of chronic inflammatory cell infiltrate and reactive fibrosis, tissue gram and silver impregnation stains of bone biopsies were informative, revealing gram-negative coccobacilli consistent in size with Brucella species. Total nucleic acids were extracted from formalin-fixed paraffin-embedded tissues and amplified by sequential real-time polymerase chain reaction, targeting genes coding (1) outer membrane protein (omp-31) of Brucella species and (2) insertion sequence (IS711) of Brucella abortus (b-abt). Polymerase chain reaction results confirmed B. abortus as the causative pathogens for presumed diagnosis of Brucella osteomyelitis.

  16. OPAD-EDIFIS Real-Time Processing

    NASA Technical Reports Server (NTRS)

    Katsinis, Constantine

    1997-01-01

    The Optical Plume Anomaly Detection (OPAD) detects engine hardware degradation of flight vehicles through identification and quantification of elemental species found in the plume by analyzing the plume emission spectra in a real-time mode. Real-time performance of OPAD relies on extensive software which must report metal amounts in the plume faster than once every 0.5 sec. OPAD software previously written by NASA scientists performed most necessary functions at speeds which were far below what is needed for real-time operation. The research presented in this report improved the execution speed of the software by optimizing the code without changing the algorithms and converting it into a parallelized form which is executed in a shared-memory multiprocessor system. The resulting code was subjected to extensive timing analysis. The report also provides suggestions for further performance improvement by (1) identifying areas of algorithm optimization, (2) recommending commercially available multiprocessor architectures and operating systems to support real-time execution and (3) presenting an initial study of fault-tolerance requirements.

  17. Quantitative identification of fecal water pollution sources by TaqMan real-time PCR assays using Bacteroidales 16S rRNA genetic markers.

    PubMed

    Lee, Dae-Young; Weir, Susan C; Lee, Hung; Trevors, Jack T

    2010-12-01

    PCR-based analysis of Bacteroidales 16S rRNA genes has emerged as a promising tool to identify sources of fecal water pollution. In this study, three TaqMan real-time PCR assays (BacGeneral, BacHuman, and BacBovine) were developed and evaluated for their ability to quantitatively detect general (total), human-specific, and bovine-specific Bacteroidales 16S rRNA genetic markers. The detection sensitivity was determined to be 6.5 copies of 16S rRNA gene for the BacGeneral and BacHuman assays and 10 copies for the BacBovine assay. The assays were capable of detecting approximately one to two cells per PCR. When tested with 70 fecal samples from various sources (human, cattle, pig, deer, dog, cat, goose, gull, horse, and raccoon), the three assays positively identified the target markers in all samples without any false-negative results. The BacHuman and BacBovine assays exhibited false-positive reactions with non-target samples in a few cases. However, the level of the false-positive reactions was about 50 times smaller than that of the true-positive ones, and therefore, these cross-reactions were unlikely to cause misidentifications of the fecal pollution sources. Microbial source-tracking capability was tested at two freshwater streams of which water quality was influenced by human and cattle feces, respectively. The assays accurately detected the presence of the corresponding host-specific markers upon fecal pollution and the persistence of the markers in downstream areas. The assays are expected to reliably determine human and bovine fecal pollution sources in environmental water samples.

  18. Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

    NASA Astrophysics Data System (ADS)

    Qiu, Reng; Sun, Boguang; Fang, Shasha; Sun, Li; Liu, Xiao

    2013-03-01

    Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Vibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues (digestive glands, foot muscle, gill, hemocyte, and mantle) were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-1-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

  19. Identification and Evaluation of Suitable Reference Genes for Normalization of MicroRNA Expression in Helicoverpa armigera (Lepidoptera: Noctuidae) Using Quantitative Real-Time PCR.

    PubMed

    Yang, Yuhui; Li, Zhen; Cao, Jinjun; Li, Yanrong; Li, Hui; Yang, Qingpo; Zhang, Qingwen; Liu, Xiaoxia

    2017-01-01

    More and more studies have focused on microRNAs (miRNAs) expression in the pest Helicoverpa armigera (Lepidoptera: Noctuidae) recently. Quantitative real-time PCR (qRT-PCR) is being widely used in miRNA expression studies. Suitable reference genes are necessary for the correct analysis of results. In this study, 10 candidate genes of H. armigera were selected and analyzed for their expression stability under different biotic and abiotic conditions with 3 statistical methods, including geNorm, NormFinder, and Bestkeeper. Combination the best number of reference genes was calculated by geNorm. One target gene, let-7, was used to validate the selection of reference genes. The suitable candidate reference genes were shown as follows: miR-9 and U6 snRNA for developmental stages, miR-100 and U6 snRNA for larval tissues, miR-100 and miR-305 for adult tissues, miR-9 and miR-279 for parasitic treatment, miR-998 and U6 snRNA for nuclear polyhedrosis virus infection, miR-9 and U6 snRNA for insecticide treatment, miR-92a, miR-100, and miR-279 for temperature treatment, miR-92a, miR-305, and miR-998 for starvation treatment, miR-9 and miR-279 for light treatment, miR-305 and miR-998 for hormone treatment, and there was not one reference gene suitable for all samples. This study could promote future research on miRNAs expression in H. armigera with optimal reference genes under different experimental conditions. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.

  20. Identification and validation of reference genes for Populus euphratica gene expression analysis during abiotic stresses by quantitative real-time PCR.

    PubMed

    Wang, Hou-Ling; Chen, Jinhuan; Tian, Qianqian; Wang, Shu; Xia, Xinli; Yin, Weilun

    2014-11-01

    Populus euphratica is the only arboreal species that is established in the world's largest shifting-sand desert in China and is well-adapted to the extreme desert environment, so it is widely considered a model system for researching into abiotic stress resistance of woody plants. However, few P. euphratica reference genes (RGs) have been identified for quantitative real-time polymerase chain reaction (qRT-PCR) until now. Validation of suitable RGs is essential for gene expression normalization research. In this study, we screened 16 endogenous candidate RGs in P. euphratica leaves in six abiotic stress treatments, including abscisic acid (ABA), cold, dehydration, drought, short-duration salt (SS) and long-duration salt (LS) treatments, each with 6 treatment gradients. After calculation of PCR efficiencies, three different software tools, NormFinder, geNorm and BestKeeper, were employed to analyze the qRT-PCR data systematically, and the outputs were merged by means of a non-weighted unsupervised rank aggregation method. The genes selected as optimal for gene expression analysis of the six treatments were RPL17 (ribosomal protein L17) in ABA, EF1α (elongation factor-1 alpha) in cold, HIS (histone superfamily protein H3) in dehydration, GIIα in drought and SS, and TUB (tubulin) in LS. The expression of 60S (the 60S ribosomal protein) varied the least during all treatments. To illustrate the suitability of these RGs, the relative quantifications of three stress-inducible genes, PePYL1, PeSCOF-1 and PeSCL7 were investigated with different RGs. The results, calculated using qBasePlus software, showed that compared with the least-appropriate RGs, the expression profiles normalized by the recommended RGs were closer to expectations. Our study provided an important RG application guideline for P. euphratica gene expression characterization. © 2014 Scandinavian Plant Physiology Society.

  1. The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

    PubMed

    Cai, Jing; Li, Tao; Huang, Bangxing; Cheng, Henghui; Ding, Hui; Dong, Weihong; Xiao, Man; Liu, Ling; Wang, Zehua

    2014-01-01

    Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

  2. Identification of Reference Genes for Quantitative Real-Time PCR in Date Palm (Phoenix dactylifera L.) Subjected to Drought and Salinity

    PubMed Central

    V. Patankar, Himanshu; M. Assaha, Dekoum V.; Al-Yahyai, Rashid; Sunkar, Ramanjulu

    2016-01-01

    Date palm is an important crop plant in the arid and semi-arid regions supporting human population in the Middle East and North Africa. These areas have been largely affected by drought and salinity due to insufficient rainfall and improper irrigation practices. Date palm is a relatively salt- and drought-tolerant plant and more recently efforts have been directed to identifying genes and pathways that confer stress tolerance in this species. Quantitative real-time PCR (qPCR) is a promising technique for the analysis of stress-induced differential gene expression, which involves the use of stable reference genes for normalizing gene expression. In an attempt to find the best reference genes for date palm’s drought and salinity research, we evaluated the stability of 12 most commonly used reference genes using the geNorm, NormFinder, BestKeeper statistical algorithms and the comparative ΔCT method. The comprehensive results revealed that HEAT SHOCK PROTEIN (HSP), UBIQUITIN (UBQ) and YTH domain-containing family protein (YT521) were stable in drought-stressed leaves whereas GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH), ACTIN and TUBULIN were stable in drought-stressed roots. On the other hand, SMALL SUBUNIT RIBOSOMAL RNA (25S), YT521 and 18S ribosomal RNA (18S); and UBQ, ACTIN and ELONGATION FACTOR 1-ALPHA (eEF1a) were stable in leaves and roots, respectively, under salt stress. The stability of these reference genes was verified by using the abiotic stress-responsive CYTOSOLIC Cu/Zn SUPEROXIDE DISMUTASE (Cyt-Cu/Zn SOD), an ABA RECEPTOR, and a PROLINE TRANSPORTER 2 (PRO) genes. A combination of top 2 or 3 stable reference genes were found to be suitable for normalization of the target gene expression and will facilitate gene expression analysis studies aimed at identifying functional genes associated with drought and salinity tolerance in date palm. PMID:27824922

  3. Real-Time Detection and Identification of Chlamydophila Species in Veterinary Specimens by Using SYBR Green-Based PCR Assays ▿

    PubMed Central

    Nordentoft, Steen; Kabell, Susanne; Pedersen, Karl

    2011-01-01

    Infections caused by members of the Chlamydiaceae family have long been underestimated due to the requirement of special laboratory facilities for the detection of this group of intracellular pathogens. Furthermore, new studies of this group of intracellular pathogens have revealed that host specificity of different species is not as clear as recently believed. As most members of the genus Chlamydophila have shown to be transmissible from animals to humans, sensitive and fast detection methods are required. In this study, SYBR green-based real-time assays were developed that detect all members of Chlamydiaceae and differentiate the most prevalent veterinary Chlamydophila species: Cp. psittaci, Cp. abortus, Cp. felis, and Cp. caviae. By adding bovine serum albumin to the master mixes, target DNA could be detected directly in crude lysates of enzymatically digested conjunctival or pharyngeal swabs or tissue specimens from heart, liver, and spleen without further purification. The assays were evaluated on veterinary specimens where all samples were screened using a family-specific PCR, and positive samples were further tested using species-specific PCRs. Cp. psittaci was detected in 47 birds, Cp. felis was found in 10 cats, Cp. caviae was found in one guinea pig, and Cp. abortus was detected in one sheep. The screening assay appeared more sensitive than traditional microscopical examination of stained tissue smears. By combining a fast, robust, and cost-effective method for sample preparation with a highly sensitive family-specific PCR, we were able to screen for Chlamydiaceae in veterinary specimens and confirm the species in positive samples with additional PCR assays. PMID:21764961

  4. Light-scattering sensor for real-time identification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae colonies on solid agar plate.

    PubMed

    Huff, Karleigh; Aroonnual, Amornrat; Littlejohn, Amy E Fleishman; Rajwa, Bartek; Bae, Euiwon; Banada, Padmapriya P; Patsekin, Valery; Hirleman, E Daniel; Robinson, J Paul; Richards, Gary P; Bhunia, Arun K

    2012-09-01

    The three most common pathogenic species of Vibrio, Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus, are of major concerns due to increased incidence of water- and seafood-related outbreaks and illness worldwide. Current methods are lengthy and require biochemical and molecular confirmation. A novel label-free forward light-scattering sensor was developed to detect and identify colonies of these three pathogens in real time in the presence of other vibrios in food or water samples. Vibrio colonies grown on agar plates were illuminated by a 635 nm laser beam and scatter-image signatures were acquired using a CCD (charge-coupled device) camera in an automated BARDOT (BActerial Rapid Detection using Optical light-scattering Technology) system. Although a limited number of Vibrio species was tested, each produced a unique light-scattering signature that is consistent from colony to colony. Subsequently a pattern recognition system analysing the collected light-scatter information provided classification in 1-2 min with an accuracy of 99%. The light-scattering signatures were unaffected by subjecting the bacteria to physiological stressors: osmotic imbalance, acid, heat and recovery from a viable but non-culturable state. Furthermore, employing a standard sample enrichment in alkaline peptone water for 6 h followed by plating on selective thiosulphate citrate bile salts sucrose agar at 30°C for ∼ 12 h, the light-scattering sensor successfully detected V. cholerae, V. parahaemolyticus and V. vulnificus present in oyster or water samples in 18 h even in the presence of other vibrios or other bacteria, indicating the suitability of the sensor as a powerful screening tool for pathogens on agar plates. © 2012 The Authors. Microbial Biotechnology © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  5. Species-specific identification of ruminant components contaminating industrial crude porcine heparin using real-time fluorescent qualitative and quantitative PCR.

    PubMed

    Huang, Qing; Xu, Ting; Wang, Gui-Yu; Huang, Jun-Fu; Xia, Han; Yin, Richard; Tang, Angie; Fu, Wei-Ling

    2012-02-01

    Ever since the emergence of bovine spongiform encephalopathy, the source of pharmaceutical heparin has been restricted to porcine intestinal mucosa. In this project, two real-time fluorescent PCR methods were developed to assist with quality control analysis. The first is a qualitative method which relies on SYBR Green I chemistry to confirm the porcine origin of industrial crude porcine heparin (ICPH), identify any ruminant contaminants, and generally control purity. The second is based on TaqMan chemistry and is able to quantitatively identify porcine, bovine, caprine, and ovine components and contaminants in ICPH. By targeting mitochondrial DNA, both PCR systems showed a detection limit of 1 pg DNA and amplification efficiencies ranging between 96% and 102%. Moreover, quantitative PCR showed a detection limit of 0.02 ppm in samples comprising porcine, bovine, caprine, and ovine DNA. The results of qualitative PCR over 27 ICPH samples showed that all samples were porcine in origin and that 17 had ruminant contaminants. The results of quantitative PCR further showed that out of all 17 samples with ruminant contaminants, seven samples had bovine, ovine, and caprine contaminants, two samples had bovine and ovine contaminants, and eight samples had only ovine contaminants. In conclusion, the qualitative PCR system was found to be a relatively inexpensive, rapid, and flexible method of identifying the porcine origin of and ruminant contaminants in ICPH, while the quantitative PCR was found suitable to accurately analyze the components and contaminants in detail. Both methods are suitable for routine control assays for the evaluation of ICPH purity and origins of contaminants.

  6. Development and Validation of LNA-Based Quantitative Real-Time PCR Assays for Detection and Identification of the Root-Knot Nematode Meloidogyne enterolobii in Complex DNA Backgrounds.

    PubMed

    Kiewnick, Sebastian; Frey, Jürg E; Braun-Kiewnick, Andrea

    2015-09-01

    Meloidogyne enterolobii is a quarantine root-knot nematode posing a major threat to agricultural production systems worldwide. It attacks many host plants, including important agricultural crops, ornamentals, and trees. M. enterolobii is a highly virulent and pathogenic root-knot nematode species, able to reproduce on plants resistant to other Meloidogyne spp. Significant crop damage has been reported in Asia, South America, Africa, the United States, France, and greenhouses in Switzerland. To identify potential introduction pathways and ensure appropriate phytosanitary measures and management strategies, accurate detection and identification tools are needed. Therefore, two real-time quantitative polymerase chain reaction (PCR) assays based on the second intergenic spacer region of the ribosomal DNA cistron and the cytochrome oxidase c subunit I (COI) gene using locked nucleic acid probes were developed and validated for fast and reliable detection and identification of M. enterolobii. Analytical specificity was confirmed with 16 M. enterolobii populations, 16 populations of eight closely related Meloidogyne spp., and four species from other nematode genera. Optimizing and testing the assays on two real-time PCR platforms revealed an analytical sensitivity of one juvenile in a background of 1,000 nematodes and the intended limit of detection of one juvenile per 100 ml of soil. Both assays performed equally well, with the COI-based assay showing a slightly better performance concerning detection of M. enterolobii target DNA in complex DNA backgrounds.

  7. Finding the Enemy: Using 3-D Laser Radar (LADAR) Imaging for Real Time Combat Identification of Ground Targets in an Obscured Environment

    DTIC Science & Technology

    2010-04-01

    Algorithms”, p. 197. 23 Ibid, p. 197. 24 http://encyclopedia2.thefreedictionary.com/Bayesian+theory, p. 1. 25 Abdallah, Mahmoud A., Tayib I. Samu , and...Bibliography Abdallah, Mahmoud A., Tayib I. Samu , and William A. Grissom. “Automatic Target Identification Using Neural Networks.” SPIE Vol

  8. HEVC real-time decoding

    NASA Astrophysics Data System (ADS)

    Bross, Benjamin; Alvarez-Mesa, Mauricio; George, Valeri; Chi, Chi Ching; Mayer, Tobias; Juurlink, Ben; Schierl, Thomas

    2013-09-01

    The new High Efficiency Video Coding Standard (HEVC) was finalized in January 2013. Compared to its predecessor H.264 / MPEG4-AVC, this new international standard is able to reduce the bitrate by 50% for the same subjective video quality. This paper investigates decoder optimizations that are needed to achieve HEVC real-time software decoding on a mobile processor. It is shown that HEVC real-time decoding up to high definition video is feasible using instruction extensions of the processor while decoding 4K ultra high definition video in real-time requires additional parallel processing. For parallel processing, a picture-level parallel approach has been chosen because it is generic and does not require bitstreams with special indication.

  9. Dependable Real-Time Systems

    DTIC Science & Technology

    1991-09-30

    and F. Wang, "On thle Competitiveness of On-Line Real-Time Task Sc~eduling," to appear. Proc. Icai - Time Systemns Symposium, Dec 1991. 6. Biyabaiii, S...Stankovic, and K. Ramrnamritham, "System Support for lRal-’Vi111C Al: A Spring Project Perspective," Workshop on Real-Time .A1, ICAI ., August 198!). 29...Informatics, Computer S,,iety ,f India , t,, aptpear. 41 . Shilh, C. and J. A. Stankovic, "Distributed Deadlock Detection in Ada IRuntinv En vi- ronments," TRI

  10. An enhanced method for the identification of Leishmania spp. using real-time polymerase chain reaction and sequence analysis of the 7SL RNA gene region.

    PubMed

    Stevenson, Lindsay G; Fedorko, Daniel P; Zelazny, Adrian M

    2010-04-01

    The accurate identification of Leishmania spp. is important for the treatment of infected patients. Molecular methods offer an alternative to time-consuming traditional laboratory techniques for species determination. We redesigned a 7SL RNA gene-based polymerase chain reaction and sequence assay for increased species identification. DNA extracted from 17 reference strains and 10 cultured clinical isolates was examined. Sequence comparison was used successfully to identify organisms to the complex level with intercomplex similarity ranging from 77.5% to 98.4%. Many species within each complex were discriminated accurately by this method including Leishmania major, Leishmania tropica, Leishmania aethiopica, Leishmania guyanensis, and the previously indistinguishable Leishmania braziliensis and Leishmania panamensis. The Leishmania donovani complex members remain indistinguishable by this method, as are the representatives of Leishmania amazonensis/Leishmania garnhami and Leishmania mexicana/Leishmania pifanoi. Published by Elsevier Inc.

  11. An Enhanced Method for the Identification of Leishmania spp. using Real-Time PCR and Sequence Analysis of the 7SL RNA Gene Region

    PubMed Central

    Stevenson, Lindsay G.; Fedorko, Daniel P.; Zelazny, Adrian M.

    2010-01-01

    The accurate identification of Leishmania species is important for the treatment of infected patients. Molecular methods offer an alternative to time consuming traditional laboratory techniques for species determination. We redesigned a 7SL rRNA gene based PCR and sequence assay for increased species identification. DNA extracted from 17 reference strains and 10 cultured clinical isolates was examined. Sequence comparison was used successfully to identify organisms to the complex level with intercomplex similarity ranging from 77.5% to 98.4%. Many species within each complex were discriminated accurately by this method including: L. major, L. tropica, L. aethiopica, L. guyanensis, and the previously indistinguishable L. brasiliensis and L. panamensis. The L. donovani complex members remain indistinguishable by this method, as are the representatives of L. amazonensis/L. garnhami and L. mexicana/L. pifanoi. PMID:20226334

  12. Identification of Burkholderia pseudomallei and Related Bacteria by Multiple-Locus Sequence Typing-Derived PCR and Real-Time PCR▿

    PubMed Central

    Wattiau, Pierre; Van Hessche, Mieke; Neubauer, Heinrich; Zachariah, Reena; Wernery, Ulrich; Imberechts, Hein

    2007-01-01

    Close relatedness and genomic plasticity characterizing the high-threat pathogens Burkholderia pseudomallei and Burkholderia mallei render the molecular diagnosis of these species hard to guarantee with a maximal confidence level. This article describes fast molecular assays derived from compiled sequences of housekeeping genes determined in more than 1,000 strains. The assays proved to be robust and appropriate for general detection as well as species identification purposes. PMID:17251403

  13. Identification of Suitable Reference Genes for Gene Expression Normalization in the Quantitative Real-Time PCR Analysis of Sweet Osmanthus (Osmanthus fragrans Lour.)

