Science.gov

Sample records for helicase-dependent isothermal amplification

  1. Detection of Group A Streptococcus in Pharyngeal Swab Specimens by Use of the AmpliVue GAS Isothermal Helicase-Dependent Amplification Assay.

    PubMed

    Faron, Matthew L; Ledeboer, Nathan A; Granato, Paul; Daly, Judy A; Pierce, Kristina; Pancholi, Preeti; Uphoff, Timothy S; Buchan, Blake W

    2015-07-01

    We evaluated the clinical performance (sensitivity and specificity) of the AmpliVue group A Streptococcus (GAS) isothermal helicase-dependent amplification assay using 1,192 pharyngeal swab specimens. AmpliVue GAS assay results were compared to the results of routine throat cultures on selective streptococcal blood agar plates. The sensitivity and specificity of the AmpliVue GAS assay were 98.3% (95% confidence interval [CI], 95 to 100%) and 93.2% (95% CI, 91 to 95%), respectively. PMID:25972419

  2. Targeting helicase-dependent amplification products with an electrochemical genosensor for reliable and sensitive screening of genetically modified organisms.

    PubMed

    Moura-Melo, Suely; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Dos Santos Junior, J Ribeiro; da Silva Fonseca, Rosana A; Lobo-Castañón, Maria Jesús

    2015-08-18

    Cultivation of genetically modified organisms (GMOs) and their use in food and feed is constantly expanding; thus, the question of informing consumers about their presence in food has proven of significant interest. The development of sensitive, rapid, robust, and reliable methods for the detection of GMOs is crucial for proper food labeling. In response, we have experimentally characterized the helicase-dependent isothermal amplification (HDA) and sequence-specific detection of a transgene from the Cauliflower Mosaic Virus 35S Promoter (CaMV35S), inserted into most transgenic plants. HDA is one of the simplest approaches for DNA amplification, emulating the bacterial replication machinery, and resembling PCR but under isothermal conditions. However, it usually suffers from a lack of selectivity, which is due to the accumulation of spurious amplification products. To improve the selectivity of HDA, which makes the detection of amplification products more reliable, we have developed an electrochemical platform targeting the central sequence of HDA copies of the transgene. A binary monolayer architecture is built onto a thin gold film where, upon the formation of perfect nucleic acid duplexes with the amplification products, these are enzyme-labeled and electrochemically transduced. The resulting combined system increases genosensor detectability up to 10(6)-fold, allowing Yes/No detection of GMOs with a limit of detection of ?30 copies of the CaMV35S genomic DNA. A set of general utility rules in the design of genosensors for detection of HDA amplicons, which may assist in the development of point-of-care tests, is also included. The method provides a versatile tool for detecting nucleic acids with extremely low abundance not only for food safety control but also in the diagnostics and environmental control areas. PMID:26198403

  3. Isothermal Amplification of Nucleic Acids.

    PubMed

    Zhao, Yongxi; Chen, Feng; Li, Qian; Wang, Lihua; Fan, Chunhai

    2015-11-25

    Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed. PMID:26551336

  4. Isothermal amplification of insect DNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The loop-mediated isothermal amplification of DNA (LAMP) method can amplify a target DNA sequence at a constant temperature in about 1 hour. LAMP has broad application in agriculture and medicine because of the need for rapid and inexpensive diagnoses. The power of LAMP is being used by researchers ...

  5. Isothermal Amplification of Insect DNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The loop-mediated isothermal amplification of DNA (LAMP) method can amplify a target DNA sequence at a constant temperature in about one hour. LAMP has broad application in agriculture and medicine because of the need for rapid and inexpensive diagnoses. LAMP eliminates the need for temperature cycl...

  6. Diagnostic Devices for Isothermal Nucleic Acid Amplification

    PubMed Central

    Chang, Chia-Chen; Chen, Chien-Cheng; Wei, Shih-Chung; Lu, Hui-Hsin; Liang, Yang-Hung; Lin, Chii-Wann

    2012-01-01

    Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development. PMID:22969402

  7. Isothermal reactions for the amplification of oligonucleotides.

    PubMed

    Van Ness, Jeffrey; Van Ness, Lori K; Galas, David J

    2003-04-15

    We have devised a class of isothermal reactions for amplifying DNA. These homogeneous reactions rapidly synthesize short oligonucleotides (8-16 bases) specified by the sequence of an amplification template. Versions of the reactions can proceed in either a linear or an exponential amplification mode. Both of these reactions require simple, constant conditions, and the rate of amplification depends entirely on the molecular parameters governing the interactions of the molecules in the reaction. The exponential version of the reaction is a molecular chain reaction that uses the oligonucleotide products of each linear reaction to create producers of more of the same oligonucleotide. It is a highly sensitive chain reaction that can be specifically triggered by given DNA sequences and can achieve amplifications of >10(6)-fold. Several similar reactions in this class are described here. The robustness, speed, and sensitivity of the exponential reaction suggest it will be useful in rapidly detecting the presence of small amounts of a specific DNA sequence in a sample, and a range of other applications, including many currently making use of the PCR. PMID:12679520

  8. Low Cost Extraction and Isothermal Amplification of DNA for Infectious Diarrhea Diagnosis

    PubMed Central

    Huang, Shichu; Do, Jaephil; Mahalanabis, Madhumita; Fan, Andy; Zhao, Lei; Jepeal, Lisa; Singh, Satish K.; Klapperich, Catherine M.

    2013-01-01

    In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10?2 pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health. PMID:23555883

  9. Detection of the 35S promoter in transgenic maize via various isothermal amplification techniques: a practical approach.

    PubMed

    Zahradnik, Celine; Kolm, Claudia; Martzy, Roland; Mach, Robert L; Krska, Rudolf; Farnleitner, Andreas H; Brunner, Kurt

    2014-11-01

    In 2003 the European Commission introduced a 0.9% threshold for food and feed products containing genetically modified organism (GMO)-derived components. For commodities containing GMO contents higher than this threshold, labelling is mandatory. To provide a DNA-based rapid and simple detection method suitable for high-throughput screening of GMOs, several isothermal amplification approaches for the 35S promoter were tested: strand displacement amplification, nicking-enzyme amplification reaction, rolling circle amplification, loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA). The assays developed were tested for specificity in order to distinguish between samples containing genetically modified (GM) maize and non-GM maize. For those assays capable of this discrimination, tests were performed to determine the lower limit of detection. A false-negative rate was determined to rule out whether GMO-positive samples were incorrectly classified as GMO-negative. A robustness test was performed to show reliable detection independent from the instrument used for amplification. The analysis of three GM maize lines showed that only LAMP and HDA were able to differentiate between the GMOs MON810, NK603, and Bt11 and non-GM maize. Furthermore, with the HDA assay it was possible to realize a detection limit as low as 0.5%. A false-negative rate of only 5% for 1% GM maize for all three maize lines shows that HDA has the potential to be used as an alternative strategy for the detection of transgenic maize. All results obtained with the LAMP and HDA assays were compared with the results obtained with a previously reported real-time PCR assay for the 35S promoter in transgenic maize. This study presents two new screening assays for detection of the 35S promoter in transgenic maize by applying the isothermal amplification approaches HDA and LAMP. PMID:24880871

  10. Coupled isothermal polynucleotide amplification and translation system

    NASA Technical Reports Server (NTRS)

    Joyce, Gerald F. (Inventor)

    1998-01-01

    A cell-free system for polynucleotide amplification and translation is disclosed. Also disclosed are methods for using the system and a composition which allows the various components of the system to function under a common set of reaction conditions.

  11. Sequence dependence of isothermal DNA amplification via EXPAR.

    PubMed

    Qian, Jifeng; Ferguson, Tanya M; Shinde, Deepali N; Ramírez-Borrero, Alissa J; Hintze, Arend; Adami, Christoph; Niemz, Angelika

    2012-06-01

    Isothermal nucleic acid amplification is becoming increasingly important for molecular diagnostics. Therefore, new computational tools are needed to facilitate assay design. In the isothermal EXPonential Amplification Reaction (EXPAR), template sequences with similar thermodynamic characteristics perform very differently. To understand what causes this variability, we characterized the performance of 384 template sequences, and used this data to develop two computational methods to predict EXPAR template performance based on sequence: a position weight matrix approach with support vector machine classifier, and RELIEF attribute evaluation with Naïve Bayes classification. The methods identified well and poorly performing EXPAR templates with 67-70% sensitivity and 77-80% specificity. We combined these methods into a computational tool that can accelerate new assay design by ruling out likely poor performers. Furthermore, our data suggest that variability in template performance is linked to specific sequence motifs. Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates. Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers. Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions. PMID:22416064

  12. Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes.

    PubMed

    Tanner, Nathan A; Zhang, Yinhua; Evans, Thomas C

    2015-02-01

    Nucleic acid amplification is the basis for many molecular diagnostic assays. In these cases, the amplification product must be detected and analyzed, typically requiring extended workflow time, sophisticated equipment, or both. Here we present a novel method of amplification detection that harnesses the pH change resulting from amplification reactions performed with minimal buffering capacity. In loop-mediated isothermal amplification (LAMP) reactions, we achieved rapid (<30 min) and sensitive (<10 copies) visual detection using pH-sensitive dyes. Additionally, the detection can be performed in real time, enabling high-throughput or quantitative applications. We also demonstrate this visual detection for another isothermal amplification method (strand-displacement amplification), PCR, and reverse transcription LAMP (RT-LAMP) detection of RNA. The colorimetric detection of amplification presented here represents a generally applicable approach for visual detection of nucleic acid amplification, enabling molecular diagnostic tests to be analyzed immediately without the need for specialized and expensive instrumentation. PMID:25652028

  13. Development of Phage Immuno-Loop-Mediated Isothermal Amplification Assays for Organophosphorus Pesticides in Agro-

    E-print Network

    Hammock, Bruce D.

    95616, United States Guangdong Provincial Key Laboratory of Food Quality and Safety, South China pesticides and their residues in food and the environment. Loop-mediated isothermal amplification (LAMP be specifically carried out under isothermal conditions, and do not require a denatured DNA template. Compared

  14. Isothermal Amplification Using a Chemical Heating Device for Point-of-Care Detection of HIV-1

    PubMed Central

    Curtis, Kelly A.; Rudolph, Donna L.; Nejad, Irene; Singleton, Jered; Beddoe, Andy; Weigl, Bernhard; LaBarre, Paul; Owen, S. Michele

    2012-01-01

    Background To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. Methodology/Significant Findings In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. Conclusion The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC. PMID:22384022

  15. Design of a New Type of Compact Chemical Heater for Isothermal Nucleic Acid Amplification

    PubMed Central

    Shah, Kamal G.; Guelig, Dylan; Diesburg, Steven; Buser, Joshua; Burton, Robert; LaBarre, Paul; Richards-Kortum, Rebecca; Weigl, Bernhard

    2015-01-01

    Previous chemical heater designs for isothermal nucleic acid amplification have been based on solid-liquid phase transition, but using this approach, developers have identified design challenges en route to developing a low-cost, disposable device. Here, we demonstrate the feasibility of a new heater configuration suitable for isothermal amplification in which one reactant of an exothermic reaction is a liquid-gas phase-change material, thereby eliminating the need for a separate phase-change compartment. This design offers potentially enhanced performance and energy density compared to other chemical and electric heaters. PMID:26430883

  16. Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices

    PubMed Central

    Zanoli, Laura Maria; Spoto, Giuseppe

    2012-01-01

    Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed. PMID:25587397

  17. A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification

    PubMed Central

    Craw, Pascal; Mackay, Ruth E.; Naveenathayalan, Angel; Hudson, Chris; Branavan, Manoharanehru; Sadiq, S. Tariq; Balachandran, Wamadeva

    2015-01-01

    Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings. PMID:26389913

  18. A Novel Real-Time DNA Detection System for Loop-Mediated Isothermal Amplification Method

    NASA Astrophysics Data System (ADS)

    Kakugawa, Koji; Yamada, Kenji; Maeda, Hiroshi; Takashiba, Shougo

    We developed a novel real-time DNA detection system for loop-mediated isothermal amplification (LAMP) method. Our prototype was composed of a thermostatic chamber, a hole slide glass, LED and a web camera. The reaction mixture was injected into the slide glass hole and the LAMP reaction was carried out at 63°C for 2 hours. To observe the DNA amplification, we monitored the fluorescence intensity of SYBR Green I that was excited by the blue LED. The captured BMP images were analyzed by NIH Image J software. The DNA amplification and amplification monitoring experiment was successful. Furthermore, quantitative accuracy was evaluated based on real-time PCR. The reaction time correlates well with the DNA concentration. These results indicate the successful development of a novel real-time DNA detection system for LAMP method.

  19. Droplet-based microfluidic systems for high-throughput single DNA molecule isothermal amplification and analysis.

    PubMed

    Mazutis, Linas; Araghi, Ali Fallah; Miller, Oliver J; Baret, Jean-Christophe; Frenz, Lucas; Janoshazi, Agnes; Taly, Valérie; Miller, Benjamin J; Hutchison, J Brian; Link, Darren; Griffiths, Andrew D; Ryckelynck, Michael

    2009-06-15

    We have developed a method for high-throughput isothermal amplification of single DNA molecules in a droplet-based microfluidic system. DNA amplification in droplets was analyzed using an intercalating fluorochrome, allowing fast and accurate "digital" quantification of the template DNA based on the Poisson distribution of DNA molecules in droplets. The clonal amplified DNA in each 2 pL droplet was further analyzed by measuring the enzymatic activity of the encoded proteins after fusion with a 15 pL droplet containing an in vitro translation system. PMID:19518143

  20. A novel multiplex isothermal amplification method for rapid detection and identification of viruses.

    PubMed

    Nyan, Dougbeh-Chris; Swinson, Kevin L

    2015-01-01

    A rapid multiplex isothermal amplification assay has been developed for detection and identification of multiple blood-borne viruses that infect millions of people world-wide. These infections may lead to chronic diseases or death if not diagnosed and treated in a timely manner. Sets of virus-specific oligonucleotides and oligofluorophores were designed and used in a reverse-transcription loop-mediated multiplexed isothermal amplification reaction for detection and gel electrophoretic identification of human Immunodeficiency virus (HIV), hepatitis-B virus (HBV), hepatitis-C virus (HCV), hepatitis-E virus (HEV), dengue virus (DENV), and West Nile (WNV) virus infection in blood plasma. Amplification was catalyzed with two thermostable enzymes for 30-60 minutes under isothermal condition, utilizing a simple digital heat source. Electrophoretic analysis of amplified products demonstrated simultaneous detection of 6 viruses that were distinctly identified by unique ladder-like banding patterns. Naked-eye fluorescent visualization of amplicons revealed intensely fluorescing products that indicated positive detection. The test demonstrated a 97% sensitivity and a 100% specificity, with no cross-reaction with other viruses observed. This portable detection tool may have clinical and field utility in the developing and developed world settings. This may enable rapid diagnosis and identification of viruses for targeted therapeutic intervention and prevention of disease transmission. PMID:26643761

  1. A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers

    NASA Astrophysics Data System (ADS)

    Du, Yan; Hughes, Randall A.; Bhadra, Sanchita; Jiang, Yu Sherry; Ellington, Andrew D.; Li, Bingling

    2015-06-01

    Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20-100 copies/?l, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device.

  2. A novel multiplex isothermal amplification method for rapid detection and identification of viruses

    PubMed Central

    Nyan, Dougbeh-Chris; Swinson, Kevin L.