    PubMed Central

    Wang, Yiguang; Bao, Zhiyi; Zhao, Hongbo

    2015-01-01

    Quantitative real-time PCR (RT-qPCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. Several studies examining the selection of reference genes have been performed in ornamental plants but none in sweet osmanthus (Osmanthus fragrans Lour.). Based on transcriptomic sequencing data from O. fragrans buds at four developmental stages, six reference genes (OfACT, OfEF1α, OfIDH, OfRAN1, OfTUB, and OfUBC2) with stable expression (0.5 to 2 fold change in expression levels between any two developmental stages), as well as the commonly used reference gene Of18S, were selected as candidates for gene expression normalization in the RT-qPCR analysis of O. fragrans. For the normalization of RT-qPCR with two dyes, SYBR Green and EvaGreen, the expressional stability of seven candidate reference genes in 43 O. fragrans samples was analyzed using geNorm, NormFinder and BestKeeper. For RT-qPCR using SYBR Green, OfRAN1 and OfUBC2 were the optimal reference genes for all samples and different cultivars, OfACT and OfEF1α were suitable for different floral developmental stages, and OfACT was the optimal reference gene for different temperature treatments. The geometric mean values of the optimal reference gene pairs for the normalization of RT-qPCR are recommended to be used for all samples, different cultivars and different floral developmental stages in O. fragrans. For RT-qPCR using EvaGreen, OfUBC2 was the optimal reference gene for all samples and different cultivars, and OfACT was the optimal reference gene for different floral developmental stages and different temperature treatments. As the worst reference gene, Of18S should not be used as a reference gene in O. fragrans in the future. Our results provide a reference gene application guideline for O. fragrans gene expression characterization using RT-qPCR. PMID:26302211

  14. A real-time data acquisition and control of gradient coil noise for fMRI identification of hearing disorder in children with history of ear infection

    PubMed Central

    Lee, Jaeseung; Holte, James

    2013-01-01

    Early ear infection and trauma, from birth to age 12 are known to have a significant effect on sensory and cognitive development. This effect can be demonstrated through the fMRI study of children who have a history of ear infection compared to a control group. A second research question is the extent to which brain plasticity at an early age can reduce the impact of infection on hearing and cognitive development. Functional Magnetic Resonance Imaging (fMRI) provides a mapping of brain activity in cognitive and sensory regions by recording the oxygenation state of the local cerebral blood flow. The gradient coils of fMRI scanners generate intense acoustic noise (GCN) - to which the subject is in close proximity - in the range of 90 to 140 db SPL during the imaging process. Clearly this noise will impress its signature on low level brain response patterns. An Active Noise Canceller (ANC) system can suppress the effect of GCN on the subject’s perception of a phonetic stimulus at the phoneme, word or phrase level. Due to a superimposition of the frequency and time domain components of the test signal and GCN for MR test, the ANC filtering system performs its function in real time - we must capture the brain’s response to the test signal AFTER the noise has been removed. This goal is achieved through the application of field programmable gate array (FPGA) technology of NI LabVIEW. The presentation (in the noisy fMRI environment) of test words and phrases to hearing impaired children can identify sources of distortion to their perceptual processes associated with GCN. Once this distortion has been identified, learning strategies may be introduced to replace the hearing function distorted by early infection as well as the short term effect of GCN. The study of speech cognition without the confounding effect of GCN and with the varying level of GCN for a repeated test signal at later age can be allowed to a measure of recovery through brain plasticity. PMID:23482910

  15. Real-time PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms: identification of model-dependent and -independent gene expression

    PubMed Central

    2010-01-01

    Background Candida albicans infections are often associated with biofilm formation. Previous work demonstrated that the expression of HWP1 (hyphal wall protein) and of genes belonging to the ALS (agglutinin-like sequence), SAP (secreted aspartyl protease), PLB (phospholipase B) and LIP (lipase) gene families is associated with biofilm growth on mucosal surfaces. We investigated using real-time PCR whether genes encoding potential virulence factors are also highly expressed in biofilms associated with abiotic surfaces. For this, C. albicans biofilms were grown on silicone in microtiter plates (MTP) or in the Centres for Disease Control (CDC) reactor, on polyurethane in an in vivo subcutaneous catheter rat (SCR) model, and on mucosal surfaces in the reconstituted human epithelium (RHE) model. Results HWP1 and genes belonging to the ALS, SAP, PLB and LIP gene families were constitutively expressed in C. albicans biofilms. ALS1-5 were upregulated in all model systems, while ALS9 was mostly downregulated. ALS6 and HWP1 were overexpressed in all models except in the RHE and MTP, respectively. The expression levels of SAP1 were more pronounced in both in vitro models, while those of SAP2, SAP4 and SAP6 were higher in the in vivo model. Furthermore, SAP5 was highly upregulated in the in vivo and RHE models. For SAP9 and SAP10 similar gene expression levels were observed in all model systems. PLB genes were not considerably upregulated in biofilms, while LIP1-3, LIP5-7 and LIP9-10 were highly overexpressed in both in vitro models. Furthermore, an elevated lipase activity was detected in supernatans of biofilms grown in the MTP and RHE model. Conclusions Our findings show that HWP1 and most of the genes belonging to the ALS, SAP and LIP gene families are upregulated in C. albicans biofilms. Comparison of the fold expression between the various model systems revealed similar expression levels for some genes, while for others model-dependent expression levels were observed

  16. A real-time data acquisition and control of gradient coil noise for fMRI identification of hearing disorder in children with history of ear infection.

    PubMed

    Lee, Jaeseung; Holte, James; Ritenour, E Russell

    2013-02-01

    Early ear infection and trauma, from birth to age 12 are known to have a significant effect on sensory and cognitive development. This effect can be demonstrated through the fMRI study of children who have a history of ear infection compared to a control group. A second research question is the extent to which brain plasticity at an early age can reduce the impact of infection on hearing and cognitive development. Functional Magnetic Resonance Imaging (fMRI) provides a mapping of brain activity in cognitive and sensory regions by recording the oxygenation state of the local cerebral blood flow. The gradient coils of fMRI scanners generate intense acoustic noise (GCN) - to which the subject is in close proximity - in the range of 90 to 140 db SPL during the imaging process. Clearly this noise will impress its signature on low level brain response patterns. An Active Noise Canceller (ANC) system can suppress the effect of GCN on the subject's perception of a phonetic stimulus at the phoneme, word or phrase level. Due to a superimposition of the frequency and time domain components of the test signal and GCN for MR test, the ANC filtering system performs its function in real time - we must capture the brain's response to the test signal AFTER the noise has been removed. This goal is achieved through the application of field programmable gate array (FPGA) technology of NI LabVIEW. The presentation (in the noisy fMRI environment) of test words and phrases to hearing impaired children can identify sources of distortion to their perceptual processes associated with GCN. Once this distortion has been identified, learning strategies may be introduced to replace the hearing function distorted by early infection as well as the short term effect of GCN. The study of speech cognition without the confounding effect of GCN and with the varying level of GCN for a repeated test signal at later age can be allowed to a measure of recovery through brain plasticity.

  17. Identification of Suitable Reference Genes for Gene Expression Normalization in the Quantitative Real-Time PCR Analysis of Sweet Osmanthus (Osmanthus fragrans Lour.).

    PubMed

    Zhang, Chao; Fu, Jianxin; Wang, Yiguang; Bao, Zhiyi; Zhao, Hongbo

    2015-01-01

    Quantitative real-time PCR (RT-qPCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. Several studies examining the selection of reference genes have been performed in ornamental plants but none in sweet osmanthus (Osmanthus fragrans Lour.). Based on transcriptomic sequencing data from O. fragrans buds at four developmental stages, six reference genes (OfACT, OfEF1α, OfIDH, OfRAN1, OfTUB, and OfUBC2) with stable expression (0.5 to 2 fold change in expression levels between any two developmental stages), as well as the commonly used reference gene Of18S, were selected as candidates for gene expression normalization in the RT-qPCR analysis of O. fragrans. For the normalization of RT-qPCR with two dyes, SYBR Green and EvaGreen, the expressional stability of seven candidate reference genes in 43 O. fragrans samples was analyzed using geNorm, NormFinder and BestKeeper. For RT-qPCR using SYBR Green, OfRAN1 and OfUBC2 were the optimal reference genes for all samples and different cultivars, OfACT and OfEF1α were suitable for different floral developmental stages, and OfACT was the optimal reference gene for different temperature treatments. The geometric mean values of the optimal reference gene pairs for the normalization of RT-qPCR are recommended to be used for all samples, different cultivars and different floral developmental stages in O. fragrans. For RT-qPCR using EvaGreen, OfUBC2 was the optimal reference gene for all samples and different cultivars, and OfACT was the optimal reference gene for different floral developmental stages and different temperature treatments. As the worst reference gene, Of18S should not be used as a reference gene in O. fragrans in the future. Our results provide a reference gene application guideline for O. fragrans gene expression characterization using RT-qPCR.

  18. Identification of human intestinal parasites affecting an asymptomatic peri-urban Argentinian population using multi-parallel quantitative real-time polymerase chain reaction.

    PubMed

    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

  19. Real Time Conference 2016 Overview

    NASA Astrophysics Data System (ADS)

    Luchetta, Adriano

    2017-06-01

    This is a special issue of the IEEE Transactions on Nuclear Science containing papers from the invited, oral, and poster presentation of the 20th Real Time Conference (RT2016). The conference was held June 6-10, 2016, at Centro Congressi Padova “A. Luciani,” Padova, Italy, and was organized by Consorzio RFX (CNR, ENEA, INFN, Università di Padova, Acciaierie Venete SpA) and the Istituto Nazionale di Fisica Nucleare. The Real Time Conference is multidisciplinary and focuses on the latest developments in real-time techniques in high-energy physics, nuclear physics, astrophysics and astroparticle physics, nuclear fusion, medical physics, space instrumentation, nuclear power instrumentation, general radiation instrumentation, and real-time security and safety. Taking place every second year, it is sponsored by the Computer Application in Nuclear and Plasma Sciences technical committee of the IEEE Nuclear and Plasma Sciences Society. RT2016 attracted more than 240 registrants, with a large proportion of young researchers and engineers. It had an attendance of 67 students from many countries.

  20. Real Time Sonic Boom Display

    NASA Technical Reports Server (NTRS)

    Haering, Ed

    2014-01-01

    This presentation will provide general information about sonic boom mitigation technology to the public in order to supply information to potential partners and licensees. The technology is a combination of flight data, atmospheric data and terrain information implemented into a control room real time display for flight planning. This research is currently being performed and as such, any results and conclusions are ongoing.

  1. POSIX real-time extensions

    NASA Technical Reports Server (NTRS)

    Robbins, Henry H.

    1992-01-01

    POSIX is an evolving set of operating system interface standards, whose parts are in varying stages of production in a number of standards working groups. This presentation separates the real-time POSIX standards work in process from the rest and provides an overview of the purpose, status, dependencies, content, and project schedule of each.

  2. Detection of Streptococcus pneumoniae and identification of pneumococcal serotypes by real-time polymerase chain reaction using blood samples from Italian children ≤ 5 years of age with community-acquired pneumonia.

    PubMed

    Marchese, Anna; Esposito, Susanna; Coppo, Erika; Rossi, Giovanni A; Tozzi, Alberto; Romano, Mariateresa; Da Dalt, Liviana; Schito, Gian Carlo; Principi, Nicola

    2011-09-01

    Streptococcus pneumoniae is a leading cause of severe life-threatening infections. Laboratory identification and serotyping of this pathogens is desirable to monitor vaccine impact and coverage; however, especially in pediatric patients, the yield of traditional microbiological diagnostic procedures can be very low. The aim of this study was to develop real-time polymerase chain reaction (PCR)-based assays to be performed directly on blood samples to identify the most common capsular serotypes causing pneumonia in Italian children (≤ 5 years of ages) after the introduction of the 7-valent conjugate vaccine. Our real-time PCR-based assays showed high sensitivity (at least 35 fg of pneumococcal DNA), and they were validated with 49 well-characterized pneumococcal isolates, 8 nonpneumococcal isolates, 13 simulated blood clinical samples loaded with S. pneumoniae of known serotypes, and 46 blood clinical samples. All the strains tested and the simulated blood clinical samples were correctly typed by the technique. Real-time PCR allowed serotyping in 37/46 children ≤ 5 years of age (80.4%) in whom pneumonia was diagnosed in four Italian hospitals. Non-PCV7 serotypes accounted for at least 47.8% (22/46) of cases, serotype 19A being the most common (34.7%, 16/46). Although, it is not known at present whether the incidence of 19A serotype is attributable to the use of PCV7 only, expanding pneumococcal serotype coverage has clearly the potential to prevent a larger number of pneumonias in Italian children less than ≤ 5 years of age. Molecular methods are of increasing importance in the diagnosis of pneumococcal pneumonia and in monitoring serotype distribution and replacement.

  3. Identification of the main malaria vectors in the Anopheles gambiae species complex using a TaqMan real-time PCR assay.

    PubMed

    Bass, Chris; Williamson, Martin S; Wilding, Craig S; Donnelly, Martin J; Field, Linda M

    2007-11-22

    The Anopheles gambiae sensu lato species complex comprises seven sibling species of mosquitoes that are morphologically indistinguishable. Rapid identification of the two main species which vector malaria, Anopheles arabiensis and An. gambiae sensu stricto, from the non-vector species Anopheles quadriannulatus is often required as part of vector control programmes. Currently the most widely used method for species identification is a multiplex PCR protocol that targets species specific differences in ribosomal DNA sequences. While this assay has proved to be reasonably robust in many studies, additional steps are required post-PCR making it time consuming. Recently, a high-throughput assay based on TaqMan single nucleotide polymorphism genotyping that detects and discriminates An. gambiae s.s and An. arabiensis has been reported. A new TaqMan assay was developed that distinguishes between the main malaria vectors (An. arabiensis and An. gambiae s.s.) and the non-vector An. quadriannulatus after it was found that the existing TaqMan assay incorrectly identified An. quadriannulatus, An. merus and An. melas as An. gambiae s.s. The performance of this new TaqMan assay was compared against the existing TaqMan assay and the standard PCR method in a blind species identification trial of over 450 samples using field collected specimens from a total of 13 countries in Sub-Saharan Africa. The standard PCR method was found to be specific with a low number of incorrect scores (<1%), however when compared to the TaqMan assays it showed a significantly higher number of failed reactions (15%). Both the new vector-specific TaqMan assay and the exisiting TaqMan showed a very low number of incorrectly identified samples (0 and 0.54%) and failed reactions (1.25% and 2.96%). In tests of analytical sensitivity the new TaqMan assay showed a very low detection threshold and can consequently be used on a single leg from a fresh or silica-dried mosquito without the need to first extract

  4. Identification and Quantification of a Toxigenic and Non-Toxigenic Aspergillus flavus Strain in Contaminated Maize Using Quantitative Real-Time PCR

    PubMed Central

    Mylroie, J. Erik; Ozkan, Seval; Shivaji, Renuka; Windham, Gary L.; Alpe, Michael N.; Williams, W. Paul

    2016-01-01

    Aflatoxins, which are produced by Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective strategies for reducing aflatoxin accumulation in maize grain. Distinguishing between the toxin and non-toxin producing strains is important in determining the effectiveness of bio-control strategies and understanding inter-strain interactions. Using polymorphisms found in the fungal rRNA intergenic spacer region (IGS) between a toxigenic strain of A. flavus (NRRL 3357) and the non-toxigenic strain used in the biological control agent Afla-Guard® (NRRL 21882), we developed a set of primers that allows for the identification and quantification of the two strains using quantitative PCR. This primer set has been used to screen maize grain that was inoculated with the two strains individually and co-inoculated with both strains, and it has been shown to be effective in both the identification and quantification of both strains. Screening of co-inoculated ears from multiple resistant and susceptible genotypic crosses revealed no significant differences in fungal biomass accumulation of either strain in the field tests from 2010 and 2011 when compared across the means of all genotypes. Only one genotype/year combination showed significant differences in strain accumulation. Aflatoxin accumulation analysis showed that, as expected, genotypes inoculated with the toxigenic strain accumulated more aflatoxin than when co-inoculated with both strains or inoculated with only the non-toxigenic strain. Furthermore, accumulation of toxigenic fungal mass was significantly correlated with aflatoxin accumulation while non-toxigenic fungal accumulation was not. This primer set will allow researchers to better determine how the two fungal strains compete on the maize ear and investigate the interaction

  5. Device-Independent Certification of a Nonprojective Qubit Measurement

    NASA Astrophysics Data System (ADS)

    Gómez, Esteban S.; Gómez, Santiago; González, Pablo; Cañas, Gustavo; Barra, Johanna F.; Delgado, Aldo; Xavier, Guilherme B.; Cabello, Adán; Kleinmann, Matthias; Vértesi, Tamás; Lima, Gustavo

    2016-12-01

    Quantum measurements on a two-level system can have more than two independent outcomes, and in this case, the measurement cannot be projective. Measurements of this general type are essential to an operational approach to quantum theory, but so far, the nonprojective character of a measurement can only be verified experimentally by already assuming a specific quantum model of parts of the experimental setup. Here, we overcome this restriction by using a device-independent approach. In an experiment on pairs of polarization-entangled photonic qubits we violate by more than 8 standard deviations a Bell-like correlation inequality that is valid for all sets of two-outcome measurements in any dimension. We combine this with a device-independent verification that the system is best described by two qubits, which therefore constitutes the first device-independent certification of a nonprojective quantum measurement.

  6. Performance of device-independent quantum key distribution

    NASA Astrophysics Data System (ADS)

    Cao, Zhu; Zhao, Qi; Ma, Xiongfeng

    2016-07-01

    Quantum key distribution provides information-theoretically-secure communication. In practice, device imperfections may jeopardise the system security. Device-independent quantum key distribution solves this problem by providing secure keys even when the quantum devices are untrusted and uncharacterized. Following a recent security proof of the device-independent quantum key distribution, we improve the key rate by tightening the parameter choice in the security proof. In practice where the system is lossy, we further improve the key rate by taking into account the loss position information. From our numerical simulation, our method can outperform existing results. Meanwhile, we outline clear experimental requirements for implementing device-independent quantum key distribution. The maximal tolerable error rate is 1.6%, the minimal required transmittance is 97.3%, and the minimal required visibility is 96.8 % .

  7. Device-Independent Certification of a Nonprojective Qubit Measurement.

    PubMed

    Gómez, Esteban S; Gómez, Santiago; González, Pablo; Cañas, Gustavo; Barra, Johanna F; Delgado, Aldo; Xavier, Guilherme B; Cabello, Adán; Kleinmann, Matthias; Vértesi, Tamás; Lima, Gustavo

    2016-12-23

    Quantum measurements on a two-level system can have more than two independent outcomes, and in this case, the measurement cannot be projective. Measurements of this general type are essential to an operational approach to quantum theory, but so far, the nonprojective character of a measurement can only be verified experimentally by already assuming a specific quantum model of parts of the experimental setup. Here, we overcome this restriction by using a device-independent approach. In an experiment on pairs of polarization-entangled photonic qubits we violate by more than 8 standard deviations a Bell-like correlation inequality that is valid for all sets of two-outcome measurements in any dimension. We combine this with a device-independent verification that the system is best described by two qubits, which therefore constitutes the first device-independent certification of a nonprojective quantum measurement.

  8. Relationship between semi- and fully-device-independent protocols

    NASA Astrophysics Data System (ADS)

    Li, Hong-Wei; Mironowicz, Piotr; Pawłowski, Marcin; Yin, Zhen-Qiang; Wu, Yu-Chun; Wang, Shuang; Chen, Wei; Hu, Hong-Gang; Guo, Guang-Can; Han, Zheng-Fu

    2013-02-01

    We study the relation between semi- and fully-device-independent protocols. As a tool, we use the correspondence between Bell inequalities and dimension witnesses. We present a method for converting the former into the latter, and vice versa. This relation provides us with interesting results for both scenarios. First, we find random-number-generation protocols with higher bit rates for both the semi- and fully-device-independent cases. As a byproduct, we obtain classes of Bell inequalities and dimension witnesses. Then, we show how optimization methods used in studies on Bell inequalities can be adopted for dimension witnesses.

  9. Device-independent certification of high-dimensional quantum systems.

    PubMed

    D'Ambrosio, Vincenzo; Bisesto, Fabrizio; Sciarrino, Fabio; Barra, Johanna F; Lima, Gustavo; Cabello, Adán

    2014-04-11

    An important problem in quantum information processing is the certification of the dimension of quantum systems without making assumptions about the devices used to prepare and measure them, that is, in a device-independent manner. A crucial question is whether such certification is experimentally feasible for high-dimensional quantum systems. Here we experimentally witness in a device-independent manner the generation of six-dimensional quantum systems encoded in the orbital angular momentum of single photons and show that the same method can be scaled, at least, up to dimension 13.

  10. Towards falls prevention: a wearable wireless and battery-less sensing and automatic identification tag for real time monitoring of human movements.

    PubMed

    Ranasinghe, Damith C; Shinmoto Torres, Roberto L; Sample, Alanson P; Smith, Joshua R; Hill, Keith; Visvanathan, Renuka

    2012-01-01

    Falls related injuries among elderly patients in hospitals or residents in residential care facilities is a significant problem that causes emotional and physical trauma to those involved while presenting a rising healthcare expense in countries such as Australia where the population is ageing. Novel approaches using low cost and privacy preserving sensor enabled Radio Frequency Identification (RFID) technology may have the potential to provide a low cost and effective technological intervention to prevent falls in hospitals. We outline the details of a wearable sensor enabled RFID tag that is battery free, low cost, lightweight, maintenance free and can be worn continuously for automatic and unsupervised remote monitoring of activities of frail patients at acute hospitals or residents in residential care. The technological developments outlined in the paper forms part of an overall technological intervention developed to reduce falls at acute hospitals or in residential care facilities. This paper outlines the details of the technology, underlying algorithms and the results (where an accuracy of 94-100% was achieved) of a successful pilot trial.

  11. Evaluation of RNA extraction methods and identification of putative reference genes for real-time quantitative polymerase chain reaction expression studies on olive (Olea europaea L.) fruits.

    PubMed

    Nonis, Alberto; Vezzaro, Alice; Ruperti, Benedetto

    2012-07-11

    Genome wide transcriptomic surveys together with targeted molecular studies are uncovering an ever increasing number of differentially expressed genes in relation to agriculturally relevant processes in olive (Olea europaea L). These data need to be supported by quantitative approaches enabling the precise estimation of transcript abundance. qPCR being the most widely adopted technique for mRNA quantification, preliminary work needs to be done to set up robust methods for extraction of fully functional RNA and for the identification of the best reference genes to obtain reliable quantification of transcripts. In this work, we have assessed different methods for their suitability for RNA extraction from olive fruits and leaves and we have evaluated thirteen potential candidate reference genes on 21 RNA samples belonging to fruit developmental/ripening series and to leaves subjected to wounding. By using two different algorithms, GAPDH2 and PP2A1 were identified as the best reference genes for olive fruit development and ripening, and their effectiveness for normalization of expression of two ripening marker genes was demonstrated.

  12. [Real time 3D echocardiography

    NASA Technical Reports Server (NTRS)

    Bauer, F.; Shiota, T.; Thomas, J. D.

    2001-01-01

    Three-dimensional representation of the heart is an old concern. Usually, 3D reconstruction of the cardiac mass is made by successive acquisition of 2D sections, the spatial localisation and orientation of which require complex guiding systems. More recently, the concept of volumetric acquisition has been introduced. A matricial emitter-receiver probe complex with parallel data processing provides instantaneous of a pyramidal 64 degrees x 64 degrees volume. The image is restituted in real time and is composed of 3 planes (planes B and C) which can be displaced in all spatial directions at any time during acquisition. The flexibility of this system of acquisition allows volume and mass measurement with greater accuracy and reproducibility, limiting inter-observer variability. Free navigation of the planes of investigation allows reconstruction for qualitative and quantitative analysis of valvular heart disease and other pathologies. Although real time 3D echocardiography is ready for clinical usage, some improvements are still necessary to improve its conviviality. Then real time 3D echocardiography could be the essential tool for understanding, diagnosis and management of patients.

  13. Real Time Data Analysis Techniques

    NASA Astrophysics Data System (ADS)

    Silberberg, George G.

    1983-03-01

    By the early 1970s, classical photo-optical range instrumentation technology (as a means of gathering weapons' system performance data) had become a costly and inefficient process. Film costs were increasing due to soaring silver prices. Time required to process, read, and produce optical data was becoming unacceptable as a means of supporting weapon system development programs. NWC investigated the feasibility of utilizing Closed Circuit Television (CCTV) technology as an alternative solution for providing optical data. In 1978 a program entitled Metric Video (measurements from video images) was formulated at the Naval Weapons Center, China Lake, California. The purpose of this program was to provide timely data, to reduce the number of operating personnel, and to lower data acquisition costs. Some of the task elements for this program included a near real-time vector miss-distance system, a weapons scoring system, a velocity measuring system, a time-space position system, and a system to replace film cameras for gathering real-time engineering sequential data. These task elements and the development of special hardware and techniques to achieve real-time data will be discussed briefly in this paper.

  14. [Real time 3D echocardiography

    NASA Technical Reports Server (NTRS)

    Bauer, F.; Shiota, T.; Thomas, J. D.

    2001-01-01

    Three-dimensional representation of the heart is an old concern. Usually, 3D reconstruction of the cardiac mass is made by successive acquisition of 2D sections, the spatial localisation and orientation of which require complex guiding systems. More recently, the concept of volumetric acquisition has been introduced. A matricial emitter-receiver probe complex with parallel data processing provides instantaneous of a pyramidal 64 degrees x 64 degrees volume. The image is restituted in real time and is composed of 3 planes (planes B and C) which can be displaced in all spatial directions at any time during acquisition. The flexibility of this system of acquisition allows volume and mass measurement with greater accuracy and reproducibility, limiting inter-observer variability. Free navigation of the planes of investigation allows reconstruction for qualitative and quantitative analysis of valvular heart disease and other pathologies. Although real time 3D echocardiography is ready for clinical usage, some improvements are still necessary to improve its conviviality. Then real time 3D echocardiography could be the essential tool for understanding, diagnosis and management of patients.

  15. Development of novel real-time TaqMan(®) PCR assays for the species and sex identification of otter (Lutra lutra) and their application to noninvasive genetic monitoring.

    PubMed

    O'Neill, David; Turner, Peter D; O'Meara, Denise B; Chadwick, Elizabeth A; Coffey, Lee; O'Reilly, Catherine

    2013-09-01

    Developing strategies to maintain biodiversity requires baseline information on the current status of each individual species. The development of genetic techniques and their application to noninvasively collected samples have the potential to yield information on the structure of elusive animal populations and so are important tools in conservation management. Using DNA isolated from faecal samples can be challenging owing to low quantity and quality. This study, however, presents the development of novel real-time polymerase chain reaction assays using fluorescently labelled TaqMan(®) MGB probes enabling species and sex identification of Eurasian otter (Lutra lutra) spraints (faeces). These assays can also be used in determining an optimum microsatellite panel and can be employed as cost-saving screening tools for downstream genetic testing including microsatellite genotyping and haplotype analysis. The techniques are shown to work efficiently with L. lutra DNA isolated from tissue, hair, spraint, blood and anal jelly samples.

  16. Memory attacks on device-independent quantum cryptography.

    PubMed

    Barrett, Jonathan; Colbeck, Roger; Kent, Adrian

    2013-01-04

    Device-independent quantum cryptographic schemes aim to guarantee security to users based only on the output statistics of any components used, and without the need to verify their internal functionality. Since this would protect users against untrustworthy or incompetent manufacturers, sabotage, or device degradation, this idea has excited much interest, and many device-independent schemes have been proposed. Here we identify a critical weakness of device-independent protocols that rely on public communication between secure laboratories. Untrusted devices may record their inputs and outputs and reveal information about them via publicly discussed outputs during later runs. Reusing devices thus compromises the security of a protocol and risks leaking secret data. Possible defenses include securely destroying or isolating used devices. However, these are costly and often impractical. We propose other more practical partial defenses as well as a new protocol structure for device-independent quantum key distribution that aims to achieve composable security in the case of two parties using a small number of devices to repeatedly share keys with each other (and no other party).

  17. Memory Attacks on Device-Independent Quantum Cryptography

    NASA Astrophysics Data System (ADS)

    Barrett, Jonathan; Colbeck, Roger; Kent, Adrian

    2013-01-01

    Device-independent quantum cryptographic schemes aim to guarantee security to users based only on the output statistics of any components used, and without the need to verify their internal functionality. Since this would protect users against untrustworthy or incompetent manufacturers, sabotage, or device degradation, this idea has excited much interest, and many device-independent schemes have been proposed. Here we identify a critical weakness of device-independent protocols that rely on public communication between secure laboratories. Untrusted devices may record their inputs and outputs and reveal information about them via publicly discussed outputs during later runs. Reusing devices thus compromises the security of a protocol and risks leaking secret data. Possible defenses include securely destroying or isolating used devices. However, these are costly and often impractical. We propose other more practical partial defenses as well as a new protocol structure for device-independent quantum key distribution that aims to achieve composable security in the case of two parties using a small number of devices to repeatedly share keys with each other (and no other party).

  18. Interactive real time flow simulations

    NASA Technical Reports Server (NTRS)

    Sadrehaghighi, I.; Tiwari, S. N.

    1990-01-01

    An interactive real time flow simulation technique is developed for an unsteady channel flow. A finite-volume algorithm in conjunction with a Runge-Kutta time stepping scheme was developed for two-dimensional Euler equations. A global time step was used to accelerate convergence of steady-state calculations. A raster image generation routine was developed for high speed image transmission which allows the user to have direct interaction with the solution development. In addition to theory and results, the hardware and software requirements are discussed.

  19. Real time infrared aerosol analyzer

    DOEpatents

    Johnson, Stanley A.; Reedy, Gerald T.; Kumar, Romesh

    1990-01-01

    Apparatus for analyzing aerosols in essentially real time includes a virtual impactor which separates coarse particles from fine and ultrafine particles in an aerosol sample. The coarse and ultrafine particles are captured in PTFE filters, and the fine particles impact onto an internal light reflection element. The composition and quantity of the particles on the PTFE filter and on the internal reflection element are measured by alternately passing infrared light through the filter and the internal light reflection element, and analyzing the light through infrared spectrophotometry to identify the particles in the sample.

  20. Real-time flutter analysis

    NASA Technical Reports Server (NTRS)

    Walker, R.; Gupta, N.

    1984-01-01

    The important algorithm issues necessary to achieve a real time flutter monitoring system; namely, the guidelines for choosing appropriate model forms, reduction of the parameter convergence transient, handling multiple modes, the effect of over parameterization, and estimate accuracy predictions, both online and for experiment design are addressed. An approach for efficiently computing continuous-time flutter parameter Cramer-Rao estimate error bounds were developed. This enables a convincing comparison of theoretical and simulation results, as well as offline studies in preparation for a flight test. Theoretical predictions, simulation and flight test results from the NASA Drones for Aerodynamic and Structural Test (DAST) Program are compared.

  1. Real-time streamflow conditions

    USGS Publications Warehouse

    Graczyk, David J.; Gebert, Warren A.

    1996-01-01

    Would you like to know streamflow conditions before you go fishing in Wisconsin or in more distant locations? Real-time streamflow data throughout Wisconsin and the United States are available on the Internet from the U.S. Geological Survey. You can see if the stream you are interested in fishing is high due to recent rain or low because of an extended dry spell. Flow conditions at more than 100 stream-gaging stations located throughout Wisconsin can be viewed by accessing the Wisconsin District Home Page at: http://wwwdwimdn.er.usgs.gov

  2. Real-time pulmonary graphics.

    PubMed

    Mammel, Mark C; Donn, Steven M

    2015-06-01

    Real-time pulmonary graphics now enable clinicians to view lung mechanics and patient-ventilator interactions on a breath-to-breath basis. Displays of pressure, volume, and flow waveforms, pressure-volume and flow-volume loops, and trend screens enable clinicians to customize ventilator settings based on the underlying pathophysiology and responses of the individual patient. This article reviews the basic concepts of pulmonary graphics and demonstrates how they contribute to our understanding of respiratory physiology and the management of neonatal respiratory failure.

  3. Real time analysis under EDS

    NASA Astrophysics Data System (ADS)

    Schneberk, D.

    1985-07-01

    The analysis component of the Enrichment Diagnostic System (EDS) developed for the Atomic Vapor Laser Isotope Separation Program (AVLIS) at Lawrence Livermore National Laboratory (LLNL) is described. Four different types of analysis are performed on data acquired through EDS: (1) absorption spectroscopy on laser-generated spectral lines, (2) mass spectrometer analysis, (3) general purpose waveform analysis, and (4) separation performance calculations. The information produced from this data includes: measures of particle density and velocity, partial pressures of residual gases, and overall measures of isotope enrichment. The analysis component supports a variety of real-time modeling tasks, a means for broadcasting data to other nodes, and a great degree of flexibility for tailoring computations to the exact needs of the process. A particular data base structure and program flow is common to all types of analysis. Key elements of the analysis component are: (1) a fast access data base which can configure all types of analysis, (2) a selected set of analysis routines, (3) a general purpose data manipulation and graphics package for the results of real time analysis.

  4. Real-time analysis keratometer

    NASA Technical Reports Server (NTRS)

    Adachi, Iwao P. (Inventor); Adachi, Yoshifumi (Inventor); Frazer, Robert E. (Inventor)

    1987-01-01

    A computer assisted keratometer in which a fiducial line pattern reticle illuminated by CW or pulsed laser light is projected on a corneal surface through lenses, a prismoidal beamsplitter quarterwave plate, and objective optics. The reticle surface is curved as a conjugate of an ideal corneal curvature. The fiducial image reflected from the cornea undergoes a polarization shift through the quarterwave plate and beamsplitter whereby the projected and reflected beams are separated and directed orthogonally. The reflected beam fiducial pattern forms a moire pattern with a replica of the first recticle. This moire pattern contains transverse aberration due to differences in curvature between the cornea and the ideal corneal curvature. The moire pattern is analyzed in real time by computer which displays either the CW moire pattern or a pulsed mode analysis of the transverse aberration of the cornea under observation, in real time. With the eye focused on a plurality of fixation points in succession, a survey of the entire corneal topography is made and a contour map or three dimensional plot of the cornea can be made as a computer readout in addition to corneal radius and refractive power analysis.

  5. Real time analysis under EDS

    SciTech Connect

    Schneberk, D.

    1985-07-01

    This paper describes the analysis component of the Enrichment Diagnostic System (EDS) developed for the Atomic Vapor Laser Isotope Separation Program (AVLIS) at Lawrence Livermore National Laboratory (LLNL). Four different types of analysis are performed on data acquired through EDS: (1) absorption spectroscopy on laser-generated spectral lines, (2) mass spectrometer analysis, (3) general purpose waveform analysis, and (4) separation performance calculations. The information produced from this data includes: measures of particle density and velocity, partial pressures of residual gases, and overall measures of isotope enrichment. The analysis component supports a variety of real-time modeling tasks, a means for broadcasting data to other nodes, and a great degree of flexibility for tailoring computations to the exact needs of the process. A particular data base structure and program flow is common to all types of analysis. Key elements of the analysis component are: (1) a fast access data base which can configure all types of analysis, (2) a selected set of analysis routines, (3) a general purpose data manipulation and graphics package for the results of real time analysis. Each of these components are described with an emphasis upon how each contributes to overall system capability. 3 figs.

  6. Screening and identification of compounds with antiviral activity against hepatitis B virus using a safe compound library and novel real-time immune-absorbance PCR-based high throughput system

    PubMed Central

    Lamontagne, Jason; Mills, Courtney; Mao, Richeng; Goddard, Cally; Cai, Dawei; Guo, Haitao; Cuconati, Andy; Block, Timothy; Lu, Xuanyong

    2013-01-01

    There are now 7 nucleoside/tide analogues, along with interferon-α, that are approved by the FDA for the management of chronic hepatitis B virus (HBV) infection, a disease affecting hundreds of millions of people worldwide. These medications, however, are limited in usefulness, and significant side effects and the emergence of viral escape mutants make the development of novel and updated therapeutics a pressing need in the treatment of HBV. With this in mind, a library containing 2,000 compounds already known to be safe in both humans and mice with known mechanisms of action in mammalian cells were tested for the possibility of either antiviral activity against HBV or selective toxicity in HBV producing cell lines. A modified real-time immune-absorbance-polymerase chain reaction (IA-PCR) assay was developed for this screen, utilizing cells that produce and secrete intact HBV virions. In this procedure, viral particles are first captured by an anti-HBs antibody immobilized on a plate. The viral load is subsequently assessed by real-time PCR directly on captured particles. Using this assay, eight compounds were shown to consistently reduce the amount of secreted HBV viral particles in the culture medium under conditions that had no detectable impact on cell viability. Two compounds, proparacaine and chlorophyllide, were shown to reduce HBV levels 4- to 6-fold with an IC50 of 1 and 1.5μM respectively, and were selected for further study. The identification of these compounds as promising antiviral drug candidates against HBV, despite a lack of previous recognition of HBV antiviral activity, supports the validity and utility of testing known compounds for “off- pathogen target” activity against HBV, and also validates this IA-PCR assay as an important tool for the detection of anti-viral activity against enveloped viruses. PMID:23415884

  7. A mitochondrial species identification assay for Australian blacktip sharks (Carcharhinus tilstoni, C. limbatus and C. amblyrhynchoides) using real-time PCR and high-resolution melt analysis.

    PubMed

    Morgan, Jess A T; Welch, David J; Harry, Alistair V; Street, Raewyn; Broderick, Damien; Ovenden, Jennifer R

    2011-09-01

    Tropical Australian shark fisheries target two morphologically indistinguishable blacktip sharks, the Australian blacktip (Carcharhinus tilstoni) and the common blacktip (C. limbatus). Their relative contributions to northern and eastern Australian coastal fisheries are unclear because of species identification difficulties. The two species differ in their number of precaudal vertebrae, which is difficult and time consuming to obtain in the field. But, the two species can be distinguished genetically with diagnostic mutations in their mitochondrial DNA ND4 gene. A third closely related sister species, the graceful shark C. amblyrhynchoides, can also be distinguished by species-specific mutations in this gene. DNA sequencing is an effective diagnostic tool, but is relatively expensive and time consuming. In contrast, real-time high-resolution melt (HRM) PCR assays are rapid and relatively inexpensive. These assays amplify regions of DNA with species-specific genetic mutations that result in PCR products with unique melt profiles. A real-time HRM PCR species-diagnostic assay (RT-HRM-PCR) has been developed based on the mtDNA ND4 gene for rapid typing of C. tilstoni, C. limbatus and C. amblyrhynchoides. The assay was developed using ND4 sequences from 66 C. tilstoni, 33. C. limbatus and five C. amblyrhynchoides collected from Indonesia and Australian states and territories; Western Australia, the Northern Territory, Queensland and New South Wales. The assay was shown to be 100% accurate on 160 unknown blacktip shark tissue samples by full mtDNA ND4 sequencing.

  8. Development of a new pentaplex real-time PCR assay for the identification of poly-microbial specimens containing Staphylococcus aureus and other staphylococci, with simultaneous detection of staphylococcal virulence and methicillin resistance markers.

    PubMed

    Okolie, Charles E; Wooldridge, Karl G; Turner, David P; Cockayne, Alan; James, Richard

    2015-06-01

    Staphylococcus aureus strains harbouring genes encoding virulence and antibiotic resistance are of public health importance. In clinical samples, pathogenic S. aureus is often mixed with putatively less pathogenic coagulase-negative staphylococci (CoNS), both of which can harbour mecA, the gene encoding staphylococcal methicillin-resistance. There have been previous attempts at distinguishing MRSA from MRCoNS, most of which were based on the detection of one of the pathognomonic markers of S. aureus, such as coa, nuc or spa. That approach might suffice for discrete colonies and mono-microbial samples; it is inadequate for identification of clinical specimens containing mixtures of S. aureus and CoNS. In the present study, a real-time pentaplex PCR assay has been developed which simultaneously detects markers for bacteria (16S rRNA), coagulase-negative staphylococcus (cns), S. aureus (spa), Panton-Valentine leukocidin (pvl) and methicillin resistance (mecA). Staphylococcal and non-staphylococcal bacterial strains (n = 283) were used to validate the new assay. The applicability of this test to clinical samples was evaluated using spiked blood cultures (n = 43) containing S. aureus and CoNS in mono-microbial and poly-microbial models, which showed that the 5 markers were all detected as expected. Cycling completes within 1 h, delivering 100% specificity, NPV and PPV with a detection limit of 1.0 × 10(1) to 3.0 × 10(1) colony forming units (CFU)/ml, suggesting direct applicability in routine diagnostic microbiology. This is the most multiplexed real-time PCR-based PVL-MRSA assay and the first detection of a unique marker for CoNS without recourse to the conventional elimination approach. There was no evidence that this new assay produced invalid/indeterminate test results.

  9. Screening and identification of compounds with antiviral activity against hepatitis B virus using a safe compound library and novel real-time immune-absorbance PCR-based high throughput system.

    PubMed

    Lamontagne, Jason; Mills, Courtney; Mao, Richeng; Goddard, Cally; Cai, Dawei; Guo, Haitao; Cuconati, Andy; Block, Timothy; Lu, Xuanyong

    2013-04-01

    There are now seven nucleoside/tide analogues, along with interferon-α, that are approved by the FDA for the management of chronic hepatitis B virus (HBV) infection, a disease affecting hundreds of millions of people worldwide. These medications, however, are limited in usefulness, and significant side effects and the emergence of viral escape mutants make the development of novel and updated therapeutics a pressing need in the treatment of HBV. With this in mind, a library containing 2000 compounds already known to be safe in both humans and mice with known mechanisms of action in mammalian cells were tested for the possibility of either antiviral activity against HBV or selective toxicity in HBV producing cell lines. A modified real-time immune-absorbance-polymerase chain reaction (IA-PCR) assay was developed for this screen, utilizing cells that produce and secrete intact HBV virions. In this procedure, viral particles are first captured by an anti-HBs antibody immobilized on a plate. The viral load is subsequently assessed by real-time PCR directly on captured particles. Using this assay, eight compounds were shown to consistently reduce the amount of secreted HBV viral particles in the culture medium under conditions that had no detectable impact on cell viability. Two compounds, proparacaine and chlorophyllide, were shown to reduce HBV levels 4- to 6-fold with an IC₅₀ of 1 and 1.5 μM, respectively, and were selected for further study. The identification of these compounds as promising antiviral drug candidates against HBV, despite a lack of previous recognition of HBV antiviral activity, supports the validity and utility of testing known compounds for "off-pathogen target" activity against HBV, and also validates this IA-PCR assay as an important tool for the detection of anti-viral activity against enveloped viruses. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Novel motB as a potential predictive tool for identification of B. cereus, B. thuringiensis and differentiation from other Bacillus species by triplex real-time PCR.

    PubMed

    Chelliah, Ramachandran; Wei, Shuai; Park, Byung-Jae; Kim, Se-Hun; Park, Dong-Suk; Kim, Soon Han; Hwan, Kim Seok; Oh, Deog-Hwan

    2017-08-01

    Quantitative triplex real-time PCR (qPCR) offers an alternative method for detection of bacterial contamination. It provides quantitation of the number of gene copies. In our study, we established a qPCR assay to detect and quantify the specificity towards Bacillus cereus and B. thuringiensis. The assay was designed to detect a 280 bp fragment of motB gene encoding the flagellar motor protein, specific for detection of B. cereus and B. thuringiensis, excluding other group species B. pseudomycoides, B. mycoides and B. weihenstephanensis. Specificity of the assay was confirmed with 111 strains belonging to Bacillus cereus group and performed against 58 B. cereus, 50 B. thuringiensis, 3 other Bacillus bacteria and 9 non-Bacillus bacteria. Detection limit was determined for each assay. Direct analysis of samples revealed the specificity towards identification and characterization of B. cereus group cultured in nutrient media. Based on results, it was observed that motB showed 97% specificity towards B. cereus strains, 98% for B. thuringiensis but other B. cereus group showed less sensitivity (0%), thus, provides an efficient tool to identify B. cereus and B. thuringiensis. Further, environmental and food samples do not require band isolation, re-amplification or sequence identification. Thus, reducing the time and cost of analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis.

    PubMed

    Knapp, Jenny; Umhang, Gérald; Poulle, Marie-Lazarine; Millon, Laurence

    2016-05-15

    Studying the environmental occurrence of parasites of concern for humans and animals based on coprosamples is an expanding field of work in epidemiology and the ecology of health. Detecting and quantifying Toxocara spp. and Echinococcus multilocularis, two predominant zoonotic helminths circulating in European carnivores, in feces may help to better target measures for prevention. A rapid, sensitive, and one-step quantitative PCR (qPCR) allowing detection of E. multilocularis and Toxocara spp. was developed in the present study, combined with a host fecal test based on the identification of three carnivores (red fox, dog, and cat) involved in the life cycles of these parasites. A total of 68 coprosamples were collected from identified specimens from Vulpes vulpes, Canis lupus familiaris, Canis lupus, Felis silvestris catus, Meles meles, Martes foina, and Martes martes With DNA coprosamples, real-time PCR was performed in duplex with a qPCR inhibitor control specifically designed for this study. All the coprosample host identifications were confirmed by qPCR combined with sequencing, and parasites were detected and confirmed (E. multilocularis in red foxes and Toxocara cati in cats; 16% of samples presented inhibition). By combining parasite detection and quantification, the host fecal test, and a new qPCR inhibitor control, we created a technique with a high sensitivity that may considerably improve environmental studies of pathogens. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  12. Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis

    PubMed Central

    Umhang, Gérald; Poulle, Marie-Lazarine; Millon, Laurence

    2016-01-01

    Studying the environmental occurrence of parasites of concern for humans and animals based on coprosamples is an expanding field of work in epidemiology and the ecology of health. Detecting and quantifying Toxocara spp. and Echinococcus multilocularis, two predominant zoonotic helminths circulating in European carnivores, in feces may help to better target measures for prevention. A rapid, sensitive, and one-step quantitative PCR (qPCR) allowing detection of E. multilocularis and Toxocara spp. was developed in the present study, combined with a host fecal test based on the identification of three carnivores (red fox, dog, and cat) involved in the life cycles of these parasites. A total of 68 coprosamples were collected from identified specimens from Vulpes vulpes, Canis lupus familiaris, Canis lupus, Felis silvestris catus, Meles meles, Martes foina, and Martes martes. With DNA coprosamples, real-time PCR was performed in duplex with a qPCR inhibitor control specifically designed for this study. All the coprosample host identifications were confirmed by qPCR combined with sequencing, and parasites were detected and confirmed (E. multilocularis in red foxes and Toxocara cati in cats; 16% of samples presented inhibition). By combining parasite detection and quantification, the host fecal test, and a new qPCR inhibitor control, we created a technique with a high sensitivity that may considerably improve environmental studies of pathogens. PMID:26969697

  13. Autonomous Real Time Requirements Tracing

    NASA Technical Reports Server (NTRS)

    Plattsmier, George; Stetson, Howard

    2014-01-01

    One of the more challenging aspects of software development is the ability to verify and validate the functional software requirements dictated by the Software Requirements Specification (SRS) and the Software Detail Design (SDD). Insuring the software has achieved the intended requirements is the responsibility of the Software Quality team and the Software Test team. The utilization of Timeliner-TLX(sup TM) Auto- Procedures for relocating ground operations positions to ISS automated on-board operations has begun the transition that would be required for manned deep space missions with minimal crew requirements. This transition also moves the auto-procedures from the procedure realm into the flight software arena and as such the operational requirements and testing will be more structured and rigorous. The autoprocedures would be required to meet NASA software standards as specified in the Software Safety Standard (NASASTD- 8719), the Software Engineering Requirements (NPR 7150), the Software Assurance Standard (NASA-STD-8739) and also the Human Rating Requirements (NPR-8705). The Autonomous Fluid Transfer System (AFTS) test-bed utilizes the Timeliner-TLX(sup TM) Language for development of autonomous command and control software. The Timeliner-TLX(sup TM) system has the unique feature of providing the current line of the statement in execution during real-time execution of the software. The feature of execution line number internal reporting unlocks the capability of monitoring the execution autonomously by use of a companion Timeliner-TLX(sup TM) sequence as the line number reporting is embedded inside the Timeliner-TLX(sup TM) execution engine. This negates I/O processing of this type data as the line number status of executing sequences is built-in as a function reference. This paper will outline the design and capabilities of the AFTS Autonomous Requirements Tracker, which traces and logs SRS requirements as they are being met during real-time execution of the

  14. Autonomous Real Time Requirements Tracing

    NASA Technical Reports Server (NTRS)

    Plattsmier, George I.; Stetson, Howard K.