    2015-01-01

    A rapid multiplex isothermal amplification assay has been developed for detection and identification of multiple blood-borne viruses that infect millions of people world-wide. These infections may lead to chronic diseases or death if not diagnosed and treated in a timely manner. Sets of virus-specific oligonucleotides and oligofluorophores were designed and used in a reverse-transcription loop-mediated multiplexed isothermal amplification reaction for detection and gel electrophoretic identification of human Immunodeficiency virus (HIV), hepatitis-B virus (HBV), hepatitis-C virus (HCV), hepatitis-E virus (HEV), dengue virus (DENV), and West Nile (WNV) virus infection in blood plasma. Amplification was catalyzed with two thermostable enzymes for 30–60 minutes under isothermal condition, utilizing a simple digital heat source. Electrophoretic analysis of amplified products demonstrated simultaneous detection of 6 viruses that were distinctly identified by unique ladder-like banding patterns. Naked-eye fluorescent visualization of amplicons revealed intensely fluorescing products that indicated positive detection. The test demonstrated a 97% sensitivity and a 100% specificity, with no cross-reaction with other viruses observed. This portable detection tool may have clinical and field utility in the developing and developed world settings. This may enable rapid diagnosis and identification of viruses for targeted therapeutic intervention and prevention of disease transmission. PMID:26643761

  3. A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers.

    PubMed

    Du, Yan; Hughes, Randall A; Bhadra, Sanchita; Jiang, Yu Sherry; Ellington, Andrew D; Li, Bingling

    2015-01-01

    Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20-100 copies/?l, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device. PMID:26050646

  4. A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers

    PubMed Central

    Du, Yan; Hughes, Randall A.; Bhadra, Sanchita; Jiang, Yu Sherry; Ellington, Andrew D.; Li, Bingling

    2015-01-01

    Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20–100 copies/?l, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device. PMID:26050646

  5. Simple System for Isothermal DNA Amplification Coupled to Lateral Flow Detection

    PubMed Central

    Roskos, Kristina; Hickerson, Anna I.; Lu, Hsiang-Wei; Ferguson, Tanya M.; Shinde, Deepali N.; Klaue, Yvonne; Niemz, Angelika

    2013-01-01

    Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF) detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb) genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP) or the Exponential Amplification Reaction (EXPAR), both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden. PMID:23922706

  6. Simple system for isothermal DNA amplification coupled to lateral flow detection.

    PubMed

    Roskos, Kristina; Hickerson, Anna I; Lu, Hsiang-Wei; Ferguson, Tanya M; Shinde, Deepali N; Klaue, Yvonne; Niemz, Angelika

    2013-01-01

    Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF) detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb) genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP) or the Exponential Amplification Reaction (EXPAR), both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden. PMID:23922706

  7. Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification

    PubMed Central

    Wang, Yi; Wang, Yan; Ma, Ai-Jing; Li, Dong-Xun; Luo, Li-Juan; Liu, Dong-Xin; Jin, Dong; Liu, Kai; Ye, Chang-Yun

    2015-01-01

    We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61–65?°C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique. PMID:26154567

  8. Loop-mediated isothermal amplification (LAMP) for the rapid detection of Mycoplasma genitalium.

    PubMed

    Edwards, Thomas; Burke, Patricia; Smalley, Helen B; Gillies, Liz; Longhurst, Denise; Vipond, Barry; Hobbs, Glyn

    2015-09-01

    Mycoplasma genitalium is a sexually transmissible, pathogenic bacterium and a significant cause of nongonococcal urethritis in both men and women. Due to the difficulty of the culture of M. genitalium from clinical samples, the laboratory diagnosis of M. genitalium infection is almost exclusively carried out using nucleic acid amplification tests. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology, utilising a set of 4 primers specific to 6 distinct regions of the target DNA sequence, in order to amplify target DNA in a highly specific and rapid manner. A LAMP assay was designed to the pdhD gene of M. genitalium, and the limit of detection of the assay was determined as 10 fg of M. genitalium genomic DNA, equating to ~16 copies of the M. genitalium genome, which was equally sensitive as a gold standard 16S rRNA polymerase chain reaction assay. PMID:26072150

  9. Tuberculosis Biomarker Extraction and Isothermal Amplification in an Integrated Diagnostic Device

    PubMed Central

    Creecy, Amy; Russ, Patricia K.; Solinas, Francesca; Wright, David W.; Haselton, Frederick R.

    2015-01-01

    In this study, we integrated magnetic bead-based sample preparation and isothermal loop mediated amplification (LAMP) of TB in a single tube. Surrogate sputum samples produced by the Program for Appropriate Technology in Health containing inactivated TB bacteria were used to test the diagnostic. In order to test the sample preparation method, samples were lysed, and DNA was manually extracted and eluted into water in the tube. In a thermal cycler, LAMP amplified TB DNA from 103 TB cells/mL of sputum at 53.5 ± 3.3 minutes, 104 cells/mL at 46.3 ± 2.2 minutes, and 105 cells/mL at 41.6 ± 1.9 minutes. Negative control samples did not amplify. Next, sample preparation was combined with in-tubing isothermal LAMP amplification by replacing the water elution chamber with a LAMP reaction chamber. In this intermediate configuration, LAMP amplified 103 cells/mL at 74 ± 10 minutes, 104 cells/mL at 60 ± 9 minutes, and 105 TB cells/mL of sputum at 54 ± 9 minutes. Two of three negative controls did not amplify; one amplified at 100 minutes. In the semi-automated system, DNA was eluted directly into an isothermal reaction solution containing the faster OptiGene DNA polymerase. The low surrogate sputum concentration, 103 TB cells/mL, amplified at 52.8 ± 3.3 minutes, 104 cells/mL at 45.4 ± 11.3 minutes, and 105 cells/mL at 31.8 ± 2.9 minutes. TB negative samples amplified at 66.4 ± 7.4 minutes. This study demonstrated the feasibility of a single tube design for integrating sample preparation and isothermal amplification, which with further development could be useful for point-of-care applications, particularly in a low-resource setting. PMID:26132307

  10. Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma.

    PubMed

    Ahmed, Sarah A; van de Sande, Wendy W J; Desnos-Ollivier, Marie; Fahal, Ahmed H; Mhmoud, Najwa A; de Hoog, G S

    2015-10-01

    Appropriate diagnosis and treatment of eumycetoma may vary significantly depending on the causative agent. To date, the most common fungus causing mycetoma worldwide is Madurella mycetomatis. This species fails to express any recognizable morphological characteristics, and reliable identification can therefore only be achieved with the application of molecular techniques. Recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) are proposed as alternatives to phenotypic methods. Species-specific primers were developed to target the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region of M. mycetomatis. Both isothermal amplification techniques showed high specificity and sufficient sensitivity to amplify fungal DNA and proved to be appropriate for detection of M. mycetomatis. Diagnostic performance of the techniques was assessed in comparison to conventional PCR using biopsy specimens from eumycetoma patients. RPA is reliable and easy to operate and has the potential to be implemented in areas where mycetoma is endemic. The techniques may be expanded to detect fungal DNA from environmental samples. PMID:26246484

  11. GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

    PubMed

    Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

    2015-06-01

    Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples. PMID:25822163

  12. Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences

    PubMed Central

    Lee, David; La Mura, Maurizio; Allnutt, Theo R; Powell, Wayne

    2009-01-01

    Background The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR). Here we have applied the loop-mediated isothermal amplification (LAMP) method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions. Results We have tested the specificity and sensitivity of the technique for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP. Conclusion This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection. PMID:19187544

  13. Loop-mediated Isothermal Amplification Assay to Rapidly Detect Wheat Streak Mosaic Virus in Quarantined Plants

    PubMed Central

    Lee, Siwon; Kim, Jin-Ho; Choi, Ji-Young; Jang, Won-Cheoul

    2015-01-01

    We developed a loop-mediated isothermal amplification (LAMP) method to rapidly diagnose Wheat streak mosaic virus (WSMV) during quarantine inspections of imported wheat, corn, oats, and millet. The LAMP method was developed as a plant quarantine inspection method for the first time, and its simplicity, quickness, specificity and sensitivity were verified compared to current reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR quarantine methods. We were able to quickly screen for WSMV at quarantine sites with many test samples; thus, this method is expected to contribute to plant quarantine inspections. PMID:26674930

  14. Monitoring the progression of loop-mediated isothermal amplification using conductivity.

    PubMed

    Zhang, Xuzhi; Liu, Wenwen; Lu, Xun; Justin Gooding, J; Li, Qiufen; Qu, Keming

    2014-12-01

    Loop-mediated isothermal amplification (LAMP) yields a large amount of DNA, as well as magnesium pyrophosphate precipitate, causing a decrease in ionic strength that can be measured with a conductivity meter. There is a clear relationship between the conductivity of the LAMP mixture solution and the duration of biochemical reaction. Moreover, there is also a clear relationship between the change in conductivity and the amount of initial template DNA over the range of 0.08 to 3.2 ng. These results demonstrate the feasibility not only for detecting the LAMP product qualitatively but also for real-time monitoring the biochemical reaction progression quantitatively using conductivity measurements. PMID:25168192

  15. Loop-mediated Isothermal Amplification Assay to Rapidly Detect Wheat Streak Mosaic Virus in Quarantined Plants.

    PubMed

    Lee, Siwon; Kim, Jin-Ho; Choi, Ji-Young; Jang, Won-Cheoul

    2015-12-01

    We developed a loop-mediated isothermal amplification (LAMP) method to rapidly diagnose Wheat streak mosaic virus (WSMV) during quarantine inspections of imported wheat, corn, oats, and millet. The LAMP method was developed as a plant quarantine inspection method for the first time, and its simplicity, quickness, specificity and sensitivity were verified compared to current reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR quarantine methods. We were able to quickly screen for WSMV at quarantine sites with many test samples; thus, this method is expected to contribute to plant quarantine inspections. PMID:26674930

  16. Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays.

    PubMed

    Glais, Laurent; Jacquot, Emmanuel

    2015-01-01

    Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay. PMID:25981257

  17. Molecular-based isothermal tests for field diagnosis of malaria and their potential contribution to malaria elimination.

    PubMed

    Oriero, Eniyou C; Jacobs, Jan; Van Geertruyden, Jean-Pierre; Nwakanma, Davis; D'Alessandro, Umberto

    2015-01-01

    In countries where malaria transmission has decreased substantially, thanks to the scale-up of control interventions, malaria elimination may be feasible. Nevertheless, this goal requires new strategies such as the active detection and treatment of infected individuals. As the detection threshold for the currently used diagnostic methods is 100 parasites/?L, most low-density, asymptomatic infections able to maintain transmission cannot be detected. Identifying them by molecular methods such as PCR is a possible option but the field deployment of these tests is problematic. Isothermal amplification of nucleic acids (at a constant temperature) offers the opportunity of addressing some of the challenges related to the field deployment of molecular diagnostic methods. One of the novel isothermal amplification methods for which a substantial amount of work has been done is the loop-mediated isothermal amplification (LAMP) assay. The present review describes LAMP and several other isothermal nucleic acid amplification methods, such as thermophilic helicase-dependent amplification, strand displacement amplification, recombinase polymerase amplification and nucleic acid sequence-based amplification, and explores their potential use as high-throughput, field-based molecular tests for malaria diagnosis. PMID:25223973

  18. Loop-mediated isothermal amplification (LAMP) based detection of Colletotrichum falcatum causing red rot in sugarcane.

    PubMed

    Chandra, Amaresh; Keizerweerd, Amber T; Que, Youxiong; Grisham, Michael P

    2015-08-01

    Red rot, caused by Colletotrichum falcatum, is a destructive disease prevalent in most sugarcane-producing countries. Disease-free sugarcane planting materials (setts) are essential as the pathogen spreads primarily through infected setts. The present study was undertaken to develop a loop-mediated isothermal amplification (LAMP) assay for the detection of C. falcatum. C. falcatum genomic DNA was isolated from pure mycelium culture and infected tissues. Four sets of primers corresponding to a unique DNA sequence specific to C. falcatum were designed. Specificity of the LAMP test was checked with DNA of another fungal pathogen of sugarcane, Puccinia melanocephala, as well as two closely-related species, Colletotrichum fructivorum and Colletotrichum acutatum. No reaction was found with the three pathogens. When C. falcatum DNA from pure culture was used in a detection limit analysis, sensitivity of the LAMP method was observed to be ten times higher than that of conventional PCR; however, sensitivity was only 5 times higher when DNA from C. falcatum-infected tissues was used. Using the LAMP assay, C. falcatum DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions. Moreover, visual judgment of color change in <1 h without further post-amplification processing makes the LAMP method convenient, economical, and useful in diagnostic laboratories and the field. PMID:25861736

  19. Detection of the food allergen celery via loop-mediated isothermal amplification technique.

    PubMed

    Zahradnik, Celine; Martzy, Roland; Mach, Robert L; Krska, Rudolf; Farnleitner, Andreas H; Brunner, Kurt

    2014-11-01

    Since 2005, celery and celery products have to be labeled according to Directive 2003/89/EC due to their allergenic potential. In order to provide a DNA-based, rapid and simple detection method suitable for high-throughput analysis, a loop-mediated isothermal amplification (LAMP) assay for the detection of celery (Apium graveolens) was developed. The assay was tested for specificity for celery since closely related species also hold food relevance. The limit of detection (LOD) for spiked food samples was found to be as low as 7.8 mg of dry celery powder per kilogram. An evaluation of different amplification and detection platforms was performed to show reliable detection independent from the instrument used for amplification (thermal cycler or heating block) and detection mechanisms (real-time fluorescence detection, agarose gel electrophoresis or nucleic acid staining). The analysis of 10 commercial food samples representing diverse and complex food matrices, and a false-negative rate of 0% for approximately 24 target copies or 0.08 ng celery DNA for three selected food matrices show that LAMP has the potential to be used as an alternative strategy for the detection of allergenic celery. The performance of the developed LAMP assay turned out to be equal or superior to the best available PCR assay for the detection of celery in food products. PMID:24880868

  20. Strand Invasion Based Amplification (SIBA®): A Novel Isothermal DNA Amplification Technology Demonstrating High Specificity and Sensitivity for a Single Molecule of Target Analyte

    PubMed Central

    Hoser, Mark J.; Mansukoski, Hannu K.; Morrical, Scott W.; Eboigbodin, Kevin E.

    2014-01-01

    Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2?-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella. PMID:25419812

  1. Development of an in situ loop-mediated isothermal amplification technique for chromosomal localization of DNA sequences

    NASA Astrophysics Data System (ADS)

    Meng, Qinglei; Wang, Shi; Zhang, Lingling; Huang, Xiaoting; Bao, Zhenmin

    2013-01-01

    In situ loop-mediated isothermal amplification (in situ LAMP) combines in situ hybridization and loop-mediated isothermal amplification (LAMP) techniques for chromosomal localization of DNA sequences. In situ LAMP is a method that is generally more specific and sensitive than conventional techniques such as fluorescence in situ hybridization (FISH), primed in situ labeling (PRINS), and cycling primed in situ labeling (C-PRINS). Here, we describe the development and application of in situ LAMP to identify the chromosomal localization of DNA sequences. To benchmark this technique, we successfully applied this technique to localize the major ribosomal RNA gene on the chromosomes of the Zhikong scallop ( Chlamys farreri).

  2. Rapid detection of Salmonella Typhi by loop-mediated isothermal amplification (LAMP) method

    PubMed Central

    Abdullah, J.; Saffie, N.; Sjasri, F.A.R.; Husin, A.; Abdul-Rahman, Z.; Ismail, A.; Aziah, I.; Mohamed, M.

    2014-01-01

    An in-house loop-mediated isothermal amplification (LAMP) reaction was established and evaluated for sensitivity and specificity in detecting the presence of Salmonella Typhi (S. Typhi) isolates from Kelantan, Malaysia. Three sets of primers consisting of two outer and 4 inner were designed based on locus STBHUCCB_38510 of chaperone PapD of S. Typhi genes. The reaction was optimised using genomic DNA of S. Typhi ATCC7251 as the template. The products were visualised directly by colour changes of the reaction. Positive results were indicated by green fluorescence and negative by orange colour. The test was further evaluated for specificity, sensitivity and application on field samples. The results were compared with those obtained by gold standard culture method and Polymerase Chain Reaction (PCR). This method was highly specific and -10 times more sensitive in detecting S. Typhi compared to the optimised conventional polymerase chain reaction (PCR) method. PMID:25763045

  3. Development of loop-mediated isothermal amplification methods for detecting Taylorella equigenitalis and Taylorella asinigenitalis

    PubMed Central

    KINOSHITA, Yuta; NIWA, Hidekazu; KATAYAMA, Yoshinari; HARIU, Kazuhisa

    2015-01-01

    ABSTRACT Taylorella equigenitalis is a causative bacterium of contagious equine metritis (CEM), and Taylorella asinigenitalis is species belonging to genus Taylorella. The authors developed two loop-mediated isothermal amplification (LAMP) methods, Te-LAMP and Ta-LAMP, for detecting T. equigenitalis and T. asinigenitalis, respectively. Using experimentally spiked samples, Te-LAMP was as sensitive as a published semi-nested PCR method, and Ta-LAMP was more sensitive than conventional PCR. Multiplex LAMP worked well without nonspecific reactions, and the analytical sensitivities of multiplex LAMP in the spiked samples were almost equivalent to those of Te-LAMP and Ta-LAMP. Therefore, the LAMP methods are considered useful tools to detect T. equigenitalis and/or T. asinigenitalis, and preventive measures will be rapidly implemented if the occurrence of CEM is confirmed by the LAMP methods. PMID:25829868

  4. Protein detection through different platforms of immuno-loop-mediated isothermal amplification

    PubMed Central

    2013-01-01

    Different immunoassay-based methods have been devised to detect protein targets. These methods have some challenges that make them inefficient for assaying ultra-low-amounted proteins. ELISA, iPCR, iRCA, and iNASBA are the common immunoassay-based methods of protein detection, each of which has specific and common technical challenges making it necessary to introduce a novel method in order to avoid their problems for detection of target proteins. Here we propose a new method nominated as ‘immuno-loop-mediated isothermal amplification’ or ‘iLAMP’. This new method is free from the problems of the previous methods and has significant advantages over them. In this paper we also offer various configurations in order to improve the applicability of this method in real-world sample analyses. Important potential applications of this method are stated as well. PMID:24237767

  5. Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25

    PubMed Central

    Xu, Junyi; Zheng, Qiuyue; Yu, Ling; Liu, Ran; Zhao, Xin; Wang, Gang; Wang, Qinghua; Cao, Jijuan

    2013-01-01

    The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. We developed and evaluated the specificity and sensitivity of the LAMP method for rapid detection of the genetically modified (GM) maize T25. A set of six specific primers was successfully designed to recognize six distinct sequences on the target gene, including a pair of inner primers, a pair of outer primers, and a pair of loop primers. The optimum reaction temperature and time were verified to be 65°C and 45 min, respectively. The detection limit of this LAMP assay was 5 g kg?1 GMO component. Comparative experiments showed that the LAMP assay was a simple, rapid, accurate, and specific method for detecting the GM maize T25. PMID:24804053