    2014-01-01

    One of the more challenging aspects of software development is the ability to verify and validate the functional software requirements dictated by the Software Requirements Specification (SRS) and the Software Detail Design (SDD). Insuring the software has achieved the intended requirements is the responsibility of the Software Quality team and the Software Test team. The utilization of Timeliner-TLX(sup TM) Auto-Procedures for relocating ground operations positions to ISS automated on-board operations has begun the transition that would be required for manned deep space missions with minimal crew requirements. This transition also moves the auto-procedures from the procedure realm into the flight software arena and as such the operational requirements and testing will be more structured and rigorous. The autoprocedures would be required to meet NASA software standards as specified in the Software Safety Standard (NASASTD- 8719), the Software Engineering Requirements (NPR 7150), the Software Assurance Standard (NASA-STD-8739) and also the Human Rating Requirements (NPR-8705). The Autonomous Fluid Transfer System (AFTS) test-bed utilizes the Timeliner-TLX(sup TM) Language for development of autonomous command and control software. The Timeliner- TLX(sup TM) system has the unique feature of providing the current line of the statement in execution during real-time execution of the software. The feature of execution line number internal reporting unlocks the capability of monitoring the execution autonomously by use of a companion Timeliner-TLX(sup TM) sequence as the line number reporting is embedded inside the Timeliner-TLX(sup TM) execution engine. This negates I/O processing of this type data as the line number status of executing sequences is built-in as a function reference. This paper will outline the design and capabilities of the AFTS Autonomous Requirements Tracker, which traces and logs SRS requirements as they are being met during real-time execution of the

  15. Experimental device-independent tests of classical and quantum entropy

    NASA Astrophysics Data System (ADS)

    Zhu, Feng; Zhang, Wei; Chen, Sijing; You, Lixing; Wang, Zhen; Huang, Yidong

    2016-12-01

    In quantum information processing, it is important to witness the entropy of the message in the device-independent way which was proposed recently [R. Chaves, J. B. Brask, and N. Brunner, Phys. Rev. Lett. 115, 110501 (2015), 10.1103/PhysRevLett.115.110501]. In this paper, we theoretically obtain the minimal quantum entropy for three widely used linear dimension witnesses, which is considered "a difficult question." Then we experimentally test the classical and quantum entropy in a device-independent manner. The experimental results agree well with the theoretical analysis, demonstrating that entropy is needed in quantum systems that is lower than the entropy needed in classical systems with the given value of the dimension witness.

  16. Measurement-device-independent entanglement-based quantum key distribution

    NASA Astrophysics Data System (ADS)

    Yang, Xiuqing; Wei, Kejin; Ma, Haiqiang; Sun, Shihai; Liu, Hongwei; Yin, Zhenqiang; Li, Zuohan; Lian, Shibin; Du, Yungang; Wu, Lingan

    2016-05-01

    We present a quantum key distribution protocol in a model in which the legitimate users gather statistics as in the measurement-device-independent entanglement witness to certify the sources and the measurement devices. We show that the task of measurement-device-independent quantum communication can be accomplished based on monogamy of entanglement, and it is fairly loss tolerate including source and detector flaws. We derive a tight bound for collective attacks on the Holevo information between the authorized parties and the eavesdropper. Then with this bound, the final secret key rate with the source flaws can be obtained. The results show that long-distance quantum cryptography over 144 km can be made secure using only standard threshold detectors.

  17. Measurement-Device-Independent Quantum Key Distribution over 200 km

    NASA Astrophysics Data System (ADS)

    Tang, Yan-Lin; Yin, Hua-Lei; Chen, Si-Jing; Liu, Yang; Zhang, Wei-Jun; Jiang, Xiao; Zhang, Lu; Wang, Jian; You, Li-Xing; Guan, Jian-Yu; Yang, Dong-Xu; Wang, Zhen; Liang, Hao; Zhang, Zhen; Zhou, Nan; Ma, Xiongfeng; Chen, Teng-Yun; Zhang, Qiang; Pan, Jian-Wei

    2014-11-01

    Measurement-device-independent quantum key distribution (MDIQKD) protocol is immune to all attacks on detection and guarantees the information-theoretical security even with imperfect single-photon detectors. Recently, several proof-of-principle demonstrations of MDIQKD have been achieved. Those experiments, although novel, are implemented through limited distance with a key rate less than 0.1 bit /s . Here, by developing a 75 MHz clock rate fully automatic and highly stable system and superconducting nanowire single-photon detectors with detection efficiencies of more than 40%, we extend the secure transmission distance of MDIQKD to 200 km and achieve a secure key rate 3 orders of magnitude higher. These results pave the way towards a quantum network with measurement-device-independent security.

  18. Real-time flood forecasting

    USGS Publications Warehouse

    Lai, C.; Tsay, T.-K.; Chien, C.-H.; Wu, I.-L.

    2009-01-01

    Researchers at the Hydroinformatic Research and Development Team (HIRDT) of the National Taiwan University undertook a project to create a real time flood forecasting model, with an aim to predict the current in the Tamsui River Basin. The model was designed based on deterministic approach with mathematic modeling of complex phenomenon, and specific parameter values operated to produce a discrete result. The project also devised a rainfall-stage model that relates the rate of rainfall upland directly to the change of the state of river, and is further related to another typhoon-rainfall model. The geographic information system (GIS) data, based on precise contour model of the terrain, estimate the regions that were perilous to flooding. The HIRDT, in response to the project's progress, also devoted their application of a deterministic model to unsteady flow of thermodynamics to help predict river authorities issue timely warnings and take other emergency measures.

  19. Device-independent security of quantum cryptography against collective attacks.

    PubMed

    Acín, Antonio; Brunner, Nicolas; Gisin, Nicolas; Massar, Serge; Pironio, Stefano; Scarani, Valerio

    2007-06-08

    We present the optimal collective attack on a quantum key distribution protocol in the "device-independent" security scenario, where no assumptions are made about the way the quantum key distribution devices work or on what quantum system they operate. Our main result is a tight bound on the Holevo information between one of the authorized parties and the eavesdropper, as a function of the amount of violation of a Bell-type inequality.

  20. Novel toggle-rate based energy-efficient scheme for heavy load real-time IM-DD OFDM-PON with ONU LLID identification in time-domain using amplitude decision.

    PubMed

    Qin, Youxiang; Zhang, Junjie

    2017-07-10

    A novel low complexity and energy-efficient scheme by controlling the toggle-rate of ONU with time-domain amplitude identification is proposed for a heavy load downlink in an intensity-modulation and direct-detection orthogonal frequency division multiplexing passive optical network (IM-DD OFDM-PON). In a conventional OFDM-PON downlink, all ONUs have to perform demodulation for all the OFDM frames in a broadcast way no matter whether the frames are targeted to or not, which causes a huge energy waste. However, in our scheme, the optical network unit (ONU) logical link identifications (LLIDs) are inserted into each downlink OFDM frame in time-domain at the optical line terminal (OLT) side. At the ONU side, the LLID is obtained with a low complexity and high precision amplitude identification method. The ONU sets the toggle-rate of demodulation module to zero when the frames are not targeted to, which avoids unnecessary digital signal processing (DSP) energy consumption. Compared with the sleep-mode methods consisting of clock recovery and synchronization, toggle-rate shows its advantage in fast changing, which is more suitable for the heavy load scenarios. Moreover, for the first time to our knowledge, the characteristics of the proposed scheme are investigated in a real-time IM-DD OFDM system, which performs well at the received optical power as low as -21dBm. The experimental results show that 25.1% energy consumption can be saved in the receiver compared to the conventional configurations.

  1. ID-CUBE direct analysis in real time high-resolution mass spectrometry and its capabilities in the identification of phenolic components from the green leaves of Bergenia crassifolia L.

    PubMed

    Chernetsova, Elena S; Crawford, Elizabeth A; Shikov, Alexander N; Pozharitskaya, Olga N; Makarov, Valery G; Morlock, Gertrud E

    2012-06-15

    Bergenia crassifolia is a plant widely used in herbal medicine. Its chemical composition has been little studied, and no studies using high-resolution mass spectrometry (HRMS) have been performed. Its phenolic components are of particular interest, due to the interest in such compounds in medicine and cosmetics. The ID-CUBE, a simplified Direct Analysis in Real Time (DART) ion source, suitable for the fast MS analysis of liquids without complex sample preparation, offers a new method of studying extracts of such plant. Coupling the ID-CUBE with a high-resolution mass spectrometer can provide identification of extract components. Mass spectral conditions were optimized for model solutions of the flavonoid naringenin and used for the identification of phenolic compounds in green leaves extracts of Bergenia crassifolia. OpenSpot sample cards with a metal grid surface were used for sample introduction into the ID-CUBE ion source on an Obitrap mass spectrometer. The samples were applied as 5-μL aliquots of the extract onto the metal grid of the card. Sample ionization was stimulated in the ion source within 20 s by applying an electric current to the metal grid to thermally desorb the analytes into the gas flow of metastable helium atoms from the ID-CUBE. Elemental compositions were assigned to abundant ions in the mass spectra of the extracts. The major phenolic components were confirmed by their [M-H](-) ions. Thirty-six other marker ions were found, and elemental compositions were suggested for 30% of them, based on a search for compounds found in herbal extracts. The ID-CUBE-Orbitrap MS coupling allowed the rapid accurate mass determination of the phenolic components (and other compounds) in herbal extracts. Higher confidence in component identification could be provided by using additional structural elucidation methods, including tandem mass spectrometry (MS/MS), and this will be the focus of future studies. Copyright © 2012 John Wiley & Sons, Ltd.

  2. Real Time Simulation of Power Grid Disruptions

    SciTech Connect

    Chinthavali, Supriya; Dimitrovski, Aleksandar D; Fernandez, Steven J; Groer, Christopher S; Nutaro, James J; Olama, Mohammed M; Omitaomu, Olufemi A; Shankar, Mallikarjun; Spafford, Kyle L; Vacaliuc, Bogdan

    2012-11-01

    DOE-OE and DOE-SC workshops (Reference 1-3) identified the key power grid problem that requires insight addressable by the next generation of exascale computing is coupling of real-time data streams (1-2 TB per hour) as the streams are ingested to dynamic models. These models would then identify predicted disruptions in time (2-4 seconds) to trigger the smart grid s self healing functions. This project attempted to establish the feasibility of this approach and defined the scientific issues, and demonstrated example solutions to important smart grid simulation problems. These objectives were accomplished by 1) using the existing frequency recorders on the national grid to establish a representative and scalable real-time data stream; 2) invoking ORNL signature identification algorithms; 3) modeling dynamically a representative region of the Eastern interconnect using an institutional cluster, measuring the scalability and computational benchmarks for a national capability; and 4) constructing a prototype simulation for the system s concept of smart grid deployment. The delivered ORNL enduring capability included: 1) data processing and simulation metrics to design a national capability justifying exascale applications; 2) Software and intellectual property built around the example solutions; 3) demonstrated dynamic models to design few second self-healing.

  3. Real-time PCR in microfluidic devices

    NASA Astrophysics Data System (ADS)

    Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Hansen-Hagge, Thomas; Gärtner, Claudia

    2014-03-01

    A central method in a standard biochemical laboratory is represented by the polymerase chain reaction (PCR), therefore many attempts have been performed so far to implement this technique in lab-on-a-chip (LOC) devices. PCR is an ideal candidate for miniaturization because of a reduction of assay time and decreased costs for expensive bio-chemicals. In case of the "classical" PCR, detection is done by identification of DNA fragments electrophoretically separated in agarose gels. This method is meanwhile frequently replaced by the so-called Real-Time-PCR because here the exponential increase of amplificates can be observed directly by measurement of DNA interacting fluorescent dyes. Two main methods for on-chip PCRs are available: traditional "batch" PCR in chambers on a chip using thermal cycling, requiring about 30 minutes for a typical PCR protocol and continuous-flow PCR, where the liquid is guided over stationary temperature zones. In the latter case, the PCR protocol can be as fast as 5 minutes. In the presented work, a proof of concept is demonstrated for a real-time-detection of PCR products in microfluidic systems.

  4. Clinical virology in real time.

    PubMed

    Niesters, Hubert G M

    2002-12-01

    The ability to detect nucleic acids has had and still has a major impact on diagnostics in clinical virology. Both quantitative and qualitative techniques, whether signal or target amplification based systems, are currently used routinely in most if not all virology laboratories. Technological improvements, from automated sample isolation to real time amplification technology, have given the ability to develop and introduce systems for most viruses of clinical interest, and to obtain clinical relevant information needed for optimal antiviral treatment options. Both polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) can currently be used together with real time detection to generate results in a short turn-around time and to determine whether variants relevant for antiviral resistance are present. These new technologies enable the introduction of an individual patient disease management concept. Within our clinical setting, we have introduced this e.g. for quantitative detection of Epstein-Barr Virus (EBV) in T-dell depleted allogeneic stem cell transplant patients. This enabled us to develop models for pre-emptive anti B-cell immunotherapy for EBV reactivation, thereby effectively reducing not the incidence of EBV-lymphoproliferative disease but the virus related mortality. Furthermore, additional clinically relevant viruses can now easily be detected simultaneously. It also becomes more feasible to introduce molecular testing for those viruses that can easily be detected using classical virological methods, like culture techniques or antigen detection. Prospective studies are needed to evaluate the clinical importance of the additional positive samples detected. It should however be made clear that a complete exchange of technologies is unlikely to occur, and that some complementary technologies should stay operational enabling the discovery of new viruses. The implementation of these molecular diagnostic technologies furthermore

  5. Identification of cagA tyrosine phosphorylation DNA motifs in Helicobacter pylori isolates from peptic ulcer patients by novel PCR-restriction fragment length polymorphism and real-time fluorescence PCR assays.

    PubMed

    Owen, Robert J; Sharp, Sally I; Chisholm, Stephanie A; Rijpkema, Sjoerd

    2003-07-01

    Cag pathogenicity island-containing Helicobacter pylori (type I) induces signal transduction pathways resulting in tyrosine phosphorylation of proteins adjacent to the site of bacterial adhesion on host gastric epithelial cells. Conventional block PCR-restriction fragment length polymorphism (RFLP) and real-time LightCycler (LC) PCR hybridization assays, validated by direct sequencing, were designed to test for the presence of three nucleotide sequences corresponding to tyrosine phosphorylation motifs (TPMs) A, B, and C in 84 isolates of H. pylori type I from patients in England. Overall, the PCR assays demonstrated that one or more TPMs were present in 62 strains (75%). Motif A was common (71% of strains), whereas motifs B and C were rarer (8% of strains). Strains lacking a TPM were typically vacuolating cytotoxin genotype vacA m2. Motif A was widely distributed in relation to disease severity and was more commonly (but not significantly [P = 0.071]) associated with gastric ulcer than with duodenal ulcer (86 versus 56%). The LC hybridization assay provided a rapid means of detecting all three motifs, but RFLP analysis was more specific for TPM-A. TPMs provide novel additional strain markers for defining cagA variation, including identification of RFLP types within TPM-A. The presence of a particular TPM was not of direct diagnostic value, either singly or in combination, but the higher proportion of TPM-A strains in gastric ulcer patients merits further investigation.

  6. Identification of cagA Tyrosine Phosphorylation DNA Motifs in Helicobacter pylori Isolates from Peptic Ulcer Patients by Novel PCR-Restriction Fragment Length Polymorphism and Real-Time Fluorescence PCR Assays

    PubMed Central

    Owen, Robert J.; Sharp, Sally I.; Chisholm, Stephanie A.; Rijpkema, Sjoerd

    2003-01-01

    Cag pathogenicity island-containing Helicobacter pylori (type I) induces signal transduction pathways resulting in tyrosine phosphorylation of proteins adjacent to the site of bacterial adhesion on host gastric epithelial cells. Conventional block PCR-restriction fragment length polymorphism (RFLP) and real-time LightCycler (LC) PCR hybridization assays, validated by direct sequencing, were designed to test for the presence of three nucleotide sequences corresponding to tyrosine phosphorylation motifs (TPMs) A, B, and C in 84 isolates of H. pylori type I from patients in England. Overall, the PCR assays demonstrated that one or more TPMs were present in 62 strains (75%). Motif A was common (71% of strains), whereas motifs B and C were rarer (8% of strains). Strains lacking a TPM were typically vacuolating cytotoxin genotype vacA m2. Motif A was widely distributed in relation to disease severity and was more commonly (but not significantly [P = 0.071]) associated with gastric ulcer than with duodenal ulcer (86 versus 56%). The LC hybridization assay provided a rapid means of detecting all three motifs, but RFLP analysis was more specific for TPM-A. TPMs provide novel additional strain markers for defining cagA variation, including identification of RFLP types within TPM-A. The presence of a particular TPM was not of direct diagnostic value, either singly or in combination, but the higher proportion of TPM-A strains in gastric ulcer patients merits further investigation. PMID:12843050

  7. Mobile real time radiography system

    SciTech Connect

    Vigil, J.; Taggart, D.; Betts, S.

    1997-11-01

    A 450-keV Mobile Real Time Radiography (RTR) System was delivered to Los Alamos National Laboratory (LANL) in January 1996. It was purchased to inspect containers of radioactive waste produced at (LANL). Since its delivery it has been used to radiograph more than 600 drums of radioactive waste at various LANL sites. It has the capability of inspecting waste containers of various sizes from <1-gal. buckets up to standard waste boxes (SWB, dimensions 54.5 in. x 71 in. x 37 in.). It has three independent x-ray acquisition formats. The primary system used is a 12- in. image intensifier, the second is a 36-in. linear diode array (LDA) and the last is an open system. It is fully self contained with on board generator, HVAC, and a fire suppression system. It is on a 53-ft long x 8-ft. wide x 14-ft. high trailer that can be moved over any highway requiring only an easily obtainable overweight permit because it weights {approximately}38 tons. It was built to conform to industry standards for a cabinet system which does not require an exclusion zone. The fact that this unit is mobile has allowed us to operate where the waste is stored, rather than having to move the waste to a fixed facility.

  8. Combined assay for two-hour identification of Streptococcus pneumoniae and Neisseria meningitidis and concomitant detection of 16S ribosomal DNA in cerebrospinal fluid by real-time PCR.

    PubMed

    Deutch, Susanna; Møller, Jens K; Ostergaard, Lars

    2008-01-01

    The main object was to examine the diagnostic performance of a novel combination of a specific real-time PCR (combined real-time PCR) for immediate and simultaneous detection of Streptococcus pneumoniae and Neisseria meningitidis and of a real-time PCR of the 16S rRNA gene (16S DNA). During 12 months, 1015 routine CSF samples were consecutively collected from patients in the County of Aarhus, Denmark. The samples were cultured, examined by microscopy, and, in parallel, CSF DNA was automatically purified and subjected to real-time PCR. Melting curve analysis discriminated between the 2 specific pathogens and 16S DNA positive samples were sequenced. Clinical data were extracted from patients having positive samples. Clinically, 35 of 46 (76%) patients with positive samples had bacterial meningitis. 18 of these 35 patients had a concomitant culture and real-time PCR-positive sample. The remaining 17 patients were either culture positive (n =7) or real-time PCR-positive (n = 10). The aetiology of bacterial meningitis was revealed by microscopy in 18/35 (51.4%), culture in 24/35 (68.6%) and combined real-time PCR in 27/35 (77.1%) patients, respectively. In conclusion, the combined real-time PCR strategy is superior to microscopy and a valuable supplement to routine culture to establish the aetiology of bacterial meningitis.

  9. Real-Time Nonlinear Optical Information Processing.

    DTIC Science & Technology

    1979-06-01

    operations aree presented. One approach realizes the halftone method of nonlinear optical processing in real time by replacing the conventional...photographic recording medium with a real-time image transducer. In the second approach halftoning is eliminated and the real-time device is used directly

  10. Students Collecting Real time Data

    NASA Astrophysics Data System (ADS)

    Miller, P.