  6. Development of Loop-Mediated Isothermal Amplification for Detection of Leifsonia xyli subsp. xyli in Sugarcane

    PubMed Central

    Liu, Jing; Guo, Jinlong; Chen, Rukai; Grisham, Michael Paul

    2013-01-01

    Ratoon stunt, caused by the xylem-limited coryneform bacterium Leifsonia xyli subsp. xyli (Lxx), is a deep bacteriosis and prevalent in most of sugarcane-producing countries. Based on loop-mediated isothermal amplification (LAMP), we developed a method for detecting Lxx. The major advantages of the LAMP method are visual judgment by color and time saving with only 60?min for identification of Lxx and without the need for costly PCR apparatus and gel scanner. In the present study, positive and negative samples detected by the LAMP method were clearly distinguishable. When total DNA extracted from internode juice was used as the template, the sensitivity of LAMP was 10 times higher than that of the conventional PCR detection. The LAMP assay is a highly specific, rapid, and sensitive method for the diagnosis of ratoon stunt caused by Lxx in sugarcane. This is the first report of LAMP-based assay for the detection of Lxx in sugarcane. PMID:23710444

  7. Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Trypanosoma brucei rhodesiense

    PubMed Central

    Njiru, Zablon Kithinji; Mikosza, Andrew Stanislaw John; Armstrong, Tanya; Enyaru, John Charles; Ndung'u, Joseph Mathu; Thompson, Andrew Richard Christopher

    2008-01-01

    Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62°C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml) and 0.1 pg (1 trypanosome/ml) using heat-treated buffy coat, while the detection limit for conventional SRA PCR was ?1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions. PMID:18253475

  8. Isothermal target and probe amplification assay for the real-time rapid detection of Staphylococcus aureus.

    PubMed

    Shin, Hyewon; Kim, Minhwan; Yoon, Eunju; Kang, Gyoungwon; Kim, Seungyu; Song, Aelee; Kim, Jeongsoon

    2015-04-01

    Staphylococcus aureus, the species most commonly associated with staphylococcal food poisoning, is one of the most prevalent causes of foodborne disease in Korea and other parts of the world, with much damage inflicted to the health of individuals and economic losses estimated at $120 million. To reduce food poisoning outbreaks by implementing prevention methods, rapid detection of S. aureus in foods is essential. Various types of detection methods for S. aureus are available. Although each method has advantages and disadvantages, high levels of sensitivity and specificity are key aspects of a robust detection method. Here, we describe a novel real-time isothermal target and probe amplification (iTPA) method that allows the rapid and simultaneous amplification of target DNA (the S. aureus nuc gene) and a fluorescence resonance energy transfer-based signal probe under isothermal conditions at 61 °C or detection of S. aureus in real time. The assay was able to specifically detect all 91 S. aureus strains tested without nonspecific detection of 51 non-S. aureus strains. The real-time iTPA assay detected S. aureus at an initial level of 10(1) CFU in overnight cultures of preenriched food samples (kiwi dressing, soybean milk, and custard cream). The advantage of this detection system is that it does not require a thermal cycler, reducing the cost of the real-time PCR and its footprint. Combined with a miniaturized fluorescence detector, this system can be developed into a simplified quantitative hand-held real-time device, which is often required. The iTPA assay was highly reliable and therefore may be used as a rapid and sensitive means of identifying S. aureus in foods. PMID:25836397

  9. EzyAmp signal amplification cascade enables isothermal detection of nucleic acid and protein targets.

    PubMed

    Linardy, Evelyn M; Erskine, Simon M; Lima, Nicole E; Lonergan, Tina; Mokany, Elisa; Todd, Alison V

    2016-01-15

    Advancements in molecular biology have improved the ability to characterize disease-related nucleic acids and proteins. Recently, there has been an increasing desire for tests that can be performed outside of centralised laboratories. This study describes a novel isothermal signal amplification cascade called EzyAmp (enzymatic signal amplification) that is being developed for detection of targets at the point of care. EzyAmp exploits the ability of some restriction endonucleases to cleave substrates containing nicks within their recognition sites. EzyAmp uses two oligonucleotide duplexes (partial complexes 1 and 2) which are initially cleavage-resistant as they lack a complete recognition site. The recognition site of partial complex 1 can be completed by hybridization of a triggering oligonucleotide (Driver Fragment 1) that is generated by a target-specific initiation event. Binding of Driver Fragment 1 generates a completed complex 1, which upon cleavage, releases Driver Fragment 2. In turn, binding of Driver Fragment 2 to partial complex 2 creates completed complex 2 which when cleaved releases additional Driver Fragment 1. Each cleavage event separates fluorophore quencher pairs resulting in an increase in fluorescence. At this stage a cascade of signal production becomes independent of further target-specific initiation events. This study demonstrated that the EzyAmp cascade can facilitate detection and quantification of nucleic acid targets with sensitivity down to aM concentration. Further, the same cascade detected VEGF protein with a sensitivity of 20nM showing that this universal method for amplifying signal may be linked to the detection of different types of analytes in an isothermal format. PMID:26296241

  10. Endpoint visual detection of three genetically modified rice events by loop-mediated isothermal amplification.

    PubMed

    Chen, Xiaoyun; Wang, Xiaofu; Jin, Nuo; Zhou, Yu; Huang, Sainan; Miao, Qingmei; Zhu, Qing; Xu, Junfeng

    2012-01-01

    Genetically modified (GM) rice KMD1, TT51-1, and KF6 are three of the most well known transgenic Bt rice lines in China. A rapid and sensitive molecular assay for risk assessment of GM rice is needed. Polymerase chain reaction (PCR), currently the most common method for detecting genetically modified organisms, requires temperature cycling and relatively complex procedures. Here we developed a visual and rapid loop-mediated isothermal amplification (LAMP) method to amplify three GM rice event-specific junction sequences. Target DNA was amplified and visualized by two indicators (SYBR green or hydroxy naphthol blue [HNB]) within 60 min at an isothermal temperature of 63 °C. Different kinds of plants were selected to ensure the specificity of detection and the results of the non-target samples were negative, indicating that the primer sets for the three GM rice varieties had good levels of specificity. The sensitivity of LAMP, with detection limits at low concentration levels (0.01%?0.005% GM), was 10- to 100-fold greater than that of conventional PCR. Additionally, the LAMP assay coupled with an indicator (SYBR green or HNB) facilitated analysis. These findings revealed that the rapid detection method was suitable as a simple field-based test to determine the status of GM crops. PMID:23203072

  11. Rapid detection of Staphylococcus aureus by loop-mediated isothermal amplification.

    PubMed

    Wang, Xin-Ru; Wu, Li-Fen; Wang, Yan; Ma, Ying-Ying; Chen, Feng-Hua; Ou, Hong-Ling

    2015-01-01

    Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is a major bacterial pathogen associated with nosocomial and community-acquired S. aureus infections all over the world. A rapid detection assay for staphylococcal gene of nuc and mecA is needed. In this study, a rapid identification assay based on the loop-mediated isothermal amplification (LAMP) method was established. PCR and LAMP assays were used to detect Staphylococcus aureus and other related species for nuc and mecA. With optimization of the primers and reaction temperature, the LAMP successfully amplified the genes under isothermal conditions at 62 °C within 60 min, of which the results were identical with those of the conventional PCR methods. The detection limits of the LAMP for nuc and mecA were 1.47 and 14.7 pg/?l DNA per tube, respectively, by naked eye inspections, while the detection limits of the PCR for nuc and mecA were 14.7 pg/?l and 147 pg/?l DNA, respectively. Finally, The LAMP method was then applied to clinical blood plaque samples. The LAMP and PCR demonstrated identical results for the plaque samples with the culture assay. Together, the LAMP offers an alternative detection assay for nuc and mecA with a great advantage of the sensitivity and rapidity. PMID:25349088

  12. Isothermal rolling circle amplification of virus genomes for rapid antigen detection and typing.

    PubMed

    Brasino, Michael D; Cha, Jennifer N

    2015-08-01

    In this work, isothermal rolling circle amplification (RCA) of the multi-kilobase genome of engineered filamentous bacteriophage is used to report the presence and identification of specific protein analytes in solution. First, bacteriophages were chosen as sensing platforms because peptides or antibodies that bind medically relevant targets can be isolated through phage display or expressed as fusions to their p3 and p8 coat proteins. Second, the circular, single-stranded genome contained within the phage serves as a natural large DNA template for a RCA reaction to rapidly generate exponential amounts of double stranded DNA in a single isothermal step that can be easily detected using low-cost fluorescent nucleic acid stains. Amplifying the entire phage genome also provides high detection sensitivities. Furthermore, since the sequence of the viral DNA can be easily modified with multiple restriction enzyme sites, a simple DNA digest can be applied to detect and identify multiple antigens simultaneously. The methods developed here will lead to protein sensors that are highly scalable to produce, can be run without complex biological equipment and do not require the use of multiple antibodies or high-cost fluorescent DNA probes or nucleotides. PMID:26040578

  13. Endpoint Visual Detection of Three Genetically Modified Rice Events by Loop-Mediated Isothermal Amplification

    PubMed Central

    Chen, Xiaoyun; Wang, Xiaofu; Jin, Nuo; Zhou, Yu; Huang, Sainan; Miao, Qingmei; Zhu, Qing; Xu, Junfeng

    2012-01-01

    Genetically modified (GM) rice KMD1, TT51-1, and KF6 are three of the most well known transgenic Bt rice lines in China. A rapid and sensitive molecular assay for risk assessment of GM rice is needed. Polymerase chain reaction (PCR), currently the most common method for detecting genetically modified organisms, requires temperature cycling and relatively complex procedures. Here we developed a visual and rapid loop-mediated isothermal amplification (LAMP) method to amplify three GM rice event-specific junction sequences. Target DNA was amplified and visualized by two indicators (SYBR green or hydroxy naphthol blue [HNB]) within 60 min at an isothermal temperature of 63 °C. Different kinds of plants were selected to ensure the specificity of detection and the results of the non-target samples were negative, indicating that the primer sets for the three GM rice varieties had good levels of specificity. The sensitivity of LAMP, with detection limits at low concentration levels (0.01%–0.005% GM), was 10- to 100-fold greater than that of conventional PCR. Additionally, the LAMP assay coupled with an indicator (SYBR green or HNB) facilitated analysis. These findings revealed that the rapid detection method was suitable as a simple field-based test to determine the status of GM crops. PMID:23203072

  14. Detection of Pathogen-Specific Antibodies by Loop-Mediated Isothermal Amplification

    PubMed Central

    Burbulis, Ian E.; Yamaguchi, Kumiko; Nikolskaia, Olga V.; Prigge, Sean T.; Magez, Stefan; Bisser, Sylvie; Reller, Megan E.

    2015-01-01

    Loop-mediated isothermal amplification (LAMP) is a method for enzymatically replicating DNA that has great utility for clinical diagnosis at the point of care (POC), given its high sensitivity, specificity, speed, and technical requirements (isothermal conditions). Here, we adapted LAMP for measuring protein analytes by creating a protein-DNA fusion (referred to here as a “LAMPole”) that attaches oligonucleotides (LAMP templates) to IgG antibodies. This fusion consists of a DNA element covalently bonded to an IgG-binding polypeptide (protein L/G domain). In our platform, LAMP is expected to provide the most suitable means for amplifying LAMPoles for clinical diagnosis at the POC, while quantitative PCR is more suitable for laboratory-based quantification of antigen-specific IgG abundance. As proof of concept, we measured serological responses to a protozoan parasite by quantifying changes in solution turbidity in real time. We observed a >6-log fold difference in signal between sera from vaccinated versus control mice and in a clinical patient sample versus a control. We assert that LAMPoles will be useful for increasing the sensitivity of measuring proteins, whether it be in a clinical laboratory or in a field setting, thereby improving acute diagnosis of a variety of infections. PMID:25651920

  15. Detection of pathogen-specific antibodies by loop-mediated isothermal amplification.

    PubMed

    Burbulis, Ian E; Yamaguchi, Kumiko; Nikolskaia, Olga V; Prigge, Sean T; Magez, Stefan; Bisser, Sylvie; Reller, Megan E; Grab, Dennis J

    2015-04-01

    Loop-mediated isothermal amplification (LAMP) is a method for enzymatically replicating DNA that has great utility for clinical diagnosis at the point of care (POC), given its high sensitivity, specificity, speed, and technical requirements (isothermal conditions). Here, we adapted LAMP for measuring protein analytes by creating a protein-DNA fusion (referred to here as a "LAMPole") that attaches oligonucleotides (LAMP templates) to IgG antibodies. This fusion consists of a DNA element covalently bonded to an IgG-binding polypeptide (protein L/G domain). In our platform, LAMP is expected to provide the most suitable means for amplifying LAMPoles for clinical diagnosis at the POC, while quantitative PCR is more suitable for laboratory-based quantification of antigen-specific IgG abundance. As proof of concept, we measured serological responses to a protozoan parasite by quantifying changes in solution turbidity in real time. We observed a >6-log fold difference in signal between sera from vaccinated versus control mice and in a clinical patient sample versus a control. We assert that LAMPoles will be useful for increasing the sensitivity of measuring proteins, whether it be in a clinical laboratory or in a field setting, thereby improving acute diagnosis of a variety of infections. PMID:25651920

  16. A loop-mediated isothermal amplification assay for the visual detection of duck circovirus

    PubMed Central

    2014-01-01

    Background Duck circovirus (DuCV) infection in farmed ducks is associated with growth problems or retardation syndromes. Rapid identification of DuCV infected ducks is essential to control DuCV effectively. Therefore, this study aims to develop of an assay for DuCV to be highly specific, sensitive, and simple without any specialized equipment. Methods A set of six specific primers was designed to target the sequences of the Rep gene of DuCV, and A loop-mediated isothermal amplification (LAMP) assay were developed and the reaction conditions were optimized for rapid detection of DuCV. Results The LAMP assay reaction was conducted in a 62°C water bath condition for 50 min. Then the amplification products were visualized directly for color changes. This LAMP assay is highly sensitive and able to detect twenty copies of DuCV DNA. The specificity of this LAMP assay was supported by no cross-reaction with other duck pathogens. Conclusion This LAMP method for DuCV is highly specific and sensitive and can be used as a rapid and direct diagnostic assay for testing clinical samples. PMID:24775810

  17. Rapid and sensitive detection of Listeria monocytogenes by loop-mediated isothermal amplification.

    PubMed

    Tang, Meng-Jun; Zhou, Sheng; Zhang, Xiao-Yan; Pu, Jun-Hua; Ge, Qing-Lian; Tang, Xiu-Jun; Gao, Yu-Shi

    2011-12-01

    Loop-mediated isothermal amplification (LAMP) was designed for detection of Listeria monocytogenes, which is an important food-borne kind of pathogenic bacteria causing human and animal disease. The primers set for the hlyA gene consist of six primers targeting eight regions on specific gene. The LAMP assay could be performed within 40 min at 65°C in a water bath. Amplification products were visualized by calcein and manganous ion and agarose gel electrophoresis. Sensitivity of the LAMP assay for detection of L. monocytogenes in pure cultures was 2.0 CFU per reaction. The LAMP assay was 100-fold higher sensitive than that of the conventional PCR assay. Taking this way, 60 chicken samples were investigated for L. monocytogenes. The accuracy of LAMP was shown to be 100% when compared to the "gold standard" culture-biotechnical, while the PCR assay failed to detect L. monocytogenes in two of the positive samples. It is shown that LAMP assay can be used as a sensitive, rapid, and simple detection tool for the detection of L. monocytogenes and will facilitate the surveillance for contamination of L. monocytogenes in food. PMID:21935669

  18. Development of loop-mediated isothermal amplification (LAMP) for detection of Babesia gibsoni infection in dogs.

    PubMed

    Mandal, Mrityunjay; Banerjee, Partha S; Kumar, Saroj; Ram, Hira; Garg, Rajat; Pawde, Abhijit M

    2015-04-15

    Diagnosis of canine babesiosis, caused by Babesia gibsoni is difficult, especially in chronically infected dogs. A loop mediated isothermal amplification (LAMP) assay was developed and standardized by using four oligonucleotide primers targeting the hypervariable region of 18S rRNA gene (GenBank Acc. no. KC461261). The primers specifically amplified B. gibsoni DNA, while no amplification was detected with DNA from non-infected dogs as well as from dogs infected with Babesia canis vogeli, Hepatozoon canis, Ehrlichia canis and Trypanosoma evansi. The assay could detect 1.35 × 10(-7) parasitaemia and 10(-4) dilution of recombinant plasmid, equivalent to 12 pg of target DNA. All the samples were tested by nested PCR as well as LAMP assay. LAMP was found to be 10 times more sensitive than nested PCR targeting the same gene. Out of 75 suspected field samples, collected from different parts of the country, LAMP could detect B. gibsoni in 43 samples, while nested PCR and microscopy could detect 37 and 23 samples, respectively. High sensitivity, specificity and rapidity of LAMP assay may be exploited for screening large number of samples in a field setting. PMID:25749021

  19. Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of BK Virus?

    PubMed Central

    Bista, Bipin Raj; Ishwad, Chandra; Wadowsky, Robert M.; Manna, Pradip; Randhawa, Parmjeet Singh; Gupta, Gaurav; Adhikari, Meena; Tyagi, Rakhi; Gasper, Gina; Vats, Abhay

    2007-01-01

    Loop-mediated isothermal amplification (LAMP) is a novel method for rapid amplification of DNA. Its advantages include rapidity and minimal equipment requirement. The LAMP assay was developed for BK virus (BKV), which is a leading cause of morbidity in renal transplant recipients. The characteristics of the assay, including its specificity and sensitivity, were evaluated. BKV LAMP was performed using various incubation times with a variety of specimens, including unprocessed urine and plasma samples. A ladder pattern on gel electrophoresis, typical of successful LAMP reactions, was observed specifically only for BKV and not for other viruses. The sensitivity of the assay with 1 h of incubation was 100 copies/tube of a cloned BKV fragment. Additionally, a positive reaction was visually ascertained by a simple color reaction using SYBR green dye. BKV LAMP was also successful for urine and plasma specimens without the need for DNA extraction. Due to its simplicity and specificity, the LAMP assay can potentially be developed for “point of care” screening of BKV. PMID:17314224

  20. Bovine embryo sex determination by multiplex loop-mediated isothermal amplification.

    PubMed

    Khamlor, Trisadee; Pongpiachan, Petai; Parnpai, Rangsun; Punyawai, Kanchana; Sangsritavong, Siwat; Chokesajjawatee, Nipa

    2015-03-15

    In cattle, the ability to determine the sex of embryos before embryo transfer is beneficial for increasing the number of animals with the desired sex. This study therefore developed a new modification of loop-mediated isothermal amplification in a multiplex format (multiplex LAMP) for highly efficient bovine embryo sexing. Two chromosomal regions, one specific for males (Y chromosome, S4 region) and the other common to both males and females (1.715 satellite DNA), were amplified in the same reaction tube. Each target was amplified by specifically designed inner primers, outer primers, and loop primers, where one of the S4 loop primers was labeled with the fluorescent dye 6-carboxyl-X-rhodamine (emitting a red color), whereas both satellite loop primers were labeled with the fluorescent dye fluorescein isothiocyanate (emitting a green color). After amplification at 63 °C for 1 hour, the amplified products were precipitated by a small volume of cationic polymer predispensed inside the reaction tube cap. Green precipitate indicated the presence of only control DNA without the Y chromosome, whereas orange precipitate indicated the presence of both target DNAs, enabling interpretation as female and male, respectively. Accuracy of the multiplex LAMP assay was evaluated using 46 bovine embryos with known sex (25 male and 21 female) generated by somatic cell nuclear transfer and confirmed by multiplex polymerase chain reaction. The multiplex LAMP showed 100% accuracy in identifying the actual sex of the embryos and provides a fast, simple, and cost-effective tool for bovine embryo sexing. PMID:25542460

  1. Sensitive Detection of Xanthomonas oryzae Pathovars oryzae and oryzicola by Loop-Mediated Isothermal Amplification

    PubMed Central

    Lang, Jillian M.; Langlois, Paul; Nguyen, Marian Hanna R.; Triplett, Lindsay R.; Purdie, Laura; Holton, Timothy A.; Djikeng, Appolinaire; Vera Cruz, Casiana M.; Verdier, Valérie

    2014-01-01

    Molecular diagnostics for crop diseases can enhance food security by enabling the rapid identification of threatening pathogens and providing critical information for the deployment of disease management strategies. Loop-mediated isothermal amplification (LAMP) is a PCR-based tool that allows the rapid, highly specific amplification of target DNA sequences at a single temperature and is thus ideal for field-level diagnosis of plant diseases. We developed primers highly specific for two globally important rice pathogens, Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight (BB) disease, and X. oryzae pv. oryzicola, the causal agent of bacterial leaf streak disease (BLS), for use in reliable, sensitive LAMP assays. In addition to pathovar distinction, two assays that differentiate X. oryzae pv. oryzae by African or Asian lineage were developed. Using these LAMP primer sets, the presence of each pathogen was detected from DNA and bacterial cells, as well as leaf and seed samples. Thresholds of detection for all assays were consistently 104 to 105 CFU ml?1, while genomic DNA thresholds were between 1 pg and 10 fg. Use of the unique sequences combined with the LAMP assay provides a sensitive, accurate, rapid, simple, and inexpensive protocol to detect both BB and BLS pathogens. PMID:24837384

  2. Evaluation of colorimetric detection methods for Shigella, Salmonella, and Vibrio cholerae by loop-mediated isothermal amplification.

    PubMed

    Soli, Kevin W; Kas, Monalisa; Maure, Tobias; Umezaki, Masahiro; Morita, Ayako; Siba, Peter M; Greenhill, Andrew R; Horwood, Paul F

    2013-12-01

    We evaluated loop-mediated isothermal amplification end-point detection methods for Salmonella, Shigella, and Vibrio cholerae. Detection sensitivities were comparable to real-time PCR methods. The colorimetric dyes hydroxynaphthol blue and SYBR Green I showed increased sensitivity when compared to visual and automated turbidity readings. End-point colorimetric dyes promise great utility in developing settings. PMID:24157057

  3. Current state and future perspectives of loop-mediated isothermal amplification (LAMP)-based diagnosis of filamentous fungi and yeasts.

    PubMed

    Niessen, Ludwig

    2015-01-01

    Loop-mediated isothermal amplification is a rather novel method of enzymatic deoxyribonucleic acid amplification which can be applied for the diagnosis of viruses, bacteria, and fungi. Although firmly established in viral and bacterial diagnosis, the technology has only recently been applied to a noteworthy number of species in the filamentous fungi and yeasts. The current review gives an overview of the literature so far published on the topic by discussing the different groups of fungal organisms to which the method has been applied. Moreover, the method is described in detail as well as the different possibilities available for signal detection and quantification and sample preparation. Future perspective of loop-mediated isothermal amplification-based assays is discussed in the light of applicability for fungal diagnostics. PMID:25492418

  4. Meat Species Identification using Loop-mediated Isothermal Amplification Assay Targeting Species-specific Mitochondrial DNA

    PubMed Central

    2014-01-01

    Meat source fraud and adulteration scandals have led to consumer demands for accurate meat identification methods. Nucleotide amplification assays have been proposed as an alternative method to protein-based assays for meat identification. In this study, we designed Loop-mediated isothermal amplification (LAMP) assays targeting species-specific mitochondrial DNA to identify and discriminate eight meat species; cattle, pig, horse, goat, sheep, chicken, duck, and turkey. The LAMP primer sets were designed and the target genes were discriminated according to their unique annealing temperature generated by annealing curve analysis. Their unique annealing temperatures were found to be 85.56±0.07? for cattle, 84.96±0.08? for pig, and 85.99±0.05? for horse in the BSE-LAMP set (Bos taurus, Sus scrofa domesticus and Equus caballus); 84.91±0.11? for goat and 83.90±0.11? for sheep in the CO-LAMP set (Capra hircus and Ovis aries); and 86.31±0.23? for chicken, 88.66±0.12? for duck, and 84.49±0.08? for turkey in the GAM-LAMP set (Gallus gallus, Anas platyrhynchos and Meleagris gallopavo). No cross-reactivity was observed in each set. The limits of detection (LODs) of the LAMP assays in raw and cooked meat were determined from 10 pg/?L to 100 fg/?L levels, and LODs in raw and cooked meat admixtures were determined from 0.01% to 0.0001% levels. The assays were performed within 30 min and showed greater sensitivity than that of the PCR assays. These novel LAMP assays provide a simple, rapid, accurate, and sensitive technology for discrimination of eight meat species.

  5. Isothermal loop-mediated amplification (lamp) for diagnosis of contagious bovine pleuro-pneumonia

    PubMed Central

    2013-01-01

    Background Contagious Bovine Pleuropneumonia (CBPP) is the most important chronic pulmonary disease of cattle on the African continent causing severe economic losses. The disease, caused by infection with Mycoplasma mycoides subsp. mycoides is transmitted by animal contact and develops slowly into a chronic form preventing an early clinical diagnosis. Because available vaccines confer a low protection rate and short-lived immunity, the rapid diagnosis of infected animals combined with traditional curbing measures is seen as the best way to control the disease. While traditional labour-intensive bacteriological methods for the detection of M. mycoides subsp. mycoides have been replaced by molecular genetic techniques in the last two decades, these latter approaches require well-equipped laboratories and specialized personnel for the diagnosis. This is a handicap in areas where CBPP is endemic and early diagnosis is essential. Results We present a rapid, sensitive and specific diagnostic tool for M. mycoides subsp. mycoides detection based on isothermal loop-mediated amplification (LAMP) that is applicable to field conditions. The primer set developed is highly specific and sensitive enough to diagnose clinical cases without prior cultivation of the organism. The LAMP assay detects M. mycoides subsp. mycoides DNA directly from crude samples of pulmonary/pleural fluids and serum/plasma within an hour using a simple dilution protocol. A photometric detection of LAMP products allows the real-time visualisation of the amplification curve and the application of a melting curve/re-association analysis presents a means of quality assurance based on the predetermined strand-inherent temperature profile supporting the diagnosis. Conclusion The CBPP LAMP developed in a robust kit format can be run on a battery-driven mobile device to rapidly detect M. mycoides subsp. mycoides infections from clinical or post mortem samples. The stringent innate quality control allows a conclusive on-site diagnosis of CBPP such as during farm or slaughter house inspections. PMID:23710975

  6. Integrated centrifugal reverse transcriptase loop-mediated isothermal amplification microdevice for influenza A virus detection.

    PubMed

    Jung, Jae Hwan; Park, Byung Hyun; Oh, Seung Jun; Choi, Goro; Seo, Tae Seok

    2015-06-15

    An integrated reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) microdevice which consists of microbead-assisted RNA purification and RT-LAMP with real-time monitoring by a miniaturized optical detector was demonstrated. The integrated RT-LAMP microdevice includes four reservoirs for a viral RNA sample (purified influenza A viral RNA or lysates), a washing solution (70% ethanol), an elution solution (RNase-free water), and an RT-LAMP cocktail, and two chambers (a waste chamber and an RT-LAMP reaction chamber). The separate reservoirs for a washing solution, an elution solution, and an RT-LAMP cocktail were designed with capillary valves for stable storage. Three influenza A virus strains (A/H1N1, A/H3N2, and A/H5N1) were used for RNA templates, and RT-LAMP primer sets were designed to detect hemagglutinin (HA) and conserved M gene. Sequential sample flow to the microbeads for RNA purification was achieved by centrifugal force with optimization of capillary valves and a siphon channel. Furthermore, the purified RNA solution was successfully isolated from the waste solution by changing the rotational direction, and combined with the RT-LAMP cocktail in the RT-LAMP reaction chamber for target gene amplification. Total process from the sample injection to the result was completed in 47 min. Influenza A H1N1 virus was confirmed on the integrated RT-LAMP microdevice even with 10 copies of viral RNAs, which revealed 10-fold higher sensitivity than that of a conventional RT-PCR. Subtyping and specificity test of influenza A H1N1 viral lysates were also performed and clinical samples were successfully genotyped to confirm influenza A virus on our proposed integrated microdevice. PMID:25569879

  7. Sensitive and Rapid Detection of Giardia lamblia Infection in Pet Dogs using Loop-Mediated Isothermal Amplification

    PubMed Central

    Li, Jie; Wang, Peiyuan; Zhang, Aiguo; Zhang, Ping; Alsarakibi, Muhamd

    2013-01-01

    Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from 10-1 to 10-5 ng/µl for LAMP and PCR assays. The LAMP assay allows the amplification to be finished within 60 min under isothermal conditions of 63? by employing 6 oligonucleotide primers designed based on G. lamblia elongation factor 1 alpha (EF1?) gene sequence. Our tests showed that the specific amplification products were obtained only with G. lamblia, while no amplification products were detected with DNA of other related protozoans. Sensitivity evaluation indicated that the LAMP assay was sensitive 10 times more than PCR. It is concluded that LAMP is a rapid, highly sensitive and specific DNA amplification technique for detection of G. lamblia, which has implications for effective control and prevention of giardiasis. PMID:23710094

  8. Rapid detection of genetically diverse tomato black ring virus isolates using reverse transcription loop-mediated isothermal amplification.

    PubMed

    Hasiów-Jaroszewska, Beata; Budzy?ska, Daria; Borodynko, Natasza; Pospieszny, Henryk

    2015-12-01

    A reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) has been developed for detection of tomato black ring virus (TBRV) isolates collected from different hosts. One-step RT-LAMP was performed with a set of four primers, the design of which was based on the coat protein gene. Results of RT-LAMP were visualized by direct staining of products with fluorescent dyes, agarose gel electrophoresis, and analysis of amplification curves. The sensitivity of RT-LAMP was 100-fold greater than that of RT-PCR. The RT-LAMP assay developed here is a useful and practical method for diagnosis of TBRV. PMID:26338092

  9. Detection of the Quarantine Species Thrips palmi by Loop-Mediated Isothermal Amplification

    PubMed Central

    Przybylska, Arnika; Fiedler, ?aneta; Kucharczyk, Halina; Obr?palska-St?plowska, Aleksandra

    2015-01-01

    Thrips palmi (from the order Thysanoptera) is a serious insect pest of various crops, including vegetables, fruits and ornamental plants, causing significant economic losses. Its presence constitutes a double threat; not only does T. palmi feed on the plants, it is also a vector for several plant viruses. T. palmi originated in Asia, but has spread to North and Central America, Africa, Oceania and the Caribbean in recent decades. This species has been sporadically noted in Europe and is under quarantine regulation in the European Union. For non-specialists its larval stages are indistinguishable morphologically from another widespread and serious insect pest Frankliniella occidentalis (a non-quarantine species in the European Union) as well as other frequently occurring thrips. In this study, we have developed a loop-mediated isothermal amplification protocol to amplify rDNA regions of T. palmi. The results were consistent whether isolated DNA or crushed insects were used as template, indicating that the DNA isolation step could be omitted. The described method is species-specific and sensitive and provides a rapid diagnostic tool to detect T. palmi in the field. PMID:25793743

  10. Centrifugal loop-mediated isothermal amplification microdevice for rapid, multiplex and colorimetric foodborne pathogen detection.

    PubMed

    Oh, Seung Jun; Park, Byung Hyun; Jung, Jae Hwan; Choi, Goro; Lee, Doh C; Kim, Do Hyun; Seo, Tae Seok

    2016-01-15

    We present a centrifugal microfluidic device which enables multiplex foodborne pathogen identification by loop-mediated isothermal amplification (LAMP) and colorimetric detection using Eriochrome Black T (EBT). Five identical structures were designed in the centrifugal microfluidic system to perform the genetic analysis of 25 pathogen samples in a high-throughput manner. The sequential loading and aliquoting of the LAMP cocktail, the primer mixtures, and the DNA sample solutions were accomplished by the optimized zigzag-shaped microchannels and RPM control. We targeted three kinds of pathogenic bacteria (Escherichia coli O157:H7, Salmonella typhimurium and Vibrio parahaemolyticus) and detected the amplicons of the LAMP reaction by the EBT-mediated colorimetric method. For the limit-of-detection (LOD) test, we carried out the LAMP reaction on a chip with serially diluted DNA templates of E. coli O157:H7, and could observe the color change with 380 copies. The used primer sets in the LAMP reaction were specific only to the genomic DNA of E. coli O157:H7, enabling the on-chip selective, sensitive, and high-throughput pathogen identification with the naked eyes. The entire process was completed in 60min. Since the proposed microsystem does not require any bulky and expensive instrumentation for end-point detection, our microdevice would be adequate for point-of-care (POC) testing with high simplicity and high speed, providing an advanced genetic analysis microsystem for foodborne pathogen detection. PMID:26322592

  11. Detection of Vibrio parahaemolyticus in food samples using in situ loop-mediated isothermal amplification method.

    PubMed

    Wang, Li; Shi, Lei; Su, Jianyu; Ye, Yuxin; Zhong, Qingping

    2013-02-25

    A novel in situ loop-mediated isothermal amplification (in situ LAMP) technique for rapid detection of the food-borne Vibrio parahaemolyticus strains had been developed and evaluated in this study. The sensitivity of the in situ LAMP assay was detected to be 10 CFU/reaction via test in serial 10-fold dilutions of V. parahaemolyticus cells, and high specificity had also been obtained through confirmation with 14 reference gram-positive and -negative strains. Application of the established in situ LAMP assay had been performed on 58 strains previously isolated from seafood samples, including 48 V. parahaemolyticus and 10 non-V. parahaemolyticus strains. Of 48 V. parahaemolyticus strains, 48, 45 and 34 strains were detected as positive by in situ LAMP, regular LAMP and PCR, respectively, with the detection rate and negative predictive value (NPV) found to be 100% vs 93.8% vs 70.8% and 100% vs 76.9% vs 41.7%. In addition, none of the tested non-V. parahaemolyticus strains showed positive result, indicating a 100% positive predictive value (PPV) for all of 3 assays. Compared with regular LAMP methods and PCR-based methods, the in situ LAMP assay is advantageous on rapidity, high specificity, less time consumption and ease in operation, and may provide a novel, useful and practical detection platform for pathogens in food safety laboratories. PMID:23266627