    2006-05-01

    Students Collecting Real-Time Data The Hawaiian Islands Humpback Whale National Marine Sanctuary has created opportunities for middle and high school students to become Student Researchers and to be involved in real-time marine data collection. It is important that we expose students to different fields of science and encourage them to enter scientific fields of study. The Humpback Whale Sanctuary has an education visitor center in Kihei, Maui. Located right on the beach, the site has become a living classroom facility. There is a traditional Hawaiian fishpond fronting the property. The fishpond wall is being restored, using traditional methods. The site has the incredible opportunity of incorporating Hawaiian cultural practices with scientific studies. The Sanctuary offers opportunities for students to get involved in monitoring and data collection studies. Invasive Seaweed Study: Students are collecting data on invasive seaweed for the University of Hawaii. They pull a large net through the shallow waters. Seaweed is sorted, identified and weighed. The invasive seaweeds are removed. The data is recorded and sent to UH. Remote controlled monitoring boats: The sanctuary has 6 boogie board sized remote controlled boats used to monitor reefs. Boats have a camera with lights on the underside. The boats have water quality monitoring devices and GPS units. The video from the underwater camera is transmitted via a wireless transmission. Students are able to monitor the fish, limu and invertebrate populations on the reef and collect water quality data via television monitors or computers. The boat can also pull a small plankton tow net. Data is being compiled into data bases. Artificial Reef Modules: The Sanctuary has a scientific permit from the state to build and deploy artificial reef modules. High school students are designing and building modules. These are deployed out in the Fishpond fronting the Sanctuary site and students are monitoring them on a weekly basis

  11. Measurement-device-independent randomness generation with arbitrary quantum states

    NASA Astrophysics Data System (ADS)

    Bischof, Felix; Kampermann, Hermann; Bruß, Dagmar

    2017-06-01

    Measurements of quantum systems can be used to generate classical data that are truly unpredictable for every observer. However, this true randomness needs to be discriminated from randomness due to ignorance or lack of control of the devices. We analyze the randomness gain of a measurement-device-independent setup, consisting of a well-characterized source of quantum states and a completely uncharacterized and untrusted detector. Our framework generalizes previous schemes as arbitrary input states and arbitrary measurements can be analyzed. Our method is used to suggest simple and realistic implementations that yield high randomness generation rates of more than one random bit per qubit for detectors of sufficient quality.

  12. Long-distance measurement-device-independent multiparty quantum communication.

    PubMed

    Fu, Yao; Yin, Hua-Lei; Chen, Teng-Yun; Chen, Zeng-Bing

    2015-03-06

    The Greenberger-Horne-Zeilinger (GHZ) entanglement, originally introduced to uncover the extreme violation of local realism against quantum mechanics, is an important resource for multiparty quantum communication tasks. But the low intensity and fragility of the GHZ entanglement source in current conditions have made the practical applications of these multiparty tasks an experimental challenge. Here we propose a feasible scheme for practically distributing the postselected GHZ entanglement over a distance of more than 100 km for experimentally accessible parameter regimes. Combining the decoy-state and measurement-device-independent protocols for quantum key distribution, we anticipate that our proposal suggests an important avenue for practical multiparty quantum communication.

  13. Long-Distance Measurement-Device-Independent Multiparty Quantum Communication

    NASA Astrophysics Data System (ADS)

    Fu, Yao; Yin, Hua-Lei; Chen, Teng-Yun; Chen, Zeng-Bing

    2015-03-01

    The Greenberger-Horne-Zeilinger (GHZ) entanglement, originally introduced to uncover the extreme violation of local realism against quantum mechanics, is an important resource for multiparty quantum communication tasks. But the low intensity and fragility of the GHZ entanglement source in current conditions have made the practical applications of these multiparty tasks an experimental challenge. Here we propose a feasible scheme for practically distributing the postselected GHZ entanglement over a distance of more than 100 km for experimentally accessible parameter regimes. Combining the decoy-state and measurement-device-independent protocols for quantum key distribution, we anticipate that our proposal suggests an important avenue for practical multiparty quantum communication.

  14. A device-independent interface for interactive image display

    NASA Technical Reports Server (NTRS)

    Perkins, D. C.; Szczur, M. R.; Owings, J.; Contractor, S.

    1984-01-01

    NASA's Goddard Space Flight Center (GSFC) has developed a Transportable Applications Executive (TAE) for use in implementing portable applications software that can be shared by different research projects. Since many of the supported disciplines require the interactive display and manipulation of remotely sensed images, a device independent Display Management Subsystem (DMS) was written as a TAE extension. The DMS attempts to abstract and standardize the device dependent functions that are used in the display and manipulation of image data on image analysis terminals. This paper explores the concept of DMS and the interface routines that are available to the applications programmer for use in developing a set of portable image display utility programs.

  15. Real-time optical tweezing

    NASA Astrophysics Data System (ADS)

    Rahman, Shah Mohammed Tamzidur

    In this thesis a new approach called ‘space-time-wavelength mapping’ has been developed for real-time electronic control of optical tweezers. The proposed technique enables precise control of optical signals in space, time, and frequency through time-domain dispersion and diffractive optics, which in turn enables generation of controlled radiation forces acting on small particles. In this study we show that 150 fs ultrafast optical pulses can be dispersed in time and space to achieve a 20 μm x 2 μm focused elliptical beam. The force field at the focal plane of the beam is dependent on local intensity gradients along the plane. The spatial intensity profile can be electronically controlled by assigning local power levels to each wavelength using time-domain RF modulation of dispersed pulses, and sending each wavelength, and hence the assigned power level, to a specific location in space through diffractive optics. We show that by choosing the appropriate RF waveform, one is able to create force fields for cell stretching and compression as well as multiple force hot-spots (of >200 pN force per pulse) for attractive and repulsive forces. A detailed theoretical model and simulation results from a proposed experimental setup are presented. This approach is significantly more advantageous in terms of flexibility and control, compared to conventional optical tweezers that require mechanical steering or holographic optical tweezers that produce undesired ‘ghost traps’. In addition, it is shown how the technique can also be extended to create tunable 2D force field distributions using a virtually-imaged phased-array (VIPA).

  16. Real-Time Data Display

    NASA Technical Reports Server (NTRS)

    Pedings, Marc

    2007-01-01

    RT-Display is a MATLAB-based data acquisition environment designed to use a variety of commercial off-the-shelf (COTS) hardware to digitize analog signals to a standard data format usable by other post-acquisition data analysis tools. This software presents the acquired data in real time using a variety of signal-processing algorithms. The acquired data is stored in a standard Operator Interactive Signal Processing Software (OISPS) data-formatted file. RT-Display is primarily configured to use the Agilent VXI (or equivalent) data acquisition boards used in such systems as MIDDAS (Multi-channel Integrated Dynamic Data Acquisition System). The software is generalized and deployable in almost any testing environment, without limitations or proprietary configuration for a specific test program or project. With the Agilent hardware configured and in place, users can start the program and, in one step, immediately begin digitizing multiple channels of data. Once the acquisition is completed, data is converted into a common binary format that also can be translated to specific formats used by external analysis software, such as OISPS and PC-Signal (product of AI Signal Research Inc.). RT-Display at the time of this reporting was certified on Agilent hardware capable of acquisition up to 196,608 samples per second. Data signals are presented to the user on-screen simultaneously for 16 channels. Each channel can be viewed individually, with a maximum capability of 160 signal channels (depending on hardware configuration). Current signal presentations include: time data, fast Fourier transforms (FFT), and power spectral density plots (PSD). Additional processing algorithms can be easily incorporated into this environment.

  17. Holin triggering in real time.

    PubMed

    White, Rebecca; Chiba, Shinobu; Pang, Ting; Dewey, Jill S; Savva, Christos G; Holzenburg, Andreas; Pogliano, Kit; Young, Ry

    2011-01-11

    During λ infections, the holin S105 accumulates harmlessly in the membrane until, at an allele-specific time, suddenly triggering to form irregular holes of unprecedented size (>300 nm), releasing the endolysin from the cytoplasm, resulting in lysis within seconds. Here we used a functional S105-GFP chimera and real-time deconvolution fluorescence microscopy to show that the S105-GFP fusion accumulated in a uniformly distributed fashion, until suddenly, within 1 min, it formed aggregates, or rafts, at the time of lethal triggering. Moreover, the isogenic fusion to a nonlethal S105 mutant remained uniformly distributed, whereas a fusion to an early-lysing mutant showed early triggering and early raft formation. Protein accumulation rates of the WT, early, and nonlethal alleles were identical. Fluorescence recovery after photobleaching (FRAP) revealed that the nonlethal mutant and untriggered WT hybrids were highly mobile in the membrane, whereas the WT raft was essentially immobile. Finally, an antiholin allele, S105(ΔTMD1)-mcherryfp, in the product of which the S105 sequence deleted for the first transmembrane domain was fused to mCherryFP. This hybrid retained full antiholin activity, in that it blocked lethal hole formation by the S105-GFP fusion, accumulated uniformly throughout the host membrane and prevented the S105-GFP protein from forming rafts. These findings suggest that phage lysis occurs when the holin reaches a critical concentration and nucleates to form rafts, analogous to the initiation of purple membrane formation after the induction of bacteriorhodopsin in halobacteria. This model for holin function may be relevant for processes in mammalian cells, including the release of nonenveloped viruses and apoptosis.

  18. Holin triggering in real time

    PubMed Central

    White, Rebecca; Chiba, Shinobu; Pang, Ting; Dewey, Jill S.; Savva, Christos G.; Holzenburg, Andreas; Pogliano, Kit; Young, Ry

    2011-01-01

    During λ infections, the holin S105 accumulates harmlessly in the membrane until, at an allele-specific time, suddenly triggering to form irregular holes of unprecedented size (>300 nm), releasing the endolysin from the cytoplasm, resulting in lysis within seconds. Here we used a functional S105–GFP chimera and real-time deconvolution fluorescence microscopy to show that the S105–GFP fusion accumulated in a uniformly distributed fashion, until suddenly, within 1 min, it formed aggregates, or rafts, at the time of lethal triggering. Moreover, the isogenic fusion to a nonlethal S105 mutant remained uniformly distributed, whereas a fusion to an early-lysing mutant showed early triggering and early raft formation. Protein accumulation rates of the WT, early, and nonlethal alleles were identical. Fluorescence recovery after photobleaching (FRAP) revealed that the nonlethal mutant and untriggered WT hybrids were highly mobile in the membrane, whereas the WT raft was essentially immobile. Finally, an antiholin allele, S105ΔTMD1–mcherryfp, in the product of which the S105 sequence deleted for the first transmembrane domain was fused to mCherryFP. This hybrid retained full antiholin activity, in that it blocked lethal hole formation by the S105–GFP fusion, accumulated uniformly throughout the host membrane and prevented the S105–GFP protein from forming rafts. These findings suggest that phage lysis occurs when the holin reaches a critical concentration and nucleates to form rafts, analogous to the initiation of purple membrane formation after the induction of bacteriorhodopsin in halobacteria. This model for holin function may be relevant for processes in mammalian cells, including the release of nonenveloped viruses and apoptosis. PMID:21187415

  19. Feasible attack on detector-device-independent quantum key distribution.

    PubMed

    Wei, Kejin; Liu, Hongwei; Ma, Haiqiang; Yang, Xiuqing; Zhang, Yong; Sun, Yongmei; Xiao, Jinghua; Ji, Yuefeng

    2017-03-27

    Recently, to bridge the gap between security of Measurement-device-independent quantum key distribution (MDI-QKD) and a high key rate, a novel protocol, the so-called detector-device-independent QKD (DDI-QKD), has been independently proposed by several groups and has attracted great interest. A higher key rate is obtained, since a single photon bell state measurement (BSM) setup is applied to DDI-QKD. Subsequently, Qi has proposed two attacks for this protocol. However, the first attack, in which Bob's BSM setup is assumed to be completely a "black box", is easily prevented by using some additional monitoring devices or by specifically characterizing the BSM. The second attack, which combines the blinding attack and the detector wavelength-dependent efficiency, is not explicitly discussed, and its feasibility is not experimentally confirmed. Here, we show that the second attack is not technically viable because of an intrinsically wavelength-dependent property of a realistic beam splitter, which is an essential component in DDI-QKD. Moreover, we propose a feasible attack that combines a well-known attack-detector blinding attack with intrinsic imperfections of single-photon detectors. The experimental measurement and proof-of-principle test results confirm that our attack can allow Eve to get a copy of quantum keys without being detected and that it is feasible with current technology.

  20. Toward Real Time Neural Net Flight Controllers

    NASA Technical Reports Server (NTRS)

    Jorgensen, C. C.; Mah, R. W.; Ross, J.; Lu, Henry, Jr. (Technical Monitor)

    1994-01-01

    NASA Ames Research Center has an ongoing program in neural network control technology targeted toward real time flight demonstrations using a modified F-15 which permits direct inner loop control of actuators, rapid switching between alternative control designs, and substitutable processors. An important part of this program is the ACTIVE flight project which is examining the feasibility of using neural networks in the design, control, and system identification of new aircraft prototypes. This paper discusses two research applications initiated with this objective in mind: utilization of neural networks for wind tunnel aircraft model identification and rapid learning algorithms for on line reconfiguration and control. The first application involves the identification of aerodynamic flight characteristics from analysis of wind tunnel test data. This identification is important in the early stages of aircraft design because complete specification of control architecture's may not be possible even though concept models at varying scales are available for aerodynamic wind tunnel testing. Testing of this type is often a long and expensive process involving measurement of aircraft lift, drag, and moment of inertia at varying angles of attack and control surface configurations. This information in turn can be used in the design of the flight control systems by applying the derived lookup tables to generate piece wise linearized controllers. Thus, reduced costs in tunnel test times and the rapid transfer of wind tunnel insights into prototype controllers becomes an important factor in more efficient generation and testing of new flight systems. NASA Ames Research Center is successfully applying modular neural networks as one way of anticipating small scale aircraft model performances prior to testing, thus reducing the number of in tunnel test hours and potentially, the number of intermediate scaled models required for estimation of surface flow effects.

  1. Real-time PCR for Strongyloides stercoralis-associated meningitis.

    PubMed

    Nadir, Eyal; Grossman, Tamar; Ciobotaro, Pnina; Attali, Malka; Barkan, Daniel; Bardenstein, Rita; Zimhony, Oren

    2016-03-01

    Four immunocompromised patients, immigrants from Ethiopia, presented with diverse clinical manifestations of meningitis associated with Strongyloides stercoralis dissemination as determined by identification of intestinal larvae. The cerebrospinal fluid of 3 patients was tested by a validated (for stool) real-time PCR for S. stercoralis and was found positive, establishing this association. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Resource-Efficient Measurement-Device-Independent Entanglement Witness

    SciTech Connect

    Verbanis, E.; Martin, A.; Rosset, D.; Lim, C. C. W.; Thew, R. T.; Zbinden, H.

    2016-05-09

    Imperfections in experimental measurement schemes can lead to falsely identifying, or over estimating, entanglement in a quantum system. A recent solution to this is to define schemes that are robust to measurement imperfections—measurement-device-independent entanglement witness (MDI-EW). This approach can be adapted to witness all entangled qubit states for a wide range of physical systems and does not depend on detection efficiencies or classical communication between devices. In this paper, we extend the theory to remove the necessity of prior knowledge about the two-qubit states to be witnessed. Moreover, we tested this model via a novel experimental implementation for MDI-EW that significantly reduces the experimental complexity. Finally, by applying it to a bipartite Werner state, we demonstrate the robustness of this approach against noise by witnessing entanglement down to an entangled state fraction close to 0.4.

  3. Resource-Efficient Measurement-Device-Independent Entanglement Witness

    DOE PAGES

    Verbanis, E.; Martin, A.; Rosset, D.; ...

    2016-05-09

    Imperfections in experimental measurement schemes can lead to falsely identifying, or over estimating, entanglement in a quantum system. A recent solution to this is to define schemes that are robust to measurement imperfections—measurement-device-independent entanglement witness (MDI-EW). This approach can be adapted to witness all entangled qubit states for a wide range of physical systems and does not depend on detection efficiencies or classical communication between devices. In this paper, we extend the theory to remove the necessity of prior knowledge about the two-qubit states to be witnessed. Moreover, we tested this model via a novel experimental implementation for MDI-EW thatmore » significantly reduces the experimental complexity. Finally, by applying it to a bipartite Werner state, we demonstrate the robustness of this approach against noise by witnessing entanglement down to an entangled state fraction close to 0.4.« less

  4. Experimental device-independent tests of classical and quantum dimensions

    NASA Astrophysics Data System (ADS)

    Ahrens, Johan; Badziag, Piotr; Cabello, Adán; Bourennane, Mohamed

    2012-08-01

    A fundamental resource in any communication and computation task is the amount of information that can be transmitted and processed. The classical information encoded in a set of states is limited by the number of distinguishable states or classical dimension dc of the set. The sets used in quantum communication and information processing contain states that are neither identical nor distinguishable, and the quantum dimension dq of the set is the dimension of the Hilbert space spanned by these states. An important challenge is to assess the (classical or quantum) dimension of a set of states in a device-independent way, that is, without referring to the internal working of the device generating the states. Here we experimentally test dimension witnesses designed to efficiently determine the minimum dimension of sets of (three or four) photonic states from the correlations originated from measurements on them, and distinguish between classical and quantum sets of states.

  5. Device-independent parallel self-testing of two singlets

    NASA Astrophysics Data System (ADS)

    Wu, Xingyao; Bancal, Jean-Daniel; McKague, Matthew; Scarani, Valerio

    2016-06-01

    Device-independent self-testing offers the possibility of certifying the quantum state and measurements, up to local isometries, using only the statistics observed by querying uncharacterized local devices. In this paper we study parallel self-testing of two maximally entangled pairs of qubits; in particular, the local tensor product structure is not assumed but derived. We prove two criteria that achieve the desired result: a double use of the Clauser-Horne-Shimony-Holt inequality and the 3 ×3 magic square game. This demonstrate that the magic square game can only be perfectly won by measuring a two-singlet state. The tolerance to noise is well within reach of state-of-the-art experiments.

  6. Experimental Measurement-Device-Independent Quantum Key Distribution

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Chen, Teng-Yun; Wang, Liu-Jun; Liang, Hao; Shentu, Guo-Liang; Wang, Jian; Cui, Ke; Yin, Hua-Lei; Liu, Nai-Le; Li, Li; Ma, Xiongfeng; Pelc, Jason S.; Fejer, M. M.; Peng, Cheng-Zhi; Zhang, Qiang; Pan, Jian-Wei

    2013-09-01

    Quantum key distribution is proven to offer unconditional security in communication between two remote users with ideal source and detection. Unfortunately, ideal devices never exist in practice and device imperfections have become the targets of various attacks. By developing up-conversion single-photon detectors with high efficiency and low noise, we faithfully demonstrate the measurement-device-independent quantum-key-distribution protocol, which is immune to all hacking strategies on detection. Meanwhile, we employ the decoy-state method to defend attacks on a nonideal source. By assuming a trusted source scenario, our practical system, which generates more than a 25 kbit secure key over a 50 km fiber link, serves as a stepping stone in the quest for unconditionally secure communications with realistic devices.

  7. Measurement-device-independent quantum communication with an untrusted source

    NASA Astrophysics Data System (ADS)

    Xu, Feihu

    2015-07-01

    Measurement-device-independent quantum key distribution (MDI-QKD) can provide enhanced security compared to traditional QKD, and it constitutes an important framework for a quantum network with an untrusted network server. Still, a key assumption in MDI-QKD is that the sources are trusted. We propose here a MDI quantum network with a single untrusted source. We have derived a complete proof of the unconditional security of MDI-QKD with an untrusted source. Using simulations, we have considered various real-life imperfections in its implementation, and the simulation results show that MDI-QKD with an untrusted source provides a key generation rate that is close to the rate of initial MDI-QKD in the asymptotic setting. Our work proves the feasibility of the realization of a quantum network. The network users need only low-cost modulation devices, and they can share both an expensive detector and a complicated laser provided by an untrusted network server.

  8. Measurement-Device-Independent Approach to Entanglement Measures

    NASA Astrophysics Data System (ADS)

    Shahandeh, Farid; Hall, Michael J. W.; Ralph, Timothy C.

    2017-04-01

    Within the context of semiquantum nonlocal games, the trust can be removed from the measurement devices in an entanglement-detection procedure. Here, we show that a similar approach can be taken to quantify the amount of entanglement. To be specific, first, we show that in this context, a small subset of semiquantum nonlocal games is necessary and sufficient for entanglement detection in the local operations and classical communication paradigm. Second, we prove that the maximum payoff for these games is a universal measure of entanglement which is convex and continuous. Third, we show that for the quantification of negative-partial-transpose entanglement, this subset can be further reduced down to a single arbitrary element. Importantly, our measure is measurement device independent by construction and operationally accessible. Finally, our approach straightforwardly extends to quantify the entanglement within any partitioning of multipartite quantum states.

  9. Resource-Efficient Measurement-Device-Independent Entanglement Witness

    SciTech Connect

    Verbanis, E.; Martin, A.; Rosset, D.; Lim, C. C. W.; Thew, R. T.; Zbinden, H.

    2016-05-09

    Imperfections in experimental measurement schemes can lead to falsely identifying, or over estimating, entanglement in a quantum system. A recent solution to this is to define schemes that are robust to measurement imperfections—measurement-device-independent entanglement witness (MDI-EW). This approach can be adapted to witness all entangled qubit states for a wide range of physical systems and does not depend on detection efficiencies or classical communication between devices. In this paper, we extend the theory to remove the necessity of prior knowledge about the two-qubit states to be witnessed. Moreover, we tested this model via a novel experimental implementation for MDI-EW that significantly reduces the experimental complexity. Finally, by applying it to a bipartite Werner state, we demonstrate the robustness of this approach against noise by witnessing entanglement down to an entangled state fraction close to 0.4.

  10. Research in Distributed Real-Time Systems

    NASA Technical Reports Server (NTRS)

    Mukkamala, R.

    1997-01-01

    This document summarizes the progress we have made on our study of issues concerning the schedulability of real-time systems. Our study has produced several results in the scalability issues of distributed real-time systems. In particular, we have used our techniques to resolve schedulability issues in distributed systems with end-to-end requirements. During the next year (1997-98), we propose to extend the current work to address the modeling and workload characterization issues in distributed real-time systems. In particular, we propose to investigate the effect of different workload models and component models on the design and the subsequent performance of distributed real-time systems.

  11. Increasing operational command and control security by the implementation of device independent quantum key distribution

    NASA Astrophysics Data System (ADS)

    Bovino, Fabio Antonio; Messina, Angelo

    2016-10-01

    In a very simplistic way, the Command and Control functions can be summarized as the need to provide the decision makers with an exhaustive, real-time, situation picture and the capability to convey their decisions down to the operational forces. This two-ways data and information flow is vital to the execution of current operations and goes far beyond the border of military operations stretching to Police and disaster recovery as well. The availability of off-the shelf technology has enabled hostile elements to endanger the security of the communication networks by violating the traditional security protocols and devices and hacking sensitive databases. In this paper an innovative approach based to implementing Device Independent Quantum Key Distribution system is presented. The use of this technology would prevent security breaches due to a stolen crypto device placed in an end-to-end communication chain. The system, operating with attenuated laser, is practical and provides the increasing of the distance between the legitimate users.

  12. Real time programming environment for Windows

    SciTech Connect

    LaBelle, D.R.