  12. Detection of capripoxvirus DNA using a novel loop-mediated isothermal amplification assay

    PubMed Central

    2013-01-01

    Background Sheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are the most serious poxviruses of ruminants. They are double stranded DNA viruses of the genus Capripoxvirus, (subfamily Chordopoxvirinae) within the family Poxviridae. The aim of this study was to develop a Loop-mediated isothermal AMPlification (LAMP) assay for the detection of Capripoxvirus (CaPV) DNA. Results A single LAMP assay targeting a conserved region of the CaPV P32 gene was selected from 3 pilot LAMP assays and optimised by adding loop primers to accelerate the reaction time. This LAMP assay successfully detected DNA prepared from representative CaPV isolates (SPPV, GTPV and LSDV), and did not cross-react with DNA extracted from other mammalian poxviruses. The analytical sensitivity of the LAMP assay was determined to be at least 163 DNA copies/?l which is equivalent to the performance reported for diagnostic real-time PCR currently used for the detection of CaPV. LAMP reactions were monitored with an intercalating dye using a real-time PCR machine, or by agarose-gel electrophoresis. Furthermore, dual labelled LAMP products (generated using internal LAMP primers that were conjugated with either biotin or fluorescein) could be readily visualised using a lateral-flow device. Conclusions This study provides a simple and rapid approach to detect CaPV DNA that may have utility for use in the field, or in non-specialised laboratories where expensive equipment is not available. PMID:23634704

  13. 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

  14. Molecular detection of Coxiella burnetii using an alternative loop-mediated isothermal amplification assay (LAMP).

    PubMed

    Raele, Donato Antonio; Garofolo, Giuliano; Galante, Domenico; Cafiero, Maria Assunta

    2015-01-01

    Q fever, caused by Coxiella burnetii, is a worldwide zoonosis with important consequences for human and animal health. In livestock, the diagnosis, using direct and indirect techniques, is challenging even if to tackle coxiellosis in domesticated animals a rapid diagnosis is crucial. In the recent years, new molecular methods have been developed to overcome these issues. Several polymerase chain reaction (PCR) assays have been studied, but loop mediated isothermal amplification (LAMP) has not been fully developed. This new methodology is emerging due to simplicity and speed in diagnosis of microbial diseases. In this study, we design a new LAMP assay against C. burnetii targeting the com1 gene as an actual alternative to conventional PCR. The assay was specific to C. burnetii reactive with sensitivity comparable to standard PCR. The application of the com1 LAMP on 10 clinical samples from water buffalo, sheep, and goats, previously tested positive, confirmed the presence of C. burnetii. To our knowledge, this study is the first report of LAMP targeting C. burnetii in Europe and the results also suggest that it may be an useful and cost-effective tool for the clinical and epidemiological surveillance of Q Fever. PMID:25842216

  15. Multicenter Clinical Evaluation of the Novel Alere i Strep A Isothermal Nucleic Acid Amplification Test

    PubMed Central

    Russo, Michael E.; Jaggi, Preeti; Kline, Jennifer; Gluckman, William; Parekh, Amisha

    2015-01-01

    Rapid detection of group A beta-hemolytic streptococcus (GAS) is used routinely to help diagnose and treat pharyngitis. However, available rapid antigen detection tests for GAS have relatively low sensitivity, and backup testing is recommended in children. Newer assays are more sensitive yet require excessive time for practical point-of-care use as well as laboratory personnel. The Alere i strep A test is an isothermal nucleic acid amplification test designed to offer highly sensitive results at the point of care within 8 min when performed by nonlaboratory personnel. The performance of the Alere i strep A test was evaluated in a multicenter prospective trial in a Clinical Laboratory Improvement Amendments (CLIA)-waived setting in comparison to bacterial culture in 481 children and adults. Compared to culture, the Aleri i strep A test had 96.0% sensitivity and 94.6% specificity. Discrepant results were adjudicated by PCR and found the Alere i strep A test to have 98.7% sensitivity and 98.5% specificity. Overall, the Alere i strep A test could provide a one-step, rapid, point-of-care testing method for GAS pharyngitis and obviate backup testing on negative results. PMID:25972418

  16. Colorimetric Detection of Dengue by Single Tube Reverse-Transcription-Loop-Mediated Isothermal Amplification

    PubMed Central

    Teoh, Boon-Teong; Abd-Jamil, Juraina; Johari, Jefree; Sam, Sing-Sin; Tan, Kim-Kee; AbuBakar, Sazaly

    2015-01-01

    Dengue is usually diagnosed by isolation of the virus, serology or molecular diagnostic methods. Several commercial kits for the diagnosis of dengue are existing, but concerns have arisen regarding to the affordability and performance characteristics of these kits. Hence, the loop-mediated isothermal amplification (LAMP) is potentially ideal to be used especially in resource limited environments. Serum was collected from healthy donors and patients diagnosed with dengue infection. RNA extracted from the serum samples were tested by reverse-transcription-LAMP assay developed based on 3?-NCR gene sequences for DENV 1–4. Results were interpreted by a turbidity meter in real time or visually at the end of the assay. Sensitivity and specificity of RT-LAMP results were calculated and compared to qRT-PCR and ELISA. RT-LAMP is highly sensitive with the detection limit of 10 RNA copies for all serotypes. Dengue virus RNA was detected in all positive samples using RT-LAMP and none of the negative samples within 30–45 minutes. With continuing efforts in the optimization of this assay, RT-LAMP may provide a simple and reliable test for detecting DENV in areas where dengue is prevalent. PMID:26384248

  17. Rapid detection of Mycoplasma synoviae by loop-mediated isothermal amplification.

    PubMed

    Kursa, Olimpia; Wo?niakowski, Grzegorz; Tomczyk, Grzegorz; Sawicka, Anna; Minta, Zenon

    2015-03-01

    Mycoplasma synoviae (MS) remains a serious concern in production of poultry and affects world production of chickens and turkeys. Loop-mediated isothermal amplification (LAMP) of DNA has been recently used for the identification of different economically important avian pathogens. The aim of this study was to develop LAMP for simple and inexpensive detection of MS strains in poultry using specifically designed primers targeting hemagglutin A (vlh) gene. The assay was conducted in a water bath for 1 h at 63 °C. The results were visualized after addition of SYBR Green(®) fluorescent dye. LAMP was specific exclusively for MS without cross-reactivity with other Mycoplasma species. The sensitivity of LAMP was determined as 10(-1) CFU/ml and was 1,000 times higher than MS-specific polymerase chain reaction. LAMP assay was conducted on 18 MS field strains to ensure its reliability and usefulness. This is the first report on LAMP development and application for the rapid detection of MS isolated from chickens. This simple method may be applied by diagnostic laboratories without access to expensive equipment. PMID:25413672

  18. Multicenter Clinical Evaluation of the Novel Alere i Strep A Isothermal Nucleic Acid Amplification Test.

    PubMed

    Cohen, Daniel M; Russo, Michael E; Jaggi, Preeti; Kline, Jennifer; Gluckman, William; Parekh, Amisha

    2015-07-01

    Rapid detection of group A beta-hemolytic streptococcus (GAS) is used routinely to help diagnose and treat pharyngitis. However, available rapid antigen detection tests for GAS have relatively low sensitivity, and backup testing is recommended in children. Newer assays are more sensitive yet require excessive time for practical point-of-care use as well as laboratory personnel. The Alere i strep A test is an isothermal nucleic acid amplification test designed to offer highly sensitive results at the point of care within 8 min when performed by nonlaboratory personnel. The performance of the Alere i strep A test was evaluated in a multicenter prospective trial in a Clinical Laboratory Improvement Amendments (CLIA)-waived setting in comparison to bacterial culture in 481 children and adults. Compared to culture, the Aleri i strep A test had 96.0% sensitivity and 94.6% specificity. Discrepant results were adjudicated by PCR and found the Alere i strep A test to have 98.7% sensitivity and 98.5% specificity. Overall, the Alere i strep A test could provide a one-step, rapid, point-of-care testing method for GAS pharyngitis and obviate backup testing on negative results. PMID:25972418

  19. Loop-mediated isothermal amplification: rapid detection of Angiostrongylus cantonensis infection in Pomacea canaliculata

    PubMed Central

    2011-01-01

    Background Angiostrongylus cantonensis is a zoonotic parasite that causes eosinophilic meningitis in humans. The most common source of infection with A. cantonensis is the consumption of raw or undercooked mollusks (e.g., snails and slugs) harbouring infectious third-stage larvae (L3). However, the parasite is difficult to identify in snails. The purpose of this study was to develop a quick, simple molecular method to survey for A. cantonensis in intermediate host snails. Findings We used a loop-mediated isothermal amplification (LAMP) assay, which was performed using Bst DNA polymerase. Reactions amplified the A. cantonensis 18S rRNA gene and demonstrated high sensitivity; as little as 1 fg of DNA was detected in the samples. Furthermore, no cross-reactivity was found with other parasites such as Toxoplasma gondii, Plasmodium falciparum, Schistosoma japonicum, Clonorchis sinensis, Paragonimus westermani and Anisakis. Pomacea canaliculata snails were exposed to A. cantonensis first-stage larvae (L1) in the laboratory, and L3 were observed in the snails thirty-five days after infection. All nine samples were positive as determined by the LAMP assay for A. cantonensis, which was identified as positive by using PCR and microscopy, this demonstrates that LAMP is sensitive and effective for diagnosis. Conclusions LAMP is an appropriate diagnostic method for the routine identification of A. cantonensis within its intermediate host snail P. canaliculata because of its simplicity, sensitivity, and specificity. It holds great promise as a useful monitoring tool for A. cantonensis in endemic regions. PMID:22023992

  20. Detection of BCR-ABL Fusion mRNA Using Reverse Transcriptase Loop-mediated Isothermal Amplification

    SciTech Connect

    Dugan, L C; Hall, S; Kohlgruber, A; Urbin, S; Torres, C; Wilson, P

    2011-12-08

    RT-PCR is commonly used for the detection of Bcr-Abl fusion transcripts in patients diagnosed with chronic myelogenous leukemia, CML. Two fusion transcripts predominate in CML, Br-Abl e13a2 and e14a2. They have developed reverse transcriptase isothermal loop-mediated amplification (RT-LAMP) assays to detect these two fusion transcripts along with the normal Bcr transcript.

  1. Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of Arcanobacterium pluranimalium.

    PubMed

    Abdulmawjood, A; Wickhorst, J; Sammra, O; Lämmler, C; Foster, G; Wragg, P N; Prenger-Berninghoff, E; Klein, G

    2015-12-01

    In the present study 28 Arcanobacterium pluranimalium strains isolated from various origins could successfully be identified with a newly designed loop-mediated isothermal amplification (LAMP) assay based on gene pla encoding pluranimaliumlysin. No comparable reaction could be observed with control strains representing five species of genus Arcanobacterium and five species of genus Trueperella. The presented pla LAMP assay might allow a reliable and low cost identification of this bacterial pathogen also in laboratories with less specified equipment. PMID:26093093

  2. Mismatch extension of DNA polymerases and high-accuracy single nucleotide polymorphism diagnostics by gold nanoparticle-improved isothermal amplification.

    PubMed

    Chen, Feng; Zhao, Yue; Fan, Chunhai; Zhao, Yongxi

    2015-09-01

    Sequence mismatches may induce nonspecific extension reaction, causing false results for SNP diagnostics. Herein, we systematically investigated the impact of various 3'-terminal mismatches on isothermal amplification catalyzed by representative DNA polymerases. Despite their diverse efficiencies depending on types of mismatch and kinds of DNA polymerase, all 12 kinds of single 3'-terminal mismatches induced the extension reaction. Generally, only several mismatches (primer-template, C-C, G-A, A-G, and A-A) present an observable inhibitory effect on the amplification reaction, whereas other mismatches trigger amplified signals as high as those of Watson-Crick pairs. The related mechanism was deeply discussed, and a primer-design guideline for specific SNP analysis was summarized. Furthermore, we found that the addition of appropriate gold nanoparticles (AuNPs) can significantly inhibit mismatch extension and enhance the amplification specificity. Also the high-accuracy SNP analysis of human blood genomic DNA has been demonstrated by AuNPs-improved isothermal amplification, the result of which was verified by sequencing (the gold standard method for SNP assay). Collectively, this work provides mechanistic insight into mismatch behavior and achieves accurate SNP diagnostics, holding great potential for the application in molecular diagnostics and personalized medicine. PMID:26249366

  3. Label-free molecular beacon-based quadratic isothermal exponential amplification: a simple and sensitive one-pot method to detect DNA methyltransferase activity.

    PubMed

    Xue, Qingwang; Wang, Lei; Jiang, Wei

    2015-09-11

    We developed a one-pot label-free molecular beacon-mediated quadratic isothermal exponential amplification strategy (LFMB-QIEA) for simple, rapid and sensitive DNA methyltransferase (MTase) activity detection. PMID:26219276

  4. Development of a loop-mediated isothermal amplification assay for rapid detection of capripoxviruses.

    PubMed

    Das, Amaresh; Babiuk, Shawn; McIntosh, Michael T

    2012-05-01

    Sheep pox (SP), goat pox (GP), and lumpy skin disease (LSD), caused by capripoxviruses (CaPVs), are economically important diseases of sheep, goats, and cattle, respectively. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of CaPVs. LAMP primers were designed to target a conserved gene encoding the poly(A) polymerase small subunit (VP39) of CaPVs. Hydroxynaphthol blue (HNB) was incorporated to monitor assay progress by color change from violet when negative to sky blue when positive, and results were verified by agarose gel electrophoresis. The LAMP assay was shown to be highly specific for CaPVs, with no apparent cross-reactivity to other related viruses (near neighbors) or viruses that cause similar clinical signs (look-a-like viruses). The performance of LAMP was compared to that of a highly sensitive quantitative real-time PCR (qPCR) assay. LAMP and qPCR exhibited similar analytical sensitivities, with limits of detection of 3 and 8 viral genome copies, respectively. Diagnostic specificity was assessed on 36 negative specimens, including swabs and EDTA blood from control sheep, goats, and cattle. Diagnostic sensitivity was assessed on 275 specimens, including EDTA blood, swabs, and tissues from experimentally infected sheep, goats, and cattle. Overall agreement on diagnostic test results between the two assays was 90 to 95% for specificity and 89 to 100% for sensitivity. The LAMP assay described in this report is simple to use, inexpensive, highly sensitive, and particularly well suited for the diagnosis of capripox in less well equipped laboratories and in rural settings where resources are limited. PMID:22357504

  5. Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Capripoxviruses

    PubMed Central

    Babiuk, Shawn; McIntosh, Michael T.

    2012-01-01

    Sheep pox (SP), goat pox (GP), and lumpy skin disease (LSD), caused by capripoxviruses (CaPVs), are economically important diseases of sheep, goats, and cattle, respectively. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of CaPVs. LAMP primers were designed to target a conserved gene encoding the poly(A) polymerase small subunit (VP39) of CaPVs. Hydroxynaphthol blue (HNB) was incorporated to monitor assay progress by color change from violet when negative to sky blue when positive, and results were verified by agarose gel electrophoresis. The LAMP assay was shown to be highly specific for CaPVs, with no apparent cross-reactivity to other related viruses (near neighbors) or viruses that cause similar clinical signs (look-a-like viruses). The performance of LAMP was compared to that of a highly sensitive quantitative real-time PCR (qPCR) assay. LAMP and qPCR exhibited similar analytical sensitivities, with limits of detection of 3 and 8 viral genome copies, respectively. Diagnostic specificity was assessed on 36 negative specimens, including swabs and EDTA blood from control sheep, goats, and cattle. Diagnostic sensitivity was assessed on 275 specimens, including EDTA blood, swabs, and tissues from experimentally infected sheep, goats, and cattle. Overall agreement on diagnostic test results between the two assays was 90 to 95% for specificity and 89 to 100% for sensitivity. The LAMP assay described in this report is simple to use, inexpensive, highly sensitive, and particularly well suited for the diagnosis of capripox in less well equipped laboratories and in rural settings where resources are limited. PMID:22357504

  6. Embryo Sexing and Sex Chromosomal Chimerism Analysis by Loop-Mediated Isothermal Amplification in Cattle and Water Buffaloes

    PubMed Central

    HIRAYAMA, Hiroki; KAGEYAMA, Soichi; MORIYASU, Satoru; SAWAI, Ken; MINAMIHASHI, Akira

    2013-01-01

    Abstract In domestic animals of the family Bovidae, sex preselection of offspring has been demanded for convenience of milk/beef production and animal breeding. Development of the nonsurgical embryo transfer technique and sexing methods of preimplantation embryos made it possible. Sexing based on detection of Y chromosome-specific DNA sequences is considered the most reliable method to date. PCR enables amplification of a target sequence from a small number of blastomeres. However, it requires technical skill and is time consuming. Furthermore, PCR has the risk of false positives because of DNA contamination during handling of the PCR products in duplicate PCR procedures and/or electrophoresis. Therefore, for embryo sexing to become widely used in the cattle embryo transfer industry, a simple, rapid and precise sexing method needs to be developed. Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method, and the reaction is carried out under isothermal conditions (range, 60 to 65 C) using DNA polymerase with strand displacement activity. When the target DNA is amplified by LAMP, a white precipitate derived from magnesium pyrophosphate (a by-product of the LAMP reaction) is observed. It is noteworthy that LAMP does not need special reagents or electrophoresis to detect the amplified DNA. This review describes the development and application of an embryo sexing method using LAMP in cattle and water buffaloes. PMID:23965599

  7. Rapid and Sensitive Identification of the Herbal Tea Ingredient Taraxacum formosanum Using Loop-Mediated Isothermal Amplification

    PubMed Central

    Lai, Guan-Hua; Chao, Jung; Lin, Ming-Kuem; Chang, Wen-Te; Peng, Wen-Huang; Sun, Fang-Chun; Lee, Meng-Shiunn; Lee, Meng-Shiou

    2015-01-01

    Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan. In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established. A set of four specific LAMP primers was designed based on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when purified genomic DNA of TF was added in the LAMP reaction under isothermal condition (65 °C) within 45 min. These specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the detection limit of the LAMP assay. In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control. PMID:25584616

  8. Detection of cucumber mosaic virus isolates from banana by one-step reverse transcription loop-mediated isothermal amplification.