    1998-04-01

    This document provides a description of the Real Time Programming Environment (RTProE). RTProE tools allow a programmer to create soft real time projects under general, multi-purpose operating systems. The basic features necessary for real time applications are provided by RTProE, leaving the programmer free to concentrate efforts on his specific project. The current version supports Microsoft Windows{trademark} 95 and NT. The tasks of real time synchronization and communication with other programs are handled by RTProE. RTProE includes a generic method for connecting a graphical user interface (GUI) to allow real time control and interaction with the programmer`s product. Topics covered in this paper include real time performance issues, portability, details of shared memory management, code scheduling, application control, Operating System specific concerns and the use of Computer Aided Software Engineering (CASE) tools. The development of RTProE is an important step in the expansion of the real time programming community. The financial costs associated with using the system are minimal. All source code for RTProE has been made publicly available. Any person with access to a personal computer, Windows 95 or NT, and C or FORTRAN compilers can quickly enter the world of real time modeling and simulation.

  13. Novel real-time PCR assays using TaqMan minor groove binder probes for identification of fecal carriage of Streptococcus bovis/Streptococcus equinus complex from rectal swab specimens.

    PubMed

    Lopes, Paulo Guilherme Markus; Cantarelli, Vlademir Vicente; Agnes, Grasiela; Costabeber, Ane Micheli; d'Azevedo, Pedro Alves

    2014-03-01

    Real-time PCR based on the recN and gyrB genes was developed to detect four Streptococcus bovis/Streptococcus equinus complex (SBEC) subspecies from rectal swab specimens. The overall prevalence was 35.2%: Streptococcus gallolyticus subsp. gallolyticus (11.1%), S. gallolyticus subsp. pasteurianus (13%), Streptococcus infantarius subsp. coli (20.4%), and S. infantarius subsp. infantarius (11.1%). To conclude, these real-time PCR assays provide a reliable molecular method to detect SBEC pathogenic subspecies from rectal swab specimens.

  14. Security of Semi-Device-Independent Random Number Expansion Protocols.

    PubMed

    Li, Dan-Dan; Wen, Qiao-Yan; Wang, Yu-Kun; Zhou, Yu-Qian; Gao, Fei

    2015-10-27

    Semi-device-independent random number expansion (SDI-RNE) protocols require some truly random numbers to generate fresh ones, with making no assumptions on the internal working of quantum devices except for the dimension of the Hilbert space. The generated randomness is certified by non-classical correlation in the prepare-and-measure test. Until now, the analytical relations between the amount of the generated randomness and the degree of non-classical correlation, which are crucial for evaluating the security of SDI-RNE protocols, are not clear under both the ideal condition and the practical one. In the paper, first, we give the analytical relation between the above two factors under the ideal condition. As well, we derive the analytical relation under the practical conditions, where devices' behavior is not independent and identical in each round and there exists deviation in estimating the non-classical behavior of devices. Furthermore, we choose a different randomness extractor (i.e., two-universal random function) and give the security proof.

  15. Loss-tolerant measurement-device-independent quantum private queries

    NASA Astrophysics Data System (ADS)

    Zhao, Liang-Yuan; Yin, Zhen-Qiang; Chen, Wei; Qian, Yong-Jun; Zhang, Chun-Mei; Guo, Guang-Can; Han, Zheng-Fu

    2017-01-01

    Quantum private queries (QPQ) is an important cryptography protocol aiming to protect both the user’s and database’s privacy when the database is queried privately. Recently, a variety of practical QPQ protocols based on quantum key distribution (QKD) have been proposed. However, for QKD-based QPQ the user’s imperfect detectors can be subjected to some detector- side-channel attacks launched by the dishonest owner of the database. Here, we present a simple example that shows how the detector-blinding attack can damage the security of QKD-based QPQ completely. To remove all the known and unknown detector side channels, we propose a solution of measurement-device-independent QPQ (MDI-QPQ) with single- photon sources. The security of the proposed protocol has been analyzed under some typical attacks. Moreover, we prove that its security is completely loss independent. The results show that practical QPQ will remain the same degree of privacy as before even with seriously uncharacterized detectors.

  16. Security of Semi-Device-Independent Random Number Expansion Protocols

    NASA Astrophysics Data System (ADS)

    Li, Dan-Dan; Wen, Qiao-Yan; Wang, Yu-Kun; Zhou, Yu-Qian; Gao, Fei

    2015-10-01

    Semi-device-independent random number expansion (SDI-RNE) protocols require some truly random numbers to generate fresh ones, with making no assumptions on the internal working of quantum devices except for the dimension of the Hilbert space. The generated randomness is certified by non-classical correlation in the prepare-and-measure test. Until now, the analytical relations between the amount of the generated randomness and the degree of non-classical correlation, which are crucial for evaluating the security of SDI-RNE protocols, are not clear under both the ideal condition and the practical one. In the paper, first, we give the analytical relation between the above two factors under the ideal condition. As well, we derive the analytical relation under the practical conditions, where devices’ behavior is not independent and identical in each round and there exists deviation in estimating the non-classical behavior of devices. Furthermore, we choose a different randomness extractor (i.e., two-universal random function) and give the security proof.

  17. High-rate measurement-device-independent quantum cryptography

    NASA Astrophysics Data System (ADS)

    Pirandola, Stefano; Ottaviani, Carlo; Spedalieri, Gaetana; Weedbrook, Christian; Braunstein, Samuel L.; Lloyd, Seth; Gehring, Tobias; Jacobsen, Christian S.; Andersen, Ulrik L.

    2015-06-01

    Quantum cryptography achieves a formidable task—the remote distribution of secret keys by exploiting the fundamental laws of physics. Quantum cryptography is now headed towards solving the practical problem of constructing scalable and secure quantum networks. A significant step in this direction has been the introduction of measurement-device independence, where the secret key between two parties is established by the measurement of an untrusted relay. Unfortunately, although qubit-implemented protocols can reach long distances, their key rates are typically very low, unsuitable for the demands of a metropolitan network. Here we show, theoretically and experimentally, that a solution can come from the use of continuous-variable systems. We design a coherent-state network protocol able to achieve remarkably high key rates at metropolitan distances, in fact three orders of magnitude higher than those currently achieved. Our protocol could be employed to build high-rate quantum networks where devices securely connect to nearby access points or proxy servers.

  18. Efficient Device-Independent Entanglement Detection for Multipartite Systems

    NASA Astrophysics Data System (ADS)

    Baccari, F.; Cavalcanti, D.; Wittek, P.; Acín, A.

    2017-04-01

    Entanglement is one of the most studied properties of quantum mechanics for its application in quantum information protocols. Nevertheless, detecting the presence of entanglement in large multipartite states continues to be a great challenge both from the theoretical and the experimental point of view. Most of the known methods either have computational costs that scale inefficiently with the number of particles or require more information on the state than what is attainable in everyday experiments. We introduce a new technique for entanglement detection that provides several important advantages in these respects. First, it scales efficiently with the number of particles, thus allowing for application to systems composed by up to few tens of particles. Second, it needs only the knowledge of a subset of all possible measurements on the state, therefore being apt for experimental implementation. Moreover, since it is based on the detection of nonlocality, our method is device independent. We report several examples of its implementation for well-known multipartite states, showing that the introduced technique has a promising range of applications.

  19. Device-independent quantum key distribution based on measurement inputs

    NASA Astrophysics Data System (ADS)

    Rahaman, Ramij; Parker, Matthew G.; Mironowicz, Piotr; Pawłowski, Marcin

    2015-12-01

    We provide an analysis of a family of device-independent quantum key distribution (QKD) protocols that has the following features. (a) The bits used for the secret key do not come from the results of the measurements on an entangled state but from the choices of settings. (b) Instead of a single security parameter (a violation of some Bell inequality) a set of them is used to estimate the level of trust in the secrecy of the key. The main advantage of these protocols is a smaller vulnerability to imperfect random number generators made possible by feature (a). We prove the security and the robustness of such protocols. We show that using our method it is possible to construct a QKD protocol which retains its security even if the source of randomness used by communicating parties is strongly biased. As a proof of principle, an explicit example of a protocol based on the Hardy's paradox is presented. Moreover, in the noiseless case, the protocol is secure in a natural way against any type of memory attack, and thus allows one to reuse the device in subsequent rounds. We also analyze the robustness of the protocol using semidefinite programming methods. Finally, we present a postprocessing method, and observe a paradoxical property that rejecting some random part of the private data can increase the key rate of the protocol.

  20. Loss-tolerant measurement-device-independent quantum private queries

    PubMed Central

    Zhao, Liang-Yuan; Yin, Zhen-Qiang; Chen, Wei; Qian, Yong-Jun; Zhang, Chun-Mei; Guo, Guang-Can; Han, Zheng-Fu

    2017-01-01

    Quantum private queries (QPQ) is an important cryptography protocol aiming to protect both the user’s and database’s privacy when the database is queried privately. Recently, a variety of practical QPQ protocols based on quantum key distribution (QKD) have been proposed. However, for QKD-based QPQ the user’s imperfect detectors can be subjected to some detector- side-channel attacks launched by the dishonest owner of the database. Here, we present a simple example that shows how the detector-blinding attack can damage the security of QKD-based QPQ completely. To remove all the known and unknown detector side channels, we propose a solution of measurement-device-independent QPQ (MDI-QPQ) with single- photon sources. The security of the proposed protocol has been analyzed under some typical attacks. Moreover, we prove that its security is completely loss independent. The results show that practical QPQ will remain the same degree of privacy as before even with seriously uncharacterized detectors. PMID:28051101

  1. Security of Semi-Device-Independent Random Number Expansion Protocols

    PubMed Central

    Li, Dan-Dan; Wen, Qiao-Yan; Wang, Yu-Kun; Zhou, Yu-Qian; Gao, Fei

    2015-01-01

    Semi-device-independent random number expansion (SDI-RNE) protocols require some truly random numbers to generate fresh ones, with making no assumptions on the internal working of quantum devices except for the dimension of the Hilbert space. The generated randomness is certified by non-classical correlation in the prepare-and-measure test. Until now, the analytical relations between the amount of the generated randomness and the degree of non-classical correlation, which are crucial for evaluating the security of SDI-RNE protocols, are not clear under both the ideal condition and the practical one. In the paper, first, we give the analytical relation between the above two factors under the ideal condition. As well, we derive the analytical relation under the practical conditions, where devices’ behavior is not independent and identical in each round and there exists deviation in estimating the non-classical behavior of devices. Furthermore, we choose a different randomness extractor (i.e., two-universal random function) and give the security proof. PMID:26503335

  2. Waste collection multi objective model with real time traceability data.

    PubMed

    Faccio, Maurizio; Persona, Alessandro; Zanin, Giorgia

    2011-12-01

    Waste collection is a highly visible municipal service that involves large expenditures and difficult operational problems, plus it is expensive to operate in terms of investment costs (i.e. vehicles fleet), operational costs (i.e. fuel, maintenances) and environmental costs (i.e. emissions, noise and traffic congestions). Modern traceability devices, like volumetric sensors, identification RFID (Radio Frequency Identification) systems, GPRS (General Packet Radio Service) and GPS (Global Positioning System) technology, permit to obtain data in real time, which is fundamental to implement an efficient and innovative waste collection routing model. The basic idea is that knowing the real time data of each vehicle and the real time replenishment level at each bin makes it possible to decide, in function of the waste generation pattern, what bin should be emptied and what should not, optimizing different aspects like the total covered distance, the necessary number of vehicles and the environmental impact. This paper describes a framework about the traceability technology available in the optimization of solid waste collection, and introduces an innovative vehicle routing model integrated with the real time traceability data, starting the application in an Italian city of about 100,000 inhabitants. The model is tested and validated using simulation and an economical feasibility study is reported at the end of the paper. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. The ALMA Real Time Control System

    NASA Astrophysics Data System (ADS)

    Kern, Jeffrey S.; Juerges, Thomas A.; Marson, Ralph G.

    2009-01-01

    The Atacama Large Millimeter Array (ALMA) is a revolutionary millimeter and submillimeter array being developed on the Atacama plateau of northern Chile. An international partnership lead by NRAO, ESO, and NAOJ this powerful and flexible telescope will provide unprecedented observations of this relatively unexplored frequency range. The control subsystem for the Atacama Large Millimeter Array must coordinate the monitor and control of at least sixty six antennas (in four different styles), two correlators, and all of the ancillary equipment (samplers, local oscillators, front ends, etc.). This equipment will be spread over tens of kilometers and operated remotely. Operation of the array requires a robust, scalable, and maintainable real time control system. The real time control system is responsible for monitoring and control of any devices where there are fixed deadlines. Examples in the ALMA context are antenna pointing and fringe tracking. Traditionally the real time portion of a large software system is an intricate and error prone portion of the software. As a result the real time portion is very expensive in terms of effort expended both during construction and during maintenance phases of a project. The ALMA real time control system uses a Linux based real time operating system to interact with the hardware and the CORBA based ALMA Common Software to communicate in the distributed computing environment. Mixing the requirements of real time computing and the non-deterministic CORBA middleware has produced an interesting design. We discuss the architecture, design, and implementation of the ALMA real time control system. Highlight some lessons learned along the way, and justify our assertion that this should be the last large scale real time control system in radio astronomy.

  4. Real-time medical applications and telecommunications.

    PubMed

    Stravs, M

    1999-01-01

    Telecommunications play an important role in telemedicine. Many forms of telecommunication services based on different telecommunication technologies are developed for various needs. The paper deals with complex real-time applications which demand high telecommunication requirements. At the beginning, medical applications are categorised and real-time applications qualified as multimedia applications. Requirements for multimedia elements are listed separately. Later on, short introduction of related telecommunication protocols is given. Real-time medical applications can show their ability in case of guaranteed quality of services delivered by telecommunication network as it is explained in the end.

  5. Achieving real-time performance in FIESTA

    NASA Technical Reports Server (NTRS)

    Wilkinson, William; Happell, Nadine; Miksell, Steve; Quillin, Robert; Carlisle, Candace

    1988-01-01

    The Fault Isolation Expert System for TDRSS Applications (FIESTA) is targeted for operation in a real-time online environment. Initial stages of the prototype development concentrated on acquisition and representation of the knowledge necessary to isolate faults in the TDRSS Network. Recent efforts focused on achieving real-time performance including: a discussion of the meaning of FIESTA real-time requirements, determination of performance levels (benchmarking) and techniques for optimization. Optimization techniques presented include redesign of critical relations, filtering of redundant data and optimization of patterns used in rules. Results are summarized.

  6. Real Time Computer Graphics From Body Motion

    NASA Astrophysics Data System (ADS)

    Fisher, Scott; Marion, Ann

    1983-10-01

    This paper focuses on the recent emergence and development of real, time, computer-aided body tracking technologies and their use in combination with various computer graphics imaging techniques. The convergence of these, technologies in our research results, in an interactive display environment. in which multipde, representations of a given body motion can be displayed in real time. Specific reference, to entertainment applications is described in the development of a real time, interactive stage set in which dancers can 'draw' with their bodies as they move, through the space. of the stage or manipulate virtual elements of the set with their gestures.

  7. Real time sensor for therapeutic radiation delivery

    DOEpatents

    Bliss, M.; Craig, R.A.; Reeder, P.L.

    1998-01-06

    The invention is a real time sensor for therapeutic radiation. A probe is placed in or near the patient that senses in real time the dose at the location of the probe. The strength of the dose is determined by either an insertion or an exit probe. The location is determined by a series of vertical and horizontal sensing elements that gives the operator a real time read out dose location relative to placement of the patient. The increased accuracy prevents serious tissue damage to the patient by preventing overdose or delivery of a dose to a wrong location within the body. 14 figs.

  8. Real-time monitoring of landslides

    USGS Publications Warehouse

    Reid, Mark E.; LaHusen, Richard G.; Baum, Rex L.; Kean, Jason W.; Schulz, William H.; Highland, Lynn M.

    2012-01-01

    Landslides cause fatalities and property damage throughout the Nation. To reduce the impact from hazardous landslides, the U.S. Geological Survey develops and uses real-time and near-real-time landslide monitoring systems. Monitoring can detect when hillslopes are primed for sliding and can provide early indications of rapid, catastrophic movement. Continuous information from up-to-the-minute or real-time monitoring provides prompt notification of landslide activity, advances our understanding of landslide behavior, and enables more effective engineering and planning efforts.

  9. Real time sensor for therapeutic radiation delivery

    DOEpatents

    Bliss, Mary; Craig, Richard A.; Reeder, Paul L.

    1998-01-01

    The invention is a real time sensor for therapeutic radiation. A probe is placed in or near the patient that senses in real time the dose at the location of the probe. The strength of the dose is determined by either an insertion or an exit probe. The location is determined by a series of vertical and horizontal sensing elements that gives the operator a real time read out dose location relative to placement of the patient. The increased accuracy prevents serious tissue damage to the patient by preventing overdose or delivery of a dose to a wrong location within the body.

  10. Achieving real-time performance in FIESTA

    NASA Technical Reports Server (NTRS)

    Wilkinson, William; Happell, Nadine; Miksell, Steve; Quillin, Robert; Carlisle, Candace

    1988-01-01

    The Fault Isolation Expert System for TDRSS Applications (FIESTA) is targeted for operation in a real-time online environment. Initial stages of the prototype development concentrated on acquisition and representation of the knowledge necessary to isolate faults in the TDRSS Network. Recent efforts focused on achieving real-time performance including: a discussion of the meaning of FIESTA real-time requirements, determination of performance levels (benchmarking) and techniques for optimization. Optimization techniques presented include redesign of critical relations, filtering of redundant data and optimization of patterns used in rules. Results are summarized.

  11. Optical Coherence Tomography (OCT) Device Independent Intraretinal Layer Segmentation

    PubMed Central

    Ehnes, Alexander; Wenner, Yaroslava; Friedburg, Christoph; Preising, Markus N.; Bowl, Wadim; Sekundo, Walter; zu Bexten, Erdmuthe Meyer; Stieger, Knut; Lorenz, Birgit

    2014-01-01

    Purpose To develop and test an algorithm to segment intraretinal layers irrespectively of the actual Optical Coherence Tomography (OCT) device used. Methods The developed algorithm is based on the graph theory optimization. The algorithm's performance was evaluated against that of three expert graders for unsigned boundary position difference and thickness measurement of a retinal layer group in 50 and 41 B-scans, respectively. Reproducibility of the algorithm was tested in 30 C-scans of 10 healthy subjects each with the Spectralis and the Stratus OCT. Comparability between different devices was evaluated in 84 C-scans (volume or radial scans) obtained from 21 healthy subjects, two scans per subject with the Spectralis OCT, and one scan per subject each with the Stratus OCT and the RTVue-100 OCT. Each C-scan was segmented and the mean thickness for each retinal layer in sections of the early treatment of diabetic retinopathy study (ETDRS) grid was measured. Results The algorithm was able to segment up to 11 intraretinal layers. Measurements with the algorithm were within the 95% confidence interval of a single grader and the difference was smaller than the interindividual difference between the expert graders themselves. The cross-device examination of ETDRS-grid related layer thicknesses highly agreed between the three OCT devices. The algorithm correctly segmented a C-scan of a patient with X-linked retinitis pigmentosa. Conclusions The segmentation software provides device-independent, reliable, and reproducible analysis of intraretinal layers, similar to what is obtained from expert graders. Translational Relevance Potential application of the software includes routine clinical practice and multicenter clinical trials. PMID:24820053

  12. Shared randomness and device-independent dimension witnessing

    NASA Astrophysics Data System (ADS)

    de Vicente, Julio I.

    2017-01-01

    It has been shown that the conditional probability distributions obtained by performing measurements on an uncharacterized physical system can be used to infer its underlying dimension in a device-independent way both in the classical and the quantum setting. We analyze several aspects of the structure of the sets of probability distributions corresponding to a certain dimension, taking into account whether shared randomness is available as a resource. We first consider the so-called prepare-and-measure scenario. We show that quantumness and shared randomness are not comparable resources. That is, on the one hand there exist behaviors that require a quantum system of arbitrarily large dimension in order to be observed while they can be reproduced with a classical physical system of minimal dimension together with shared randomness. On the other hand, there exist behaviors that require exponentially larger dimensions classically than quantumly even if the former is supplemented with shared randomness. We also show that in the absence of shared randomness, the sets corresponding to a sufficiently small dimension are negligible (zero measure and nowhere dense) both classically and quantumly. This is in sharp contrast to the situation in which this resource is available, and it explains the exceptional robustness of dimension witnesses in the setting in which devices can be taken to be uncorrelated. We finally consider the Bell scenario in the absence of shared randomness, and we prove some nonconvexity and negligibility properties of these sets for sufficiently small dimensions. This shows again the enormous difference induced by the availability (or lack thereof) of this resource.

  13. Interferometer real time control development for SIM

    NASA Astrophysics Data System (ADS)

    Bell, Charles E.

    2003-02-01

    Real Time Control (RTC) for the Space Interferometry Mission will build on the real time core interferometer control technology under development at JPL since the mid 1990s, with heritage from the ground based MKII and Palomar Testbed Interferometer projects developed in the late '80s and early '90s. The core software and electronics technology for SIM interferometer real time control is successfully operating on several SIM technology demonstration testbeds, including the Real-time Interferometer Control System Testbed, System Testbed-3, and the Microarcsecond Metrology testbed. This paper provides an overview of the architecture, design, integration, and test of the SIM flight interferometer real time control to meet challenging flight system requirements for the high processor throughput, low-latency interconnect, and precise synchronization to support microarcsecond-level astrometric measurements for greater than five years at 1 AU in Earth-trailing orbit. The electronics and software architecture of the interferometer real time control core and its adaptation to a flight design concept are described. Control loops for pointing and pathlength control within each of four flight interferometers and for coordination of control and data across interferometers are illustrated. The nature of onboard data processing to fit average downlink rates while retaining post-processed astrometric measurement precision and accuracy is also addressed. Interferometer flight software will be developed using a software simulation environment incorporating models of the metrology and starlight sensors and actuators to close the real time control loops. RTC flight software and instrument flight electronics will in turn be integrated utilizing the same simulation architecture for metrology and starlight component models to close real time control loops and verify RTC functionality and performance prior to delivery to flight interferometer system integration at Lockheed Martin

  14. Real-Time Ada Problem Study

    DTIC Science & Technology

    1989-03-24

    define this set of problems. The authors were chosen because of their proven expertise in real-time development with Ada. They could enrich the results of...for Real-Time Embedded Systems". LabTek Corporation, the author , had proven expertise in embedded system design utilizing Motorola MC680XO- based...processors. The second report is entitledSoftware Enineering Problems Using Ada in Computers Integral to Weapons Systems. Its author , Sonicraft, had

  15. Real-time scheduling using minimum search

    NASA Technical Reports Server (NTRS)

    Tadepalli, Prasad; Joshi, Varad

    1992-01-01

    In this paper we consider a simple model of real-time scheduling. We present a real-time scheduling system called RTS which is based on Korf's Minimin algorithm. Experimental results show that the schedule quality initially improves with the amount of look-ahead search and tapers off quickly. So it sppears that reasonably good schedules can be produced with a relatively shallow search.