    PubMed

    Peng, Jun; Shi, Minjing; Xia, Zihao; Huang, Junsheng; Fan, Zaifeng

    2012-11-01

    Cucumber mosaic virus (CMV) is one of the most devastating threats to the banana industry. A single-tube, one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid detection of CMV-infected banana and plantain (Musa spp.). The reaction was performed in a single tube at 63 °C for 90 min using a real-time turbidimeter, with an improved closed-tube visual detection system in which fluorescent dye was added to the inside of the lid prior to amplification. This RT-LAMP assay is an alternative method for the rapid detection of CMV in banana plants and tissue culture materials. PMID:22782136

  9. Increased Robustness of Single-Molecule Counting with Microfluidics, Digital Isothermal Amplification, and a Mobile Phone

    E-print Network

    Ismagilov, Rustem F.

    Amplification, and a Mobile Phone versus Real-Time Kinetic Measurements David A. Selck,¶ Mikhail A. Karymov with a consumer cell- phone camera, and to automated cloud-based processing of these images (R2 = 0.9997 vs true be imaged with the cell phone camera using flash as the excitation source. Many nonlinear amplification

  10. One-step reverse transcription loop-mediated isothermal amplification for the rapid detection of cucumber green mottle mosaic virus.

    PubMed

    Li, Jin-yu; Wei, Qi-wei; Liu, Yong; Tan, Xin-qiu; Zhang, Wen-na; Wu, Jian-yan; Charimbu, Miriam Karwitha; Hu, Bai-shi; Cheng, Zhao-bang; Yu, Cui; Tao, Xiao-rong

    2013-11-01

    Cucumber green mottle mosaic virus (CGMMV) has caused serious damage to Cucurbitaceae crops worldwide. The virus is considered one of the most serious Cucurbitaceae quarantine causes in many countries. In this study, a highly efficient and practical one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of CGMMV. The total RNA or crude RNA extracted from watermelon plants or seeds could be detected easily by this RT-LAMP assay. The RT-LAMP assay was conducted in isothermal (63°C) conditions within 1h. The amplified products of CGMMV could be detected as ladder-like bands using agarose gel electrophoresis or visualized in-tube under UV light with the addition of a fluorescent dye. The RT-LAMP amplification was specific to CGMMV, as no cross-reaction was observed with other viruses. The RT-LAMP assay was 100-fold more sensitive than that of reverse-transcription polymerase chain reaction (RT-PCR). This is the first report of the application of the RT-LAMP assay to detect CGMMV. The sensitive, specific and rapid RT-LAMP assay developed in this study can be applied widely in laboratories, the field and quarantine surveillance of CGMMV. PMID:23933076

  11. On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria.

    PubMed

    Kunze, A; Dilcher, M; Abd El Wahed, A; Hufert, F; Niessner, R; Seidel, M

    2016-01-01

    This work presents an on-chip isothermal nucleic acid amplification test (iNAAT) for the multiplex amplification and detection of viral and bacterial DNA by a flow-based chemiluminescence microarray. In a principle study, on-chip recombinase polymerase amplification (RPA) on defined spots of a DNA microarray was used to spatially separate the amplification reaction of DNA from two viruses (Human adenovirus 41, Phi X 174) and the bacterium Enterococcus faecalis, which are relevant for water hygiene. By establishing the developed assay on the microarray analysis platform MCR 3, the automation of isothermal multiplex-amplification (39 °C, 40 min) and subsequent detection by chemiluminescence imaging was realized. Within 48 min, the microbes could be identified by the spot position on the microarray while the generated chemiluminescence signal correlated with the amount of applied microbe DNA. The limit of detection (LOD) determined for HAdV 41, Phi X 174, and E. faecalis was 35 GU/?L, 1 GU/?L, and 5 × 10(3) GU/?L (genomic units), which is comparable to the sensitivity reported for qPCR analysis, respectively. Moreover the simultaneous amplification and detection of DNA from all three microbes was possible. The presented assay shows that complex enzymatic reactions like an isothermal amplification can be performed in an easy-to-use experimental setup. Furthermore, iNAATs can be potent candidates for multipathogen detection in clinical, food, or environmental samples in routine or field monitoring approaches. PMID:26624222

  12. GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use

    PubMed Central

    2012-01-01

    Background There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM) crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR) and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART) occurs at a constant temperature and only requires a simple light detection and integration device. Results Loop mediated isothermal amplification (LAMP) shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART) for determination of genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation. Conclusions LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must be controlled to ensure quantification at low target concentrations. PMID:22546148

  13. [Evaluating the Stability of Loop-Mediated Isothermal Amplification Reagents at Irregular Storage Temperatures for On-Site Diagnosis].

    PubMed

    Inoshima, Yasuo; Ishiguro, Naotaka

    2015-11-11

    Temperature-stability of loop-mediated isothermal amplification (LAMP) reagents was determined for their use in on-site diagnosis, such as in farms/pastures. Bst and Csa DNA polymerases and the reagents that were stored at different temperatures (4 or 25°C) for 1, 2, or 4 days were used for the LAMP assay to detect orf virus DNA as a model. After storage at 4 and 25°C for 2 days, the enzymes and reagents were found to retain sufficient activity to carry out successful DNA amplification. Visual diagnosis was also possible with the reagents (Loopamp Fluorescent Detection Reagent or hydroxy naphthol blue, as well as DNA amplification checker, D-Quick) that were stored for 2 days at different temperatures. Although the time taken to obtain the positive/negative results were delayed, the enzymes and reagents, stored at 25°C for 4 days, were active and had the ability to efficiently amplify DNA in less than 50 min. These results indicate that LAMP assay can be successfully utilized for the diagnosis of infectious diseases under non-clinical settings such as for on-site diagnosis in farms/pastures, owing to the fact that the relevant enzymes and reagents does not require restricted temperature storage. PMID:26635003

  14. Point of care nucleic acid detection of viable pathogenic bacteria with isothermal RNA amplification based paper biosensor

    NASA Astrophysics Data System (ADS)

    Liu, Hongxing; Xing, Da; Zhou, Xiaoming

    2014-09-01

    Food-borne pathogens such as Listeria monocytogenes have been recognized as a major cause of human infections worldwide, leading to substantial health problems. Food-borne pathogen identification needs to be simpler, cheaper and more reliable than the current traditional methods. Here, we have constructed a low-cost paper biosensor for the detection of viable pathogenic bacteria with the naked eye. In this study, an effective isothermal amplification method was used to amplify the hlyA mRNA gene, a specific RNA marker in Listeria monocytogenes. The amplification products were applied to the paper biosensor to perform a visual test, in which endpoint detection was performed using sandwich hybridization assays. When the RNA products migrated along the paper biosensor by capillary action, the gold nanoparticles accumulated at the designated Test line and Control line. Under optimized experimental conditions, as little as 0.5 pg/?L genomic RNA from Listeria monocytogenes could be detected. The whole assay process, including RNA extraction, amplification, and visualization, can be completed within several hours. The developed method is suitable for point-of-care applications to detect food-borne pathogens, as it can effectively overcome the false-positive results caused by amplifying nonviable Listeria monocytogenes.

  15. Real-time isothermal detection of Shiga toxin-producing Escherichia coli using recombinase polymerase amplification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin (Stx) producing E. coli (STEC) are a major family of foodborne pathogens of immense public health, zoonotic and economic significance in the US and worldwide. To date, there are no published reports on use of recombinase polymerase amplification (RPA) for STEC detection. The primary goal...

  16. Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Trichosporon asahii in Experimental and Clinical Samples

    PubMed Central

    Zhou, Jianfeng; Liao, Yong; Li, Haitao; Lu, Xuelian; Han, Xiufeng; Tian, Yanli; Chen, Shanshan; Yang, Rongya

    2015-01-01

    Invasive trichosporonosis is a deep mycosis found mainly in immunocompromised hosts, and the major pathogen is Trichosporon asahii. We detected the species-specific intergenic spacers (IGS) of rRNA gene of T. asahii using a loop-mediated isothermal amplification (LAMP) assay in 15 isolates with 3 different visualization methods, including SYBR green detection, gel electrophoresis, and turbidimetric methods. The LAMP assay displayed superior rapidity to other traditional methods in the detection time; that is, only 1?h was needed for detection and identification of the pathogen DNA. Furthermore, the detection limit of the LAMP assay was more sensitive than the PCR assay. We also successfully detect the presence of T. asahii in samples from experimentally infected mice and samples from patients with invasive trichosporonosis caused by T. asahii, suggesting that this method may become useful in clinical applications in the near future. PMID:25692144

  17. A rapid and precise diagnostic method for detecting the Pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification.

    PubMed

    Kikuchi, Taisei; Aikawa, Takuya; Oeda, Yuka; Karim, Nurul; Kanzaki, Natsumi

    2009-12-01

    ABSTRACT Bursaphelenchus xylophilus is the causal agent of pine wilt disease, which is a major forest disease in Japan, Korea, China, Taiwan, and Portugal. A diagnostic method which is rapid, precise, and simple could greatly help the proper management of this disease. Here, we present a novel detection method using loop-mediated isothermal amplification (LAMP) targeting the internal transcribed spacer region of ribosomal DNA of the nematode. Specificity of the primers and LAMP was confirmed using DNA from various nematode species related to B. xylophilus. Our experimental results suggest that LAMP can detect B. xylophilus faster and with higher sensitivity than the traditional diagnostic method. Moreover, because it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions. PMID:19900002

  18. Determination of ABO blood group genotypes using the real-time loop-mediated isothermal amplification method

    PubMed Central

    ZHANG, CHAO; ZHU, JUANLI; YANG, JIANGCUN; WAN, YINSHENG; MA, TING; CUI, YALI

    2015-01-01

    ABO genotyping is commonly used in several situations, including blood transfusion, personal identification and disease detection. The present study developed a novel method for ABO genotyping, using loop-mediated isothermal amplification (LAMP). This method allows the simultaneous determination of six ABO genotypes under 40 min at a constant temperature of 62°C. The genotypes of 101 blood samples were determined to be AA (n=6), AO (n=38), BB (n=12), BO (n =29), AB (n=8) and OO (n=8) by the LAMP assay. The results were compared with the phenotypes determined by serological assay and the genotypes determined by direct sequencing, and no discrepancies were observed. This novel and rapid method, with good accuracy and reasonably cost effective, provides a supplement to routine serological ABO typing and may also be useful in other point-of-care testing. PMID:26238310

  19. Detection of methicillin-resistant Staphylococcus aureus directly by loop-mediated isothermal amplification and direct cefoxitin disk diffusion tests.

    PubMed

    Metwally, L; Gomaa, N; Hassan, R

    2014-04-01

    We evaluated the utility of 2 methods for detection of methicillin-resistant Staphylococcus aureus (MRSA) directly from signal-positive blood culture bottles: loop-mediated isothermal amplification (LAMP) assay, and direct cefoxitin disk diffusion (DCDD) test using a 30 ?g cefoxitin disk. In parallel, standard microbiological identification and oxacillin susceptibility testing with MecA PCR was performed. Of 60 blood cultures positive for Gram-positive cocci in clusters, LAMP (via detection of the FemA and MecA genes) showed 100% sensitivity and specificity for identification of MRSA/MSSA. When coagulase-negative staphylococci were tested, sensitivity for detection of methicillin resistance was 91.7% and specificity was 100%. DCDD along with direct tube coagulase assay detected only 80.6% of MRSA/MSSA. LAMP showed higher diagnostic accuracy although DCDD was more cost-effective and did not require additional reagents or supplies. PMID:24952125

  20. Point-of-care multiplexed assays of nucleic acids using microcapillary-based loop-mediated isothermal amplification.

    PubMed

    Zhang, Yi; Zhang, Lu; Sun, Jiashu; Liu, Yulei; Ma, Xingjie; Cui, Shangjin; Ma, Liying; Xi, Jianzhong Jeff; Jiang, Xingyu

    2014-07-15

    This report demonstrates a straightforward, robust, multiplexed and point-of-care microcapillary-based loop-mediated isothermal amplification (cLAMP) for assaying nucleic acids. This assay integrates capillaries (glass or plastic) to introduce and house sample/reagents, segments of water droplets to prevent contamination, pocket warmers to provide heat, and a hand-held flashlight for a visual readout of the fluorescent signal. The cLAMP system allows the simultaneous detection of two RNA targets of human immunodeficiency virus (HIV) from multiple plasma samples, and achieves a high sensitivity of two copies of standard plasmid. As few nucleic acid detection methods can be wholly independent of external power supply and equipment, our cLAMP holds great promise for point-of-care applications in resource-poor settings. PMID:24937125

  1. Non-instrumented DNA isolation, amplification and microarray-based hybridization for a rapid on-site detection of devastating Phytophthora kernoviae.

    PubMed

    Schwenkbier, Lydia; Pollok, Sibyll; Rudloff, Anne; Sailer, Sebastian; Cialla-May, Dana; Weber, Karina; Popp, Jürgen

    2015-10-01

    A rapid and simple instrument-free detection system was developed for the identification of the plant pathogen Phytophthora kernoviae (P. kernoviae). The on-site operable analysis steps include magnetic particle based DNA isolation, helicase-dependent amplification (HDA) and chip-based DNA hybridization. The isothermal approach enabled the convenient amplification of the yeast GTP-binding protein (Ypt1) target gene in a miniaturized HDA-zeolite-heater (HZH) by an exothermic reaction. The amplicon detection on the chip was performed under room temperature conditions – either by successive hybridization and enzyme binding or by a combined step. A positive signal is displayed by enzymatically generated silver nanoparticle deposits, which serve as robust endpoint signals allowing an immediate visual readout. The hybridization assay enabled the reliable detection of 10 pg ?L(-1) target DNA. This is the first report of an entirely electricity-free, field applicable detection approach for devastating Phytophthora species, exemplarily shown for P. kernoviae. PMID:26331157

  2. Loop-mediated isothermal amplification with the Procedure for Ultra Rapid Extraction kit for the diagnosis of pneumocystis pneumonia.

    PubMed

    Kawano, Shuichi; Maeda, Takuya; Suzuki, Takefumi; Abe, Tatsuhiro; Mikita, Kei; Hamakawa, Yusuke; Ono, Takeshi; Sonehara, Wataru; Miyahira, Yasushi; Kawana, Akihiko

    2015-03-01

    Loop-mediated isothermal amplification (LAMP) is an innovative molecular technique requiring only a heating device and isothermal conditions to amplify a specific target gene. The results of current microscopic diagnostic tools for pneumocystis pneumonia are not sufficiently consistent for detecting infection with a low-density of Pneumocystis jirovecii. Although polymerase chain reaction (PCR) is highly sensitive, it is not suitable for resource-limited facilities. LAMP is a potential diagnostic replacement for PCR in such settings but a critical disadvantage of DNA extraction was still remained. Therefore, we employed the Procedure for Ultra Rapid Extraction (PURE) kit, which uses a porous material, to isolate the DNA from clinical samples in a simple way in combination with previously reported LAMP procedure for diagnosing PCP. The detection limit of the PURE-LAMP method applied to artificial bronchoalveolar lavage fluid samples was 100 copies/tube, even with the use of massive blood-contaminated solutions. In addition, we concluded the diagnostic procedure within 1 h without the need for additional equipment. PURE-LAMP coupled with suitable primers for specific pathogens has good potential for diagnosing various infectious diseases. PMID:25455747

  3. Development of reverse transcription loop-mediated isothermal amplification assays to detect Hantaan virus and Seoul virus.