  16. Real-time interferometric synthetic aperture microscopy

    PubMed Central

    Ralston, Tyler S.; Marks, Daniel L.; Carney, P. Scott; Boppart, Stephen A.

    2010-01-01

    An interferometric synthetic aperture microscopy (ISAM) system design with real-time 2D cross-sectional processing is described in detail. The system can acquire, process, and display the ISAM reconstructed images at frame rates of 2.25 frames per second for 512 × 1024 pixel images. This system provides quantitatively meaningful structural information from previously indistinguishable scattering intensities and provides proof of feasibility for future real-time ISAM systems. PMID:18542337

  17. The LAA real-time benchmarks

    SciTech Connect

    Block, R.K.; Krischer, W.; Lone, S.

    1989-04-01

    In the context of the LAA detector development program a subgroup Real Time Data Processing has tackled the problem of intelligent triggering. The main goal of this group is to show how fast digital devices, implemented as custom-made or commercial processors, can execute some basic algorithms, and how they can be embedded in the data flow between detector readout components and fully programmable commercial processors, which are expected to be the final data processing filter in real time.

  18. Real Time Cockpit Resource Management (CRM) Training

    DTIC Science & Technology

    2010-10-01

    i AFRL-RH-AZ-TR-2011-0005 Real Time Cockpit Resource Management ( CRM ) Training David Kaiser Jeffery Eberhart Chris Butler Gregg...Resource Management ( CRM ) Training 5a. CONTRACT NUMBER FA8650-08-C-6848 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Kaiser, David...283 Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std. Z39-18 Real Time Cockpit Resource Management ( CRM ) Training 4 Table of

  19. Development and validation of a TaqMan real-time PCR assay for the identification and quantification of roe deer (Capreolus capreolus) in food to detect food adulteration.

    PubMed

    Druml, Barbara; Mayer, Walter; Cichna-Markl, Margit; Hochegger, Rupert

    2015-07-01

    In order to protect the consumer from meat adulteration it is necessary to identify and quantify the meat content in foodstuffs. Game meat is particularly susceptible for fraudulent labeling since it is more valuable than meat from domestic animals. The paper presents a TaqMan real-time PCR assay for the quantitative determination of roe deer in meat products. The assay developed does not show cross-reactivity with 23 animal and 43 plant species tested and is therefore specific for roe deer. The amplification efficiency determined by analyzing serially diluted roe deer DNA extracts was found to be 93.9%. For quantifying the roe deer content in % (w/w), a reference system based on the myostatin gene was used. The quantification strategy was validated by determining the roe deer content in model meat mixtures and a model sausage. In addition, the real-time PCR assay was applied to the analysis of commercially available meat products.

  20. Real-time {sup 90}Sr Counter

    SciTech Connect

    Kaneko, Naomi; Kawai, Hideyuki; Kodama, Satoshi; Kobayashi, Atsushi; Tabata, Makoto; Ito, Hiroshi; Han, Soorim

    2015-07-01

    Radioisotopes have been emitted around Japan due to a nuclear accident at the Fukushima Daiichi nuclear power station in March 2011. A problem is the contaminated water including the atomic nucleus which relatively has a long half- life time and soluble such as {sup 90}Sr, {sup 137}Cs. Internal exposures by {sup 90}Sr are more dangerous than {sup 137}Cs's because Sr has effective half-life time of 18 years and property of accumulation in a born. We have developed real-time {sup 90}Sr counter which is sensitive beta-ray of maximum kinematic energy of 2.28 MeV from {sup 90}Sr and insensitive of beta-ray of maximum kinematic energy of 1.17 MeV and gamma-ray from {sup 90}Sr by Cherenkov detection. This counter composes of Cerenkov counter, trigger scintillation counter and veto counter. Silica aerogel for Cherenkov counter can obtain refractive index between 1.017 and 1.049 easily. And wavelength shifting fiber (WLSF) is used as a light guide for extending effective area and producing lower cost. A mechanism of the identification of {sup 90}Sr is explained in following. In case of {sup 90}Sr, when the trigger counter reacts on the beta-ray from {sup 90}Sr, aerogel emits the Cherenkov light and WLSF reacts and read the Cherenkov light. On the other hand, in case of {sup 137}Cs, the trigger counter reacts on the beta-ray, aerogel stops the beta- ray and Cherenkov light is not emitted. Therefore, aerogel has a function as a radiator and shielding material. the gamma-ray is not reacted on the lower density detector. Cosmic rays would be also reacted by the veto counter. A prototype counter whose the effective area is 30 cm x 10 cm was obtained (2.0±1.2){sup 3} of mis-identification as {sup 137}Cs/{sup 90}Sr. Detection limit in the surface contamination inspection depends on measurement time and effective area mainly. The sensitivity of wide range, 10{sup -2} - 10{sup 4} Bq/cm{sup 2}, is obtained by adjustment of detection level in circuit of this counter. A lower

  1. REAL TIME SYSTEM OPERATIONS 2006-2007

    SciTech Connect

    Eto, Joseph H.; Parashar, Manu; Lewis, Nancy Jo

    2008-08-15

    The Real Time System Operations (RTSO) 2006-2007 project focused on two parallel technical tasks: (1) Real-Time Applications of Phasors for Monitoring, Alarming and Control; and (2) Real-Time Voltage Security Assessment (RTVSA) Prototype Tool. The overall goal of the phasor applications project was to accelerate adoption and foster greater use of new, more accurate, time-synchronized phasor measurements by conducting research and prototyping applications on California ISO's phasor platform - Real-Time Dynamics Monitoring System (RTDMS) -- that provide previously unavailable information on the dynamic stability of the grid. Feasibility assessment studies were conducted on potential application of this technology for small-signal stability monitoring, validating/improving existing stability nomograms, conducting frequency response analysis, and obtaining real-time sensitivity information on key metrics to assess grid stress. Based on study findings, prototype applications for real-time visualization and alarming, small-signal stability monitoring, measurement based sensitivity analysis and frequency response assessment were developed, factory- and field-tested at the California ISO and at BPA. The goal of the RTVSA project was to provide California ISO with a prototype voltage security assessment tool that runs in real time within California ISO?s new reliability and congestion management system. CERTS conducted a technical assessment of appropriate algorithms, developed a prototype incorporating state-of-art algorithms (such as the continuation power flow, direct method, boundary orbiting method, and hyperplanes) into a framework most suitable for an operations environment. Based on study findings, a functional specification was prepared, which the California ISO has since used to procure a production-quality tool that is now a part of a suite of advanced computational tools that is used by California ISO for reliability and congestion management.

  2. Hard Real-Time: C++ Versus RTSJ

    NASA Technical Reports Server (NTRS)

    Dvorak, Daniel L.; Reinholtz, William K.

    2004-01-01

    In the domain of hard real-time systems, which language is better: C++ or the Real-Time Specification for Java (RTSJ)? Although ordinary Java provides a more productive programming environment than C++ due to its automatic memory management, that benefit does not apply to RTSJ when using NoHeapRealtimeThread and non-heap memory areas. As a result, RTSJ programmers must manage non-heap memory explicitly. While that's not a deterrent for veteran real-time programmers-where explicit memory management is common-the lack of certain language features in RTSJ (and Java) makes that manual memory management harder to accomplish safely than in C++. This paper illustrates the problem for practitioners in the context of moving data and managing memory in a real-time producer/consumer pattern. The relative ease of implementation and safety of the C++ programming model suggests that RTSJ has a struggle ahead in the domain of hard real-time applications, despite its other attractive features.

  3. Visualization of Real-Time Data

    NASA Technical Reports Server (NTRS)

    Stansifer, Ryan; Engrand, Peter

    1996-01-01

    In this project we explored various approaches to presenting real-time data from the numerous systems monitored on the space shuttle to computer users. We examined the approach that several projects at the Kennedy Space Center (KSC) used to accomplish this. We undertook to build a prototype system to demonstrate that the Internet and the Java programming language could be used to present the real-time data conveniently. Several Java programs were developed that presented real-time data in different forms including one form that emulated the display screens of the PC GOAL system which is familiar to many at KSC. Also, we developed several communications programs to supply the data continuously. Furthermore, a framework was created using the World Wide Web (WWW) to organize the collection and presentation of the real-time data. We believe our demonstration project shows the great flexibility of the approach. We had no particular use of the data in mind, instead we wanted the most general and the least complex framework possible. People who wish to view data need only know how to use a WWW browser and the address (the URL). People wanting to build WWW documents containing real-time data need only know the values of a few parameters, they do not need to program in Java or any other language. These are stunning advantages over more monolithic systems.

  4. Personnel real time dosimetry in interventional radiology.

    PubMed

    Servoli, L; Bissi, L; Fabiani, S; Magalotti, D; Placidi, P; Scorzoni, A; Calandra, A; Cicioni, R; Chiocchini, S; Dipilato, A C; Forini, N; Paolucci, M; Di Lorenzo, R; Cappotto, F P; Scarpignato, M; Maselli, A; Pentiricci, A

    2016-12-01

    Interventional radiology and hemodynamic procedures have rapidly grown in number in the past decade, increasing the importance of personnel dosimetry not only for patients but also for medical staff. The optimization of the absorbed dose during operations is one of the goals that fostered the development of real-time dosimetric systems. Indeed, introducing proper procedure optimization, like correlating dose rate measurements with medical staff position inside the operating room, the absorbed dose could be reduced. Real-time dose measurements would greatly facilitate this task through real-time monitoring and automatic data recording. Besides real-time dose monitoring could allow automatic data recording. In this work, we will describe the calibration and validation of a wireless real-time prototype dosimeter based on a new sensor device (CMOS imager). The validation measurement campaign in clinical conditions has demonstrated the prototype capability of measuring dose-rates with a frequency in the range of few Hz, and an uncertainty smaller than 10%. Copyright © 2016 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.

  5. Real-time Enhanced Vision System

    NASA Technical Reports Server (NTRS)

    Hines, Glenn D.; Rahman, Zia-Ur; Jobson, Daniel J.; Woodell, Glenn A.; Harrah, Steven D.

    2005-01-01

    Flying in poor visibility conditions, such as rain, snow, fog or haze, is inherently dangerous. However these conditions can occur at nearly any location, so inevitably pilots must successfully navigate through them. At NASA Langley Research Center (LaRC), under support of the Aviation Safety and Security Program Office and the Systems Engineering Directorate, we are developing an Enhanced Vision System (EVS) that combines image enhancement and synthetic vision elements to assist pilots flying through adverse weather conditions. This system uses a combination of forward-looking infrared and visible sensors for data acquisition. A core function of the system is to enhance and fuse the sensor data in order to increase the information content and quality of the captured imagery. These operations must be performed in real-time for the pilot to use while flying. For image enhancement, we are using the LaRC patented Retinex algorithm since it performs exceptionally well for improving low-contrast range imagery typically seen during poor visibility conditions. In general, real-time operation of the Retinex requires specialized hardware. To date, we have successfully implemented a single-sensor real-time version of the Retinex on several different Digital Signal Processor (DSP) platforms. In this paper we give an overview of the EVS and its performance requirements for real-time enhancement and fusion and we discuss our current real-time Retinex implementations on DSPs.

  6. Characterization of real-time computers

    NASA Technical Reports Server (NTRS)

    Shin, K. G.; Krishna, C. M.

    1984-01-01

    A real-time system consists of a computer controller and controlled processes. Despite the synergistic relationship between these two components, they have been traditionally designed and analyzed independently of and separately from each other; namely, computer controllers by computer scientists/engineers and controlled processes by control scientists. As a remedy for this problem, in this report real-time computers are characterized by performance measures based on computer controller response time that are: (1) congruent to the real-time applications, (2) able to offer an objective comparison of rival computer systems, and (3) experimentally measurable/determinable. These measures, unlike others, provide the real-time computer controller with a natural link to controlled processes. In order to demonstrate their utility and power, these measures are first determined for example controlled processes on the basis of control performance functionals. They are then used for two important real-time multiprocessor design applications - the number-power tradeoff and fault-masking and synchronization.

  7. Real-Time Visualization of Tissue Ischemia

    NASA Technical Reports Server (NTRS)

    Bearman, Gregory H. (Inventor); Chrien, Thomas D. (Inventor); Eastwood, Michael L. (Inventor)

    2000-01-01

    A real-time display of tissue ischemia which comprises three CCD video cameras, each with a narrow bandwidth filter at the correct wavelength is discussed. The cameras simultaneously view an area of tissue suspected of having ischemic areas through beamsplitters. The output from each camera is adjusted to give the correct signal intensity for combining with, the others into an image for display. If necessary a digital signal processor (DSP) can implement algorithms for image enhancement prior to display. Current DSP engines are fast enough to give real-time display. Measurement at three, wavelengths, combined into a real-time Red-Green-Blue (RGB) video display with a digital signal processing (DSP) board to implement image algorithms, provides direct visualization of ischemic areas.

  8. Real-time inspection by submarine images

    NASA Astrophysics Data System (ADS)

    Tascini, Guido; Zingaretti, Primo; Conte, Giuseppe

    1996-10-01

    A real-time application of computer vision concerning tracking and inspection of a submarine pipeline is described. The objective is to develop automatic procedures for supporting human operators in the real-time analysis of images acquired by means of cameras mounted on underwater remotely operated vehicles (ROV) Implementation of such procedures gives rise to a human-machine system for underwater pipeline inspection that can automatically detect and signal the presence of the pipe, of its structural or accessory elements, and of dangerous or alien objects in its neighborhood. The possibility of modifying the image acquisition rate in the simulations performed on video- recorded images is used to prove that the system performs all necessary processing with an acceptable robustness working in real-time up to a speed of about 2.5 kn, widely greater than that the actual ROVs and the security features allow.

  9. Network protocols for real-time applications

    NASA Technical Reports Server (NTRS)

    Johnson, Marjory J.

    1987-01-01

    The Fiber Distributed Data Interface (FDDI) and the SAE AE-9B High Speed Ring Bus (HSRB) are emerging standards for high-performance token ring local area networks. FDDI was designed to be a general-purpose high-performance network. HSRB was designed specifically for military real-time applications. A workshop was conducted at NASA Ames Research Center in January, 1987 to compare and contrast these protocols with respect to their ability to support real-time applications. This report summarizes workshop presentations and includes an independent comparison of the two protocols. A conclusion reached at the workshop was that current protocols for the upper layers of the Open Systems Interconnection (OSI) network model are inadequate for real-time applications.

  10. Continuous, real time microwave plasma element sensor

    DOEpatents

    Woskov, P.P.; Smatlak, D.L.; Cohn, D.R.; Wittle, J.K.; Titus, C.H.; Surma, J.E.

    1995-12-26

    Microwave-induced plasma is described 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. 3 figs.

  11. Continuous, real time microwave plasma element sensor

    DOEpatents

    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.

  12. Real-time, high frequency QRS electrocardiograph

    NASA Technical Reports Server (NTRS)

    Schlegel, Todd T. (Inventor); DePalma, Jude L. (Inventor); Moradi, Saeed (Inventor)

    2006-01-01

    Real time cardiac electrical data are received from a patient, manipulated to determine various useful aspects of the ECG signal, and displayed in real time in a useful form on a computer screen or monitor. The monitor displays the high frequency data from the QRS complex in units of microvolts, juxtaposed with a display of conventional ECG data in units of millivolts or microvolts. The high frequency data are analyzed for their root mean square (RMS) voltage values and the discrete RMS values and related parameters are displayed in real time. The high frequency data from the QRS complex are analyzed with imbedded algorithms to determine the presence or absence of reduced amplitude zones, referred to herein as RAZs. RAZs are displayed as go, no-go signals on the computer monitor. The RMS and related values of the high frequency components are displayed as time varying signals, and the presence or absence of RAZs may be similarly displayed over time.

  13. Multicolor combinatorial probe coding for real-time PCR.

    PubMed

    Huang, Qiuying; Zheng, Linlin; Zhu, Yumei; Zhang, Jiafeng; Wen, Huixin; Huang, Jianwei; Niu, Jianjun; Zhao, Xilin; Li, Qingge

    2011-01-14

    The target volume of multiplex real-time PCR assays is limited by the number of fluorescent dyes available and the number of fluorescence acquisition channels present in the PCR instrument. We hereby explored a probe labeling strategy that significantly increased the target volume of real-time PCR detection in one reaction. The labeling paradigm, termed "Multicolor Combinatorial Probe Coding" (MCPC), uses a limited number (n) of differently colored fluorophores in various combinations to label each probe, enabling one of 2(n)-1 genetic targets to be detected in one reaction. The proof-of-principle of MCPC was validated by identification of one of each possible 15 human papillomavirus types, which is the maximum target number theoretically detectable by MCPC with a 4-color channel instrument, in one reaction. MCPC was then improved from a one-primer-pair setting to a multiple-primer-pair format through Homo-Tag Assisted Non-Dimer (HAND) system to allow multiple primer pairs to be included in one reaction. This improvement was demonstrated via identification of one of the possible 10 foodborne pathogen candidates with 10 pairs of primers included in one reaction, which had limit of detection equivalent to the uniplex PCR. MCPC was further explored in detecting combined genotypes of five β-globin gene mutations where multiple targets were co-amplified. MCPC strategy could expand the scope of real-time PCR assays in applications which are unachievable by current labeling strategy.

  14. NASADIG - NASA DEVICE INDEPENDENT GRAPHICS LIBRARY (AMDAHL VERSION)

    NASA Technical Reports Server (NTRS)

    Rogers, J. E.

    1994-01-01

    The NASA Device Independent Graphics Library, NASADIG, can be used with many computer-based engineering and management applications. The library gives the user the opportunity to translate data into effective graphic displays for presentation. The software offers many features which allow the user flexibility in creating graphics. These include two-dimensional plots, subplot projections in 3D-space, surface contour line plots, and surface contour color-shaded plots. Routines for three-dimensional plotting, wireframe surface plots, surface plots with hidden line removal, and surface contour line plots are provided. Other features include polar and spherical coordinate plotting, world map plotting utilizing either cylindrical equidistant or Lambert equal area projection, plot translation, plot rotation, plot blowup, splines and polynomial interpolation, area blanking control, multiple log/linear axes, legends and text control, curve thickness control, and multiple text fonts (18 regular, 4 bold). NASADIG contains several groups of subroutines. Included are subroutines for plot area and axis definition; text set-up and display; area blanking; line style set-up, interpolation, and plotting; color shading and pattern control; legend, text block, and character control; device initialization; mixed alphabets setting; and other useful functions. The usefulness of many routines is dependent on the prior definition of basic parameters. The program's control structure uses a serial-level construct with each routine restricted for activation at some prescribed level(s) of problem definition. NASADIG provides the following output device drivers: Selanar 100XL, VECTOR Move/Draw ASCII and PostScript files, Tektronix 40xx, 41xx, and 4510 Rasterizer, DEC VT-240 (4014 mode), IBM AT/PC compatible with SmartTerm 240 emulator, HP Lasergrafix Film Recorder, QMS 800/1200, DEC LN03+ Laserprinters, and HP LaserJet (Series III). NASADIG is written in FORTRAN and is available for several

  15. NASADIG - NASA DEVICE INDEPENDENT GRAPHICS LIBRARY (CRAY VERSION)

    NASA Technical Reports Server (NTRS)

    Rogers, J. E.

    1994-01-01

    The NASA Device Independent Graphics Library, NASADIG, can be used with many computer-based engineering and management applications. The library gives the user the opportunity to translate data into effective graphic displays for presentation. The software offers many features which allow the user flexibility in creating graphics. These include two-dimensional plots, subplot projections in 3D-space, surface contour line plots, and surface contour color-shaded plots. Routines for three-dimensional plotting, wireframe surface plots, surface plots with hidden line removal, and surface contour line plots are provided. Other features include polar and spherical coordinate plotting, world map plotting utilizing either cylindrical equidistant or Lambert equal area projection, plot translation, plot rotation, plot blowup, splines and polynomial interpolation, area blanking control, multiple log/linear axes, legends and text control, curve thickness control, and multiple text fonts (18 regular, 4 bold). NASADIG contains several groups of subroutines. Included are subroutines for plot area and axis definition; text set-up and display; area blanking; line style set-up, interpolation, and plotting; color shading and pattern control; legend, text block, and character control; device initialization; mixed alphabets setting; and other useful functions. The usefulness of many routines is dependent on the prior definition of basic parameters. The program's control structure uses a serial-level construct with each routine restricted for activation at some prescribed level(s) of problem definition. NASADIG provides the following output device drivers: Selanar 100XL, VECTOR Move/Draw ASCII and PostScript files, Tektronix 40xx, 41xx, and 4510 Rasterizer, DEC VT-240 (4014 mode), IBM AT/PC compatible with SmartTerm 240 emulator, HP Lasergrafix Film Recorder, QMS 800/1200, DEC LN03+ Laserprinters, and HP LaserJet (Series III). NASADIG is written in FORTRAN and is available for several

  16. NASADIG - NASA DEVICE INDEPENDENT GRAPHICS LIBRARY (UNIX VERSION)

    NASA Technical Reports Server (NTRS)

    Rogers, J. E.

    1994-01-01

    The NASA Device Independent Graphics Library, NASADIG, can be used with many computer-based engineering and management applications. The library gives the user the opportunity to translate data into effective graphic displays for presentation. The software offers many features which allow the user flexibility in creating graphics. These include two-dimensional plots, subplot projections in 3D-space, surface contour line plots, and surface contour color-shaded plots. Routines for three-dimensional plotting, wireframe surface plots, surface plots with hidden line removal, and surface contour line plots are provided. Other features include polar and spherical coordinate plotting, world map plotting utilizing either cylindrical equidistant or Lambert equal area projection, plot translation, plot rotation, plot blowup, splines and polynomial interpolation, area blanking control, multiple log/linear axes, legends and text control, curve thickness control, and multiple text fonts (18 regular, 4 bold). NASADIG contains several groups of subroutines. Included are subroutines for plot area and axis definition; text set-up and display; area blanking; line style set-up, interpolation, and plotting; color shading and pattern control; legend, text block, and character control; device initialization; mixed alphabets setting; and other useful functions. The usefulness of many routines is dependent on the prior definition of basic parameters. The program's control structure uses a serial-level construct with each routine restricted for activation at some prescribed level(s) of problem definition. NASADIG provides the following output device drivers: Selanar 100XL, VECTOR Move/Draw ASCII and PostScript files, Tektronix 40xx, 41xx, and 4510 Rasterizer, DEC VT-240 (4014 mode), IBM AT/PC compatible with SmartTerm 240 emulator, HP Lasergrafix Film Recorder, QMS 800/1200, DEC LN03+ Laserprinters, and HP LaserJet (Series III). NASADIG is written in FORTRAN and is available for several

  17. NASADIG - NASA DEVICE INDEPENDENT GRAPHICS LIBRARY (VAX VMS VERSION)

    NASA Technical Reports Server (NTRS)

    Rogers, J. E.