    PubMed

    Hu, Dan; Hao, Lina; Zhang, Jinhai; Yao, Pingping; Zhang, Qi; Lv, Heng; Gong, Xiufang; Pan, Xiuzhen; Cao, Min; Zhu, Jin; Zhang, Yun; Feng, Youjun; Wang, Changjun

    2015-09-01

    We developed two assays based on one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) to identify Hantaan virus (HTNV) and Seoul virus (SEOV), members of the Hantavirus genus that cause hemorrhagic fever with renal syndrome (HFRS). Our results showed that these assays can be conducted within 30min under isothermal conditions. The detection limit for HTNV was around 10 copies per reaction, similar to detection levels for quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. The detection limit for SEOV was 100 copies per reaction, a sensitivity that was 10-fold lower than that for qRT-PCR assays but 10-fold higher than that for RT-PCR assays. The method we developed was specific for both HTNV and SEOV without any cross-reaction with other pathogens. We conclude that RT-LAMP assays could be useful for the rapid and direct detection of HTNV and SEOV clinically, and for the epidemiological investigation of HFRS. PMID:25920565

  4. Analysis of a Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) for yellow fever diagnostic.

    PubMed

    Nunes, Marcio R T; Vianez, João Lídio; Nunes, Keley N B; da Silva, Sandro Patroca; Lima, Clayton P S; Guzman, Hilda; Martins, Lívia C; Carvalho, Valéria L; Tesh, Robert B; Vasconcelos, Pedro F C

    2015-12-15

    Yellow Fever virus (YFV) is an important human pathogen in tropical areas of Africa and South America. Although an efficient vaccine is available and has been used since the early 1940s, sylvatic YFV transmission still occurs in forested areas where anthropogenic actions are present, such as mineral extraction, rearing livestock and agriculture, and ecological tourism. In this context, two distinct techniques based on the RT-PCR derived method have been previously developed, however both methods are expensive due to the use of thermo cyclers and labeled probes. We developed isothermal genome amplification, which is a rapid, sensitive, specific and low cost molecular approach for YFV genome detection. This assay used a set of degenerate primers designed for the NS1 gene and was able to amplify, within 30min in isothermal conditions, the YFV 17D vaccine strain derived from an African wild prototype strain (Asibi), as well as field strains from Brazil, other endemic countries from South and Central America, and the Caribbean. The generic RT-LAMP assay could be helpful for YFV surveillance in field and rapid response during outbreaks in endemic areas. PMID:26459206

  5. Loop-mediated isothermal amplification: rapid visual and real-time methods for detection of genetically modified crops.

    PubMed

    Randhawa, Gurinder Jit; Singh, Monika; Morisset, Dany; Sood, Payal; Zel, Jana

    2013-11-27

    A rapid, reliable, and sensitive loop-mediated isothermal amplification (LAMP) system was developed for screening of genetically modified organisms (GMOs). The optimized LAMP assays using designed primers target commonly employed promoters, i.e., Cauliflower Mosaic Virus 35S (P-35S) and Figwort Mosaic Virus promoter (P-FMV), and marker genes, i.e., aminoglycoside 3'-adenyltransferase (aadA), neomycin phosphotransferase II (nptII), and ?-glucuronidase (uidA). The specificity and performance of the end-point and real-time LAMP assays were confirmed using eight genetically modified (GM) cotton events on four detection systems, employing two chemistries. LAMP assays on the isothermal real-time system were found to be most sensitive, detecting up to four target copies, within 35 min. The LAMP assays herein presented using alternate detection systems can be effectively utilized for rapid and cost-effective screening of the GM status of a sample, irrespective of the crop species or GM trait. These assays coupled with a fast and simple DNA extraction method may further facilitate on-site GMO screening. PMID:24188249

  6. Rapid visual detection of phytase gene in genetically modified maize using loop-mediated isothermal amplification method.

    PubMed

    Huang, Xin; Chen, Lili; Xu, Jiangmin; Ji, Hai-Feng; Zhu, Shuifang; Chen, Hongjun

    2014-08-01

    Transgenic maize plant expressing high phytase activity has been reported and approved by Chinese government in 2009. Here, we report a highly specific loop-mediated isothermal amplification (LAMP) method to detect the phytase gene in the GMO maize. The LAMP reaction takes less than 20min and the amplification is visible without gel electrophoresis. The detection sensitivity of the LAMP method is about 30 copies of phytase genomic DNA, which is 33.3 times greater than the conventional PCR method with gel electrophoresis. The quantitative detection results showed that the LAMP method has a good linear correlation between the DNA copy number and the associated Tt values over a large dynamic range of template concentration from 6×10(1) to 6×10(7) copies, with a quantification limit of 60 copies. Therefore, the LAMP method is visual, faster, and more sensitive, and does not need special equipment compared to traditional PCR technique, which is very useful for field tests and fast screening of GMO feeds. PMID:24629956

  7. Development of a loop-mediated isothermal amplification assay for rapid detection of Yersinia enterocolitica via targeting a conserved locus

    PubMed Central

    Ranjbar, Reza; Afshar, Davoud

    2015-01-01

    Background and Objectives: Loop-mediated isothermal amplification is a novel nucleic acid amplification assay providing as a simple diagnostic tool for rapid identification of microbial diseases in developing countries. In this study, a LAMP assay was established for Yersinia enterocolitica, a leading cause of acute enterocolitis in young children. Materials and Methods: LAMP assay was established with four primers targeting a specific locus for the detection of Y. enterocolitica. The assay was conducted at 65°C in thermo block for 90min. The sensitivity of LAMP was evaluated in comparison to conventional PCR using pTZ57R containing the target locus. Finally, specificity was assessed using DNA from common enteropathogenic bacteria. Results: Results showed that the sensitivity of LAMP assay was 44-copy number, which was 10-fold higher than that of PCR. No cross-reactivity was observed when testing against other enteropathogenic pathogens. Conclusion: This study showed that LAMP assay is an alternative molecular diagnostic tool for infections with Y. enterocolitica. In addition, this method may be useful in diagnosis at field or in laboratories without PCR machine.

  8. Rapid and Sensitive Detection of Listeria ivanovii by Loop-Mediated Isothermal Amplification of the smcL Gene

    PubMed Central

    Wang, Yi; Wang, Yan; Xu, Huaqing; Dai, Hang; Meng, Shuang; Ye, Changyun

    2014-01-01

    A loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of the L. ivanovii strains had been developed and evaluated in this study. Oligonucleotide primers specific for L. ivanovii species were designed corresponding to smcL gene sequences. The primers set comprise six primers targeting eight regions on the species-specific gene smcL. The LAMP assay could be completed within 1 h at 64°C in a water bath. Amplification products were directly observed by the Loopamp Fluorescent Detection Reagent (FD) or detected by agarose gel electrophoresis. Moreover, the LAMP reactions were also detected by real-time measurement of turbidity. The exclusivity of 77 non-L. ivanovii and the inclusivity of 17 L. ivanovii were both 100% in the assay. Sensitivity of the LAMP assay was 250 fg DNA and 16 CFU per reaction for detection of L. ivanovii in pure cultures and simulated human stool. The LAMP assay was 10 and 100-fold more sensitive than quantitative PCR (qPCR) and conventional PCR assays,respectively. When applied to human stool samples spiked with low level (8 CFU/0.5 g) of L. ivanovii strains, the new LAMP assay described here achieved positive detection after 6 hours enrichment. In conclusion, the new LAMP assay in this study can be used as a valuable, rapid and sensitive detection tool for the detection of L. ivanovii in field, medical and veterinary laboratories. PMID:25549337

  9. Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification.

    PubMed

    Duan, Ya-Bing; Ge, Chang-Yan; Zhang, Xiao-Ke; Wang, Jian-Xin; Zhou, Ming-Guo

    2014-01-01

    Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB). The LAMP reaction was optimal at 63 °C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10(-3) ng µL(-1) of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10(-2) ng µL(-1)). Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2%) were confirmed as positive by LAMP, 172 (90.1%) positive by the tissue separation, while 147 (77.0%) positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables. PMID:25329402

  10. Detection of Bacillus anthracis from spores and cells by loop-mediated isothermal amplification without sample preparation.

    PubMed

    Dugan, Lawrence; Bearinger, Jane; Hinckley, Aubree; Strout, Cheryl; Souza, Brian

    2012-09-01

    Loop-mediated isothermal amplification (LAMP) is a technique capable of rapidly amplifying specific nucleic acid sequences without specialized thermal cycling equipment. In addition, several detection methods that include dye fluorescence, gel electrophoresis, turbidity and colorimetric change, can be used to measure or otherwise detect target amplification. To date, publications have described the requirement for some form of sample nucleic acid extraction (boiling, lysis, DNA purification, etc.) prior to initiating a LAMP reaction. We demonstrate here, the first LAMP positive results obtained from vegetative cells and spores of Bacillus anthracis without nucleic acid extraction. Our data show that the simple addition of cells or spores to the reaction mixture, followed by heating at 63°C is all that is required to reproducibly amplify and detect target plasmid and chromosomal DNA via colorimetric change. The use of three primer sets targeting both plasmids and the chromosome of B. anthracis allows for the rapid discrimination of non-pathogenic bacteria from pathogenic bacteria within 30 min of sampling. Our results indicate that direct testing of B. anthracis spores and cells via LAMP assay will greatly simplify and shorten the detection process by eliminating nucleic acid purification. These results may allow more rapid detection of DNA from pathogenic organisms present in field and environmental samples. PMID:22677603

  11. Evaluation of Loop-Mediated Isothermal Amplification Assay for the Detection of Pneumocystis jirovecii in Immunocompromised Patients

    PubMed Central

    Singh, Preeti; Singh, Sundeep; Mirdha, Bijay Ranjan; Guleria, Randeep; Agarwal, Sanjay Kumar; Mohan, Anant

    2015-01-01

    Pneumocystis pneumonia (PCP) is one of the common opportunistic infection among HIV and non-HIV immunocompromised patients. The lack of a rapid and specific diagnostic test necessitates a more reliable laboratory diagnostic test for PCP. In the present study, the loop-mediated isothermal amplification (LAMP) assay was evaluated for the detection of Pneumocystis jirovecii. 185 clinical respiratory samples, including both BALF and IS, were subjected to GMS staining, nested PCR, and LAMP assay. Of 185 respiratory samples, 12/185 (6.5%), 41/185 (22.2%), and 49/185 (26.5%) samples were positive by GMS staining, nested PCR, and LAMP assay, respectively. As compared to nested PCR, additional 8 samples were positive by LAMP assay and found to be statistically significant (p < 0.05) with the detection limit of 1?pg. Thus, the LAMP assay may serve as a better diagnostic tool for the detection of P. jirovecii with high sensitivity and specificity, less turn-around time, operational simplicity, single-step amplification, and immediate visual detection. PMID:26664746

  12. Shewanella putrefaciens in cultured tilapia detected by a new calcein-loop-mediated isothermal amplification (Ca-LAMP) method.

    PubMed

    Suebsing, Rungkarn; Kampeera, Jantana; Sirithammajak, Sarawut; Pradeep, Padmaja Jayaprasad; Jitrakorn, Sarocha; Arunrut, Narong; Sangsuriya, Pakkakul; Saksmerprome, Vanvimon; Senapin, Saengchan; Withyachumnarnkul, Boonsirm; Kiatpathomchai, Wansika

    2015-12-01

    Shewanella putrefaciens is being increasingly isolated from a wide variety of sources and is pathogenic to many marine and freshwater fish. For better control of this pathogen, there is a need for the development of simple and inexpensive but highly specific, sensitive, and rapid detection methods suitable for application in field laboratories. Our colorogenic loop-mediated isothermal amplification (LAMP) assay combined with calcein (Ca-LAMP) for unaided visual confirmation of LAMP amplicons is a simple method for fish pathogen detection in cultured tilapia. Here, we describe the detection of S. putrefaciens using the same platform. As before, the method gave positive results (orange to green color change) in 45 min at 63°C with sensitivity 100 times higher than that of a conventional PCR assay, with no cross-amplification of other known fish bacterial pathogens tested. Using the assay with 389 samples of gonads, fertilized eggs, and fry of farmed Nile and red tilapia Oreochromis spp., 35% of samples were positive for S. putrefaciens. The highest prevalence was found in samples of gonads (55%) and fertilized eggs (55%) from adult breeding stocks, indicating that S. putrefaciens could be passed on easily to fry used for stocking production ponds. Tissue tropism assays revealed that the spleen showed the highest colonization by S. putrefaciens in naturally infected tilapia and that it would be the most suitable organ for screening and monitoring fish stocks for presence of the bacteria. PMID:26648105

  13. Development of a pan-serotype reverse transcription loop-mediated isothermal amplification assay for the detection of dengue virus.

    PubMed

    Dauner, Allison L; Mitra, Indrani; Gilliland, Theron; Seales, Sajeewane; Pal, Subhamoy; Yang, Shih-Chun; Guevara, Carolina; Chen, Jiann-Hwa; Liu, Yung-Chuan; Kochel, Tadeusz J; Wu, Shuenn-Jue L

    2015-09-01

    During dengue outbreaks, acute diagnosis at the patient's point of need followed by appropriate supportive therapy reduces morbidity and mortality. To facilitate needed diagnosis, we developed and optimized a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that detects all 4 serotypes of dengue virus (DENV). We used a quencher to reduce nonspecific amplification. The assay does not require expensive thermocyclers, utilizing a simple water bath to maintain the reaction at 63 °C. Results can be visualized using UV fluorescence, handheld readers, or lateral flow immunochromatographic tests. We report a sensitivity of 86.3% (95% confidence interval [CI], 72.7-94.8%) and specificity of 93.0% (95% CI, 83.0-98.1%) using a panel of clinical specimens characterized by DENV quantitative reverse transcription-polymerase chain reaction. This pan-serotype DENV RT-LAMP can be adapted to field-expedient formats where it can provide actionable diagnosis near the patient's point of need. PMID:26032430

  14. Tip-enhanced fluorescence with radially polarized illumination for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA

    NASA Astrophysics Data System (ADS)

    Wei, Shih-Chung; Chuang, Tsung-Liang; Wang, Da-Shin; Lu, Hui-Hsin; Gu, Frank X.; Sung, Kung-Bin; Lin, Chii-Wann

    2015-02-01

    A tip nanobiosensor for monitoring DNA replication was presented. The effects of excitation power and polarization on tip-enhanced fluorescence (TEF) were assessed with the tip immersed in fluorescein isothiocyanate solution first. The photon count rose on average fivefold with radially polarized illumination at 50 mW. We then used polymerase-functionalized tips for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA. The amplicon-SYBR Green I complex was detected and compared to real-time loop-mediated isothermal amplification. The signals of the reaction using 4 and 0.004 ng/?l templates were detected 10 and 30 min earlier, respectively. The results showed the potential of TEF in developing a nanobiosensor for real-time DNA amplification.

  15. A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification

    PubMed Central

    Kouzaki, Yuji; Maeda, Takuya; Sasaki, Hiroaki; Tamura, Shinsuke; Hamamoto, Takaaki; Yuki, Atsushi; Sato, Akinori; Miyahira, Yasushi; Kawana, Akihiko

    2015-01-01

    Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64°C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 103 cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 103 cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation involving immunocompromised patients because of its convenience, rapidity, and cost effectiveness. PMID:26208001

  16. Visual detection of Potato Leafroll virus by loop-mediated isothermal amplification of DNA with the GeneFinder™ dye.

    PubMed

    Almasi, Mohammad Amin; Erfan Manesh, Maryam; Jafary, Hossein; Dehabadi, Seyed Mohammad Hosseini

    2013-09-01

    The most common virus affecting potatoes in the field worldwide is Potato Leafroll virus (PLRV), belonging to the family Luteoviridae, genius Plerovirus. There are several molecular methods to detect PLRV including polymerase chain reaction (PCR), Multiplex AmpliDet RNA and double antibody sandwich ELISA (DAS-ELISA). But these techniques take a long time for 3h to two days, requiring sophisticated tools. The aim of this study was to reduce the time required to detect PLRV, using a newly designed loop-mediated isothermal amplification (LAMP) technique requiring only an ordinary water bath or thermoblock. PLRV RNA was extracted from overall 80 infected naturally potato leaves. A set of six novel primers for the LAMP reaction was designed according to the highly conserved sequence of the viral coat protein (CP) gene. LAMP was carried out under isothermal conditions, applying the Bst DNA polymerase enzyme; the LAMP products were detected visually using the GeneFinder™ florescence dye. A positive result using the GeneFinder™ dye was a color change from the original orange to green. Results confirmed LAMP with GeneFinder™ provides a rapid and safe assay for detection of PLRV. Since with other molecular methods, equipping laboratories with a thermocycler or expensive detector systems is unavoidable, this assay was found to be a simple, cost-effective molecular method that has the potential to replace other diagnostic methods in primary laboratories without the need for expensive equipment or specialized techniques. It can also be considered as a reliable alternative viral detection system in further investigations. PMID:23680094

  17. Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide.