    1994-01-01

    The NASA Device Independent Graphics Library, NASADIG, can be used with many computer-based engineering and management applications. The library gives the user the opportunity to translate data into effective graphic displays for presentation. The software offers many features which allow the user flexibility in creating graphics. These include two-dimensional plots, subplot projections in 3D-space, surface contour line plots, and surface contour color-shaded plots. Routines for three-dimensional plotting, wireframe surface plots, surface plots with hidden line removal, and surface contour line plots are provided. Other features include polar and spherical coordinate plotting, world map plotting utilizing either cylindrical equidistant or Lambert equal area projection, plot translation, plot rotation, plot blowup, splines and polynomial interpolation, area blanking control, multiple log/linear axes, legends and text control, curve thickness control, and multiple text fonts (18 regular, 4 bold). NASADIG contains several groups of subroutines. Included are subroutines for plot area and axis definition; text set-up and display; area blanking; line style set-up, interpolation, and plotting; color shading and pattern control; legend, text block, and character control; device initialization; mixed alphabets setting; and other useful functions. The usefulness of many routines is dependent on the prior definition of basic parameters. The program's control structure uses a serial-level construct with each routine restricted for activation at some prescribed level(s) of problem definition. NASADIG provides the following output device drivers: Selanar 100XL, VECTOR Move/Draw ASCII and PostScript files, Tektronix 40xx, 41xx, and 4510 Rasterizer, DEC VT-240 (4014 mode), IBM AT/PC compatible with SmartTerm 240 emulator, HP Lasergrafix Film Recorder, QMS 800/1200, DEC LN03+ Laserprinters, and HP LaserJet (Series III). NASADIG is written in FORTRAN and is available for several

  18. NASADIG - NASA DEVICE INDEPENDENT GRAPHICS LIBRARY (VAX VMS VERSION)

    NASA Technical Reports Server (NTRS)

    Rogers, J. E.

    1994-01-01

    The NASA Device Independent Graphics Library, NASADIG, can be used with many computer-based engineering and management applications. The library gives the user the opportunity to translate data into effective graphic displays for presentation. The software offers many features which allow the user flexibility in creating graphics. These include two-dimensional plots, subplot projections in 3D-space, surface contour line plots, and surface contour color-shaded plots. Routines for three-dimensional plotting, wireframe surface plots, surface plots with hidden line removal, and surface contour line plots are provided. Other features include polar and spherical coordinate plotting, world map plotting utilizing either cylindrical equidistant or Lambert equal area projection, plot translation, plot rotation, plot blowup, splines and polynomial interpolation, area blanking control, multiple log/linear axes, legends and text control, curve thickness control, and multiple text fonts (18 regular, 4 bold). NASADIG contains several groups of subroutines. Included are subroutines for plot area and axis definition; text set-up and display; area blanking; line style set-up, interpolation, and plotting; color shading and pattern control; legend, text block, and character control; device initialization; mixed alphabets setting; and other useful functions. The usefulness of many routines is dependent on the prior definition of basic parameters. The program's control structure uses a serial-level construct with each routine restricted for activation at some prescribed level(s) of problem definition. NASADIG provides the following output device drivers: Selanar 100XL, VECTOR Move/Draw ASCII and PostScript files, Tektronix 40xx, 41xx, and 4510 Rasterizer, DEC VT-240 (4014 mode), IBM AT/PC compatible with SmartTerm 240 emulator, HP Lasergrafix Film Recorder, QMS 800/1200, DEC LN03+ Laserprinters, and HP LaserJet (Series III). NASADIG is written in FORTRAN and is available for several

  19. NASADIG - NASA DEVICE INDEPENDENT GRAPHICS LIBRARY (UNIX VERSION)

    NASA Technical Reports Server (NTRS)

    Rogers, J. E.

    1994-01-01

    The NASA Device Independent Graphics Library, NASADIG, can be used with many computer-based engineering and management applications. The library gives the user the opportunity to translate data into effective graphic displays for presentation. The software offers many features which allow the user flexibility in creating graphics. These include two-dimensional plots, subplot projections in 3D-space, surface contour line plots, and surface contour color-shaded plots. Routines for three-dimensional plotting, wireframe surface plots, surface plots with hidden line removal, and surface contour line plots are provided. Other features include polar and spherical coordinate plotting, world map plotting utilizing either cylindrical equidistant or Lambert equal area projection, plot translation, plot rotation, plot blowup, splines and polynomial interpolation, area blanking control, multiple log/linear axes, legends and text control, curve thickness control, and multiple text fonts (18 regular, 4 bold). NASADIG contains several groups of subroutines. Included are subroutines for plot area and axis definition; text set-up and display; area blanking; line style set-up, interpolation, and plotting; color shading and pattern control; legend, text block, and character control; device initialization; mixed alphabets setting; and other useful functions. The usefulness of many routines is dependent on the prior definition of basic parameters. The program's control structure uses a serial-level construct with each routine restricted for activation at some prescribed level(s) of problem definition. NASADIG provides the following output device drivers: Selanar 100XL, VECTOR Move/Draw ASCII and PostScript files, Tektronix 40xx, 41xx, and 4510 Rasterizer, DEC VT-240 (4014 mode), IBM AT/PC compatible with SmartTerm 240 emulator, HP Lasergrafix Film Recorder, QMS 800/1200, DEC LN03+ Laserprinters, and HP LaserJet (Series III). NASADIG is written in FORTRAN and is available for several

  20. NASADIG - NASA DEVICE INDEPENDENT GRAPHICS LIBRARY (CRAY VERSION)

    NASA Technical Reports Server (NTRS)

    Rogers, J. E.

    1994-01-01

    The NASA Device Independent Graphics Library, NASADIG, can be used with many computer-based engineering and management applications. The library gives the user the opportunity to translate data into effective graphic displays for presentation. The software offers many features which allow the user flexibility in creating graphics. These include two-dimensional plots, subplot projections in 3D-space, surface contour line plots, and surface contour color-shaded plots. Routines for three-dimensional plotting, wireframe surface plots, surface plots with hidden line removal, and surface contour line plots are provided. Other features include polar and spherical coordinate plotting, world map plotting utilizing either cylindrical equidistant or Lambert equal area projection, plot translation, plot rotation, plot blowup, splines and polynomial interpolation, area blanking control, multiple log/linear axes, legends and text control, curve thickness control, and multiple text fonts (18 regular, 4 bold). NASADIG contains several groups of subroutines. Included are subroutines for plot area and axis definition; text set-up and display; area blanking; line style set-up, interpolation, and plotting; color shading and pattern control; legend, text block, and character control; device initialization; mixed alphabets setting; and other useful functions. The usefulness of many routines is dependent on the prior definition of basic parameters. The program's control structure uses a serial-level construct with each routine restricted for activation at some prescribed level(s) of problem definition. NASADIG provides the following output device drivers: Selanar 100XL, VECTOR Move/Draw ASCII and PostScript files, Tektronix 40xx, 41xx, and 4510 Rasterizer, DEC VT-240 (4014 mode), IBM AT/PC compatible with SmartTerm 240 emulator, HP Lasergrafix Film Recorder, QMS 800/1200, DEC LN03+ Laserprinters, and HP LaserJet (Series III). NASADIG is written in FORTRAN and is available for several

  1. NASADIG - NASA DEVICE INDEPENDENT GRAPHICS LIBRARY (AMDAHL VERSION)

    NASA Technical Reports Server (NTRS)

    Rogers, J. E.

    1994-01-01

    The NASA Device Independent Graphics Library, NASADIG, can be used with many computer-based engineering and management applications. The library gives the user the opportunity to translate data into effective graphic displays for presentation. The software offers many features which allow the user flexibility in creating graphics. These include two-dimensional plots, subplot projections in 3D-space, surface contour line plots, and surface contour color-shaded plots. Routines for three-dimensional plotting, wireframe surface plots, surface plots with hidden line removal, and surface contour line plots are provided. Other features include polar and spherical coordinate plotting, world map plotting utilizing either cylindrical equidistant or Lambert equal area projection, plot translation, plot rotation, plot blowup, splines and polynomial interpolation, area blanking control, multiple log/linear axes, legends and text control, curve thickness control, and multiple text fonts (18 regular, 4 bold). NASADIG contains several groups of subroutines. Included are subroutines for plot area and axis definition; text set-up and display; area blanking; line style set-up, interpolation, and plotting; color shading and pattern control; legend, text block, and character control; device initialization; mixed alphabets setting; and other useful functions. The usefulness of many routines is dependent on the prior definition of basic parameters. The program's control structure uses a serial-level construct with each routine restricted for activation at some prescribed level(s) of problem definition. NASADIG provides the following output device drivers: Selanar 100XL, VECTOR Move/Draw ASCII and PostScript files, Tektronix 40xx, 41xx, and 4510 Rasterizer, DEC VT-240 (4014 mode), IBM AT/PC compatible with SmartTerm 240 emulator, HP Lasergrafix Film Recorder, QMS 800/1200, DEC LN03+ Laserprinters, and HP LaserJet (Series III). NASADIG is written in FORTRAN and is available for several

  2. Machine vision for real time orbital operations

    NASA Technical Reports Server (NTRS)

    Vinz, Frank L.

    1988-01-01

    Machine vision for automation and robotic operation of Space Station era systems has the potential for increasing the efficiency of orbital servicing, repair, assembly and docking tasks. A machine vision research project is described in which a TV camera is used for inputing visual data to a computer so that image processing may be achieved for real time control of these orbital operations. A technique has resulted from this research which reduces computer memory requirements and greatly increases typical computational speed such that it has the potential for development into a real time orbital machine vision system. This technique is called AI BOSS (Analysis of Images by Box Scan and Syntax).

  3. Automated real-time software development

    NASA Technical Reports Server (NTRS)

    Jones, Denise R.; Walker, Carrie K.; Turkovich, John J.

    1993-01-01

    A Computer-Aided Software Engineering (CASE) system has been developed at the Charles Stark Draper Laboratory (CSDL) under the direction of the NASA Langley Research Center. The CSDL CASE tool provides an automated method of generating source code and hard copy documentation from functional application engineering specifications. The goal is to significantly reduce the cost of developing and maintaining real-time scientific and engineering software while increasing system reliability. This paper describes CSDL CASE and discusses demonstrations that used the tool to automatically generate real-time application code.

  4. Axial Tomography from Digitized Real Time Radiography

    DOE R&D Accomplishments Database

    Zolnay, A. S.; McDonald, W. M.; Doupont, P. A.; McKinney, R. L.; Lee, M. M.

    1985-01-18

    Axial tomography from digitized real time radiographs provides a useful tool for industrial radiography and tomography. The components of this system are: x-ray source, image intensifier, video camera, video line extractor and digitizer, data storage and reconstruction computers. With this system it is possible to view a two dimensional x-ray image in real time at each angle of rotation and select the tomography plane of interest by choosing which video line to digitize. The digitization of a video line requires less than a second making data acquisition relatively short. Further improvements on this system are planned and initial results are reported.

  5. Software Package For Real-Time Graphics

    NASA Technical Reports Server (NTRS)

    Malone, Jacqueline C.; Moore, Archie L.

    1991-01-01

    Software package for master graphics interactive console (MAGIC) at Western Aeronautical Test Range (WATR) of NASA Ames Research Center provides general-purpose graphical display system for real-time and post-real-time analysis of data. Written in C language and intended for use on workstation of interactive raster imaging system (IRIS) equipped with level-V Unix operating system. Enables flight researchers to create their own displays on basis of individual requirements. Applicable to monitoring of complicated processes in chemical industry.

  6. Real-Time, Interactive Sonic Boom Display

    NASA Technical Reports Server (NTRS)

    Haering, Jr., Edward A. (Inventor); Plotkin, Kenneth J. (Inventor)

    2012-01-01

    The present invention is an improved real-time, interactive sonic boom display for aircraft. By using physical properties obtained via various sensors and databases, the invention determines, in real-time, sonic boom impacts locations and intensities for aircraft traveling at supersonic speeds. The information is provided to a pilot via a display that lists a selectable set of maneuvers available to the pilot to mitigate sonic boom issues. Upon selection of a maneuver, the information as to the result of the maneuver is displayed and the pilot may proceed with making the maneuver, or provide new data to the system in order to calculate a different maneuver.

  7. Imaging of living cells in real time

    NASA Astrophysics Data System (ADS)

    Tychinsky, Vladimir P.; Koufal, Georgy E.; Perevedentseva, Elena V.; Vyshenskaia, Tatiana V.; Nikandrov, Serguei L.

    1996-12-01

    Parameters of intrinsic cell motility is one of the cell activity characteristics which can be measured in real-time. For evaluation of certain organelles velocity we propose to use high sensitivity of computer-aided phase microscope airyscan to local phase changes connected with refractive index. This method is based on periodical scanning of cell profile in direction perpendicular to organelles movement. Analysis of the obtained 2-dimensional time-coordinate matrix allows us to define organelle velocity in quasi-real time and areas of cell activity. The experiments with onion cells confirm the method applicability for cell activity investigation.

  8. Robotic real-time radiographic inspection system

    SciTech Connect

    McNair, J.

    1987-01-01

    A computer-controlled real-time radiographic system with remote robotic material handling has been developed and installed at the US Army's Yuma Proving Ground. This system is used for the nondestructive examination of a variety of munition types tested at the proving ground. This paper describes the system and its capabilities. The system consists of an overhead robot for material handling, a five-axis manipulator for positioning the item being inspected, and the real-time radiographic image acquisition and analysis equipment. The system is fully automated and uses a single minicomputer as the system controller.

  9. Real-time elastography of the prostate.

    PubMed

    Junker, D; De Zordo, T; Quentin, M; Ladurner, M; Bektic, J; Horniger, W; Jaschke, W; Aigner, F

    2014-01-01

    Palpation of organs is one of the oldest clinical examination techniques, for instance, if you think of the palpation of the breast or the digital rectal examination of the prostate, where hard palpable regions are suspicious for cancer. This is the basic principle of real-time elastography, an ultrasound technique, which is able to visualise tissue elasticity. Since prostate cancer features an increased stiffness due to the higher cell and vessel density than the normal surrounding tissue, real-time elastography has been used for several years for prostate cancer detection. This review introduces the different techniques of ultrasound elastography and furthermore summarises its limitations and potentials.

  10. Real-Time Elastography of the Prostate

    PubMed Central

    Junker, D.; De Zordo, T.; Quentin, M.; Ladurner, M.; Bektic, J.; Horniger, W.; Jaschke, W.; Aigner, F.

    2014-01-01

    Palpation of organs is one of the oldest clinical examination techniques, for instance, if you think of the palpation of the breast or the digital rectal examination of the prostate, where hard palpable regions are suspicious for cancer. This is the basic principle of real-time elastography, an ultrasound technique, which is able to visualise tissue elasticity. Since prostate cancer features an increased stiffness due to the higher cell and vessel density than the normal surrounding tissue, real-time elastography has been used for several years for prostate cancer detection. This review introduces the different techniques of ultrasound elastography and furthermore summarises its limitations and potentials. PMID:24967334

  11. Quantitative Real-Time PCR: Recent Advances.

    PubMed

    Singh, Charanjeet; Roy-Chowdhuri, Sinchita

    2016-01-01

    Quantitative real-time polymerase chain reaction is a technique for simultaneous amplification and product quantification of a target DNA as the process takes place in real time in a "closed-tube" system. Although this technique can provide an absolute quantification of the initial template copy number, quantification relative to a control sample or second sequence is typically adequate. The quantification process employs melting curve analysis and/or fluorescent detection systems and can provide amplification and genotyping in a relatively short time. Here we describe the properties and uses of various fluorescent detection systems used for quantification.

  12. Software Package For Real-Time Graphics

    NASA Technical Reports Server (NTRS)

    Malone, Jacqueline C.; Moore, Archie L.

    1991-01-01

    Software package for master graphics interactive console (MAGIC) at Western Aeronautical Test Range (WATR) of NASA Ames Research Center provides general-purpose graphical display system for real-time and post-real-time analysis of data. Written in C language and intended for use on workstation of interactive raster imaging system (IRIS) equipped with level-V Unix operating system. Enables flight researchers to create their own displays on basis of individual requirements. Applicable to monitoring of complicated processes in chemical industry.

  13. Machine vision for real time orbital operations

    NASA Technical Reports Server (NTRS)

    Vinz, Frank L.

    1988-01-01

    Machine vision for automation and robotic operation of Space Station era systems has the potential for increasing the efficiency of orbital servicing, repair, assembly and docking tasks. A machine vision research project is described in which a TV camera is used for inputing visual data to a computer so that image processing may be achieved for real time control of these orbital operations. A technique has resulted from this research which reduces computer memory requirements and greatly increases typical computational speed such that it has the potential for development into a real time orbital machine vision system. This technique is called AI BOSS (Analysis of Images by Box Scan and Syntax).

  14. Real-time Shakemap implementation in Austria

    NASA Astrophysics Data System (ADS)

    Weginger, Stefan; Jia, Yan; Papi Isaba, Maria; Horn, Nikolaus

    2017-04-01

    ShakeMaps provide near-real-time maps of ground motion and shaking intensity following significant earthquakes. They are automatically generated within a few minutes after occurrence of an earthquake. We tested and included the USGS ShakeMap 4.0 (experimental code) based on python in the Antelope real-time system with local modified GMPE and Site Effects based on the conditions in Austria. The ShakeMaps are provided in terms of Intensity, PGA, PGV and PSA. Future presentation of ShakeMap contour lines and Ground Motion Parameter with interactive maps and data exchange over Web-Services are shown.

  15. Mobile waste inspection real time radiography system

    SciTech Connect

    Vigil, J.; Taggart, D.; Betts, S.; Rael, C.; Martinez, F.; Mendez, J.

    1995-10-01

    The 450-KeV Mobile Real Time Radiography System was designed and purchased to inspect containers of radioactive waste produced at Los Alamos National Laboratory (LANL). The Mobile Real Time Radiography System has the capability of inspecting waste containers of various sizes from 5-gal. buckets to standard waste boxes (SWB, dimensions 54.5 in. x 71 in. x 37 in.). The fact that this unit is mobile makes it an attractive alternative to the costly road closures associated with moving waste from the waste generator to storage or disposal facilities.

  16. Real-time ISEE data system

    NASA Technical Reports Server (NTRS)

    Tsurutani, B. T.; Baker, D. N.

    1979-01-01

    A real-time ISEE data system directed toward predicting geomagnetic substorms and storms is discussed. Such a system may allow up to 60+ minutes advance warning of magnetospheric substorms and up to 30 minute warnings of geomagnetic storms (and other disturbances) induced by high-speed streams and solar flares. The proposed system utilizes existing capabilities of several agencies (NASA, NOAA, USAF), and thereby minimizes costs. This same concept may be applicable to data from other spacecraft, and other NASA centers; thus, each individual experimenter can receive quick-look data in real time at his or her base institution.

  17. Axial tomography from digitized real time radiography

    SciTech Connect

    Zolnay, A.S.; McDonald, W.M.; Doupont, P.A.; McKinney, R.L.; Lee, M.M.

    1985-01-18

    Axial tomography from digitized real time radiographs provides a useful tool for industrial radiography and tomography. The components of this system are: x-ray source, image intensifier, video camera, video line extractor and digitizer, data storage and reconstruction computers. With this system it is possible to view a two dimensional x-ray image in real time at each angle of rotation and select the tomography plane of interest by choosing which video line to digitize. The digitization of a video line requires less than a second making data acquisition relatively short. Further improvements on this system are planned and initial results are reported.

  18. Real-Time Occupancy Change Analyzer

    SciTech Connect

    2005-03-30

    The Real-Time Occupancy Change Analyzer (ROCA) produces an occupancy grid map of an environment around the robot, scans the environment to generate a current obstacle map relative to a current robot position, and converts the current obstacle map to a current occupancy grid map. Changes in the occupancy grid can be reported in real time to support a number of tracking capabilities. The benefit of ROCA is that rather than only providing a vector to the detected change, it provides the actual x,y position of the change.

  19. Real time analysis of multichannel data in tokamaks

    NASA Astrophysics Data System (ADS)

    Wijnands, T.; Parlange, F.; Couturier, B.; Moulin, D.

    1996-10-01

    Four different techniques for the fast analysis of multichannel data in plasma physics are discussed. All four of these techniques are general and sufficiently fast to be used in real time applications. Function parametrization, canonical correlation analysis and a neural network of the multilayer perceptron (MLP) type are compared with a unique linear mapping based on a singular value decomposition, which is used as a reference. Applications deal with the identification of the plasma boundary and some global plasma parameters in the DIII-D and the Tore Supra tokamaks by using magnetic measurements. The results of an MLP-1 neural network, employed for the real time plasma position determination in Tore Supra, are presented

  20. Real-time detection of optical transients with RAPTOR

    SciTech Connect

    Borozdin, K. N.; Brumby, Steven P.; Galassi, M. C.; McGowan, K. E.; Starr, D. L.; Vestrand, W. T.; White, R. R.; Wozniak, P. R.; Wren, J.

    2002-01-01

    Fast variability of optical objects is an interesting though poorly explored subject in modern astronomy. Real-time data processing and identification of transient, celestial events in the images is very important, for such study as it allows rapid follow-up with more sensitive instruments, We discuss an approach which we have chosen for the RAPTOR project which is a pioneering close-loop system combining real-time transient detection with rapid follow-up. Our data processing pipeline is able to identify and localize an optical transient within seconds after the observation. We describe the challenges we met, solutions we found and some results obtained in our search for fast optical transients. The software pipeline we have developed for RAPTOR can easily be applied to the data from other experiments.

  1. Device-Independent Randomness Generation in the Presence of Weak Cross-Talk

    NASA Astrophysics Data System (ADS)

    Silman, J.; Pironio, S.; Massar, S.

    2013-03-01

    Device-independent protocols use nonlocality to certify that they are performing properly. This is achieved via Bell experiments on entangled quantum systems, which are kept isolated from one another during the measurements. However, with present-day technology, perfect isolation comes at the price of experimental complexity and extremely low data rates. Here we argue that for device-independent randomness generation—and other device-independent protocols where the devices are in the same lab—we can slightly relax the requirement of perfect isolation and still retain most of the advantages of the device-independent approach, by allowing a little cross-talk between the devices. This opens up the possibility of using existent experimental systems with high data rates, such as Josephson phase qubits on the same chip, thereby bringing device-