    PubMed

    Li, Wei; Hou, Ting; Wu, Min; Li, Feng

    2016-02-01

    MicroRNAs (miRNAs) play an important role in many biological processes, and have been regarded as potential targets and biomarkers in cancer diagnosis and therapy. Also, to meet the big challenge imposed by the characteristics of miRNAs, such as small size and vulnerability to enzymatic digestion, it is of great importance to develop accurate, sensitive and simple miRNA assays. Herein, we developed a label-free fluorescence strategy for sensitive miRNA detection by combining isothermal exponential amplification and the unique features of SYBR Green I (SG) and graphene oxide (GO), in which SG gives significantly enhanced fluorescence upon intercalation into double-stranded DNAs (dsDNAs), and GO selectively adsorbs miRNA, single-stranded DNA and SG, to protect miRNA from enzymatic digestion, and to quench the fluorescence of the adsorbed SG. In the presence of the target miRNA, the ingeniously designed hairpin probe (HP) is unfolded and the subsequent polymerization and strand displacement reaction takes place to initiate the target recycling process. The newly formed dsDNAs are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNAs are formed to generate significantly enhanced fluorescence upon the intercalation of SG. Thus sensitive and selective fluorescence miRNA detection is realized, and the detection limit of 3fM is obtained. Besides, this method exhibits additional advantages of simplicity and low cost, since expensive and tedious labeling process is avoided. Therefore, the as-proposed label-free fluorescence strategy has great potential in the applications in miRNA-related clinical practices and biochemical researches. PMID:26653431

  18. Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick

    PubMed Central

    2014-01-01

    Background Citrus Huanglongbing (HLB) is the most devastating bacterial citrus disease worldwide. Three Candidatus Liberibacter species are associated with different forms of the disease: Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus and Candidatus Liberibacter africanus. Amongst them, Candidatus Liberibacter asiaticus is the most widespread and economically important. These Gram-negative bacterial plant pathogens are phloem-limited and vectored by citrus psyllids. The current management strategy of HLB is based on early and accurate detection of Candidatus Liberibacter asiaticus in both citrus plants and vector insects. Nowadays, real time PCR is the method of choice for this task, mainly because of its sensitivity and reliability. However, this methodology has several drawbacks, namely high equipment costs, the need for highly trained personnel, the time required to conduct the whole process, and the difficulty in carrying out the detection reactions in field conditions. Results A recent DNA amplification technique known as Loop Mediated Isothermal Amplification (LAMP) was adapted for the detection of Candidatus Liberibacter asiaticus. This methodology was combined with a Lateral Flow Dipstick (LFD) device for visual detection of the resulting amplicons, eliminating the need for gel electrophoresis. The assay was highly specific for the targeted bacterium. No cross-reaction was observed with DNA from any of the other phytopathogenic bacteria or fungi assayed. By serially diluting purified DNA from an infected plant, the sensitivity of the assay was found to be 10 picograms. This sensitivity level was proven to be similar to the values obtained running a real time PCR in parallel. This methodology was able to detect Candidatus Liberibacter asiaticus from different kinds of samples including infected citrus plants and psyllids. Conclusions Our results indicate that the methodology here reported constitutes a step forward in the development of new tools for the management, control and eradication of this destructive citrus disease. This system constitutes a potentially field-capable approach for the detection of the most relevant HLB-associated bacteria in plant material and psyllid vectors. PMID:24708539

  19. Sensitive and rapid detection of genetic modified soybean (Roundup Ready) by loop-mediated isothermal amplification.

    PubMed

    Liu, Mei; Luo, Yan; Tao, Ran; He, Ru; Jiang, Keyong; Wang, Baojie; Wang, Lei

    2009-11-01

    Using the LAMP method, a highly specific and sensitive detection system for genetically modified soybean (Roundup Ready) was designed. In this detection system, a set of four primers was designed by targeting the exogenous 35S epsps gene. Target DNA was amplified and visualized on agarose gel within 45 min under isothermal conditions at 65 degrees C. Without gel electrophoresis, the LAMP amplicon was visualized directly in the reaction tube by the addition of SYBR Green I for naked-eye inspection. The detection sensitivity of LAMP was 10-fold higher than the nested PCR established in our laboratory. Moreover, the LAMP method was much quicker, taking only 70 min, as compared with 300 min for nested PCR to complete the analysis of the GM soybean. Compared with traditional PCR approaches, the LAMP procedure is faster and more sensitive, and there is no need for a special PCR machine or electrophoresis equipment. Hence, this method can be a very useful tool for GMO detection and is particularly convenient for fast screening. PMID:19897926

  20. A Portable Automatic Endpoint Detection System for Amplicons of Loop Mediated Isothermal Amplification on Microfluidic Compact Disk Platform

    PubMed Central

    Uddin, Shah Mukim; Ibrahim, Fatimah; Sayad, Abkar Ahmed; Thiha, Aung; Pei, Koh Xiu; Mohktar, Mas S.; Hashim, Uda; Cho, Jongman; Thong, Kwai Lin

    2015-01-01

    In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10?3 ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment. PMID:25751077

  1. Development and application of loop-mediated isothermal amplification for detecting the highly benzimidazole-resistant isolates in Sclerotinia sclerotiorum.

    PubMed

    Duan, Ya Bing; Yang, Ying; Wang, Jian Xin; Liu, Cong Chao; He, Ling Ling; Zhou, Ming Guo

    2015-01-01

    Resistance of benzimidazole fungicides is related to the point mutation of the ?-tubulin gene in Sclerotinia sclerotiorum. The point mutation at codon 198 (GAG???GCG, E198A) occurs in more than 90% of field resistant populations in China. Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient. To establish a suitable and rapid detection of benzimidazole-resistant mutants of S. sclerotiorum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). Eight sets of LAMP primers were designed and four sets were optimized to specially distinguish benzimidazole-resistant mutants of S. sclerotiorum. With the optimal LAMP primers, the concentration of LAMP components was optimized and the reaction conditions were set as 60-64?°C for 60?min. This method had a good specificity, sensitivity, stability and repeatability. In the 1491 sclerotia, 614 (41.18%) were positive by LAMP, and 629 (42.19%) positive by MIC. Therefore, the LAMP assay is more feasible to detect benzimidazole-resistant mutants of S. sclerotiorum than traditional detection methods. PMID:26606972

  2. Evaluation of Loop-Mediated Isothermal Amplification Suitable for Molecular Monitoring of Schistosome-Infected Snails in Field Laboratories

    PubMed Central

    Hamburger, Joseph; Abbasi, Ibrahim; Kariuki, Curtis; Wanjala, Atsabina; Mzungu, Elton; Mungai, Peter; Muchiri, Eric; King, Charles H.

    2013-01-01

    We previously described loop-mediated isothermal amplification (LAMP) for detection of Schistosoma haematobium and S. mansoni DNA in infected snails. In the present study, we adapted the LAMP assay for application in field laboratories in schistosomiasis-endemic areas. Isolation of DNA was simplified by blotting snail tissue (extracted in NaOH/sodium dodecyl sulfate) onto treated membranes, which enabled preservation at ambient temperatures. A ready-mix of LAMP reagents, suitable for shipment at ambient temperature and storage in minimal refrigeration, was used. Local survey teams without experience in molecular biology acquired operational expertise with this test within a few hours. Fifty-four field-caught snails were tested locally by LAMP and 59 were tested at similar conditions in Jerusalem. The LAMP results were consistent with those of a polymerase chain reaction; only four samples showed false-negative results. Results indicate that LAMP assays are suitable for detection of S. haematobium and S. mansoni in low-technology parasitology laboratories in which schistosomiasis elimination activities are undertaken. PMID:23208875

  3. Development of Phage Immuno-Loop-Mediated Isothermal Amplification Assays for Organophosphorus Pesticides in Agro-products

    PubMed Central

    2015-01-01

    Two immuno-loop-mediated isothermal amplification assays (iLAMP) were developed by using a phage-borne peptide that was isolated from a cyclic eight-peptide phage library. One assay was used to screen eight organophosphorus (OP) pesticides with limits of detection (LOD) between 2 and 128 ng mL–1. The iLAMP consisted of the competitive immuno-reaction coupled to the LAMP reaction for detection. This method provides positive results in the visual color of violet, while a negative response results in a sky blue color; therefore, the iLAMP allows one to rapidly detect analytes in yes or no fashion. We validated the iLAMP by detecting parathion-methyl, parathion, and fenitrothion in Chinese cabbage, apple, and greengrocery, and the detection results were consistent with the enzyme-linked immunosorbent assay (ELISA). In conclusion, the iLAMP is a simple, rapid, sensitive, and economical method for detecting OP pesticide residues in agro-products with no instrumental requirement. PMID:25135320

  4. Development of phage immuno-loop-mediated isothermal amplification assays for organophosphorus pesticides in agro-products.

    PubMed

    Hua, Xiude; Yin, Wei; Shi, Haiyan; Li, Ming; Wang, Yanru; Wang, Hong; Ye, Yonghao; Kim, Hee Joo; Gee, Shirley J; Wang, Minghua; Liu, Fengquan; Hammock, Bruce D

    2014-08-19

    Two immuno-loop-mediated isothermal amplification assays (iLAMP) were developed by using a phage-borne peptide that was isolated from a cyclic eight-peptide phage library. One assay was used to screen eight organophosphorus (OP) pesticides with limits of detection (LOD) between 2 and 128 ng mL(-1). The iLAMP consisted of the competitive immuno-reaction coupled to the LAMP reaction for detection. This method provides positive results in the visual color of violet, while a negative response results in a sky blue color; therefore, the iLAMP allows one to rapidly detect analytes in yes or no fashion. We validated the iLAMP by detecting parathion-methyl, parathion, and fenitrothion in Chinese cabbage, apple, and greengrocery, and the detection results were consistent with the enzyme-linked immunosorbent assay (ELISA). In conclusion, the iLAMP is a simple, rapid, sensitive, and economical method for detecting OP pesticide residues in agro-products with no instrumental requirement. PMID:25135320

  5. Graphene oxide based fluorescence resonance energy transfer and loop-mediated isothermal amplification for white spot syndrome virus detection.

    PubMed

    Waiwijit, U; Phokaratkul, D; Kampeera, J; Lomas, T; Wisitsoraat, A; Kiatpathomchai, W; Tuantranont, A

    2015-10-20

    Graphene oxide (GO) is attractived for biological or medical applications due to its unique electrical, physical, optical and biological properties. In particular, GO can adsorb DNA via ?-? stacking or non-covalent interactions, leading to fluorescence quenching phenomenon applicable for bio-molecular detection. In this work, a new method for white spot syndrome virus (WSSV)-DNA detection is developed based on loop-mediated isothermal amplification (LAMP) combined with fluorescence resonance energy transfer (FRET) between GO and fluorescein isothiocyanate-labeled probe (FITC-probe). The fluorescence quenching efficiency of FITC-probe was found to increase with increasing GO concentration and reached 98.7% at a GO concentration of 50?g/ml. The fluorescence intensity of FITC-probe was recovered after hybridization with WSSV LAMP product with an optimal hybridization time of 10min and increased accordingly with increasing amount of LAMP products. The detection limit was estimated to be as low as 10 copies of WSSV plasmid DNA or 0.6fg of the total DNA extracted from shrimp infected with WSSV. In addition, no cross reaction was observed with other common shrimp viral pathogens. Therefore, the GO-FRET-LAMP technique is promising for fast, sensitive and specific detection of DNAs. PMID:26277651

  6. Rapid detection of aflatoxin producing fungi in food by real-time quantitative loop-mediated isothermal amplification.

    PubMed

    Luo, Jie; Vogel, Rudi F; Niessen, Ludwig

    2014-12-01

    Aflatoxins represent a serious risk for human and animal health. They are mainly produced by Aspergillus flavus and Aspergillus parasiticus but also by Aspergillus nomius. Three species specific turbidimeter based real-time LAMP (loop-mediated isothermal amplification) assays were developed to quantify the three species individually in conidial solutions and to define contamination levels in samples of shelled Brazil nuts, maize, and peanuts. Standard curves relating spore numbers to time to threshold (Tt) values were set up for each of the species. Assays had detection limits of 10, 100 and 100 conidia per reaction of A. flavus, A. parasiticus, and A. nomius, respectively. Analysis of contaminated sample materials revealed that the A. nomius specific real-time LAMP assay detected a minimum of 10 conidia/g in Brazil nuts while assays specific for A. flavus and A. parasiticus had detection limits of 10(2) conidia/g and 10(5) conidia/g, respectively in peanut samples as well as 10(4) conidia/g and 10(4) conidia/g, respectively in samples of maize. The real-time LAMP assays developed here appear to be promising tools for the prediction of potential aflatoxigenic risk at an early stage and in all critical control points of the food and feed production chain. PMID:25084656

  7. Loop Mediated Isothermal Amplification for Detection of Trypanosoma brucei gambiense in Urine and Saliva Samples in Nonhuman Primate Model

    PubMed Central

    Ngotho, Maina; Kagira, John Maina; Gachie, Beatrice Muthoni; Karanja, Simon Muturi; Waema, Maxwell Wambua; Maranga, Dawn Nyawira; Maina, Naomi Wangari

    2015-01-01

    Human African trypanosomiasis (HAT) is a vector-borne parasitic zoonotic disease. The disease caused by Trypanosoma brucei gambiense is the most prevalent in Africa. Early diagnosis is hampered by lack of sensitive diagnostic techniques. This study explored the potential of loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) in the detection of T. b. gambiense infection in a vervet monkey HAT model. Six vervet monkeys were experimentally infected with T. b. gambiense IL3253 and monitored for 180 days after infection. Parasitaemia was scored daily. Blood, cerebrospinal fluid (CSF), saliva, and urine samples were collected weekly. PCR and LAMP were performed on serum, CSF, saliva, and urine samples. The detection by LAMP was significantly higher than that of parasitological methods and PCR in all the samples. The performance of LAMP varied between the samples and was better in serum followed by saliva and then urine samples. In the saliva samples, LAMP had 100% detection between 21 and 77?dpi, whereas in urine the detection it was slightly lower, but there was over 80% detection between 28 and 91?dpi. However, LAMP could not detect trypanosomes in either saliva or urine after 140 and 126?dpi, respectively. The findings of this study emphasize the importance of LAMP in diagnosis of HAT using saliva and urine samples. PMID:26504841

  8. A portable automatic endpoint detection system for amplicons of loop mediated isothermal amplification on microfluidic compact disk platform.

    PubMed

    Uddin, Shah Mukim; Ibrahim, Fatimah; Sayad, Abkar Ahmed; Thiha, Aung; Pei, Koh Xiu; Mohktar, Mas S; Hashim, Uda; Cho, Jongman; Thong, Kwai Lin

    2015-01-01

    In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10(-3) ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment. PMID:25751077

  9. Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of ostreid herpesvirus 1 DNA.

    PubMed

    Ren, Weicheng; Renault, Tristan; Cai, Yuyong; Wang, Chongming

    2010-12-01

    A loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of ostreid herpesvirus 1 (OsHV-1) DNA. A set of four primers was designed, based on the sequence of the ATPase subunit of the OsHV-1 DNA-packaging terminase gene. The reaction temperature and time were optimized to 64°C and 60min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change due to addition of fluorescent dye. The developed method was highly specific for detection of OsHV-1, and no cross-reaction was observed with other DNA viruses, such as White spot syndrome virus (WSSV), Penaeus stylirostris densovirus (PstDNV), Turbot reddish body iridovirus (TRBIV) and Lymphocystis disease virus (LCDV) found commonly in China. The lower detection limit of the LAMP assay was approximately 20 copies per reaction, and it was 100 times more sensitive than that of conventional PCR. A comparative evaluation of 10 oyster samples using LAMP and PCR assays showed overall correlation in positive and negative results for OsHV-1. These results indicate that the LAMP assay is a simple, rapid, sensitive, specific and reliable technique for the detection of OsHV-1. The LAMP technique has capacity for use for the detection of OsHV-1 both in the laboratory and on farms. PMID:20813133

  10. Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid and Sensitive Identification of Ostrich Meat

    PubMed Central

    Abdulmawjood, Amir; Grabowski, Nils; Fohler, Svenja; Kittler, Sophie; Nagengast, Helga; Klein, Guenter

    2014-01-01

    Animal species identification is one of the primary duties of official food control. Since ostrich meat is difficult to be differentiated macroscopically from beef, therefore new analytical methods are needed. To enforce labeling regulations for the authentication of ostrich meat, it might be of importance to develop and evaluate a rapid and reliable assay. In the present study, a loop-mediated isothermal amplification (LAMP) assay based on the cytochrome b gene of the mitochondrial DNA of the species Struthio camelus was developed. The LAMP assay was used in combination with a real-time fluorometer. The developed system allowed the detection of 0.01% ostrich meat products. In parallel, a direct swab method without nucleic acid extraction using the HYPLEX LPTV buffer was also evaluated. This rapid processing method allowed detection of ostrich meat without major incubation steps. In summary, the LAMP assay had excellent sensitivity and specificity for detecting ostrich meat and could provide a sampling-to-result identification-time of 15 to 20 minutes. PMID:24963709

  11. Efficient and Specific Detection of Salmonella in Food Samples Using a stn-Based Loop-Mediated Isothermal Amplification Method

    PubMed Central

    Srisawat, Mevaree; Panbangred, Watanalai

    2015-01-01

    The Salmonella enterotoxin (stn) gene exhibits high homology among S. enterica serovars and S. bongori. A set of 6 specific primers targeting the stn gene were designed for detection of Salmonella spp. using the loop-mediated isothermal amplification (LAMP) method. The primers amplified target sequences in all 102 strains of 87 serovars of Salmonella tested and no products were detected in 57 non-Salmonella strains. The detection limit in pure cultures was 5?fg DNA/reaction when amplified at 65°C for 25?min. The LAMP assay could detect Salmonella in artificially contaminated food samples as low as 220?cells/g of food without a preenrichment step. However, the sensitivity was increased 100-fold (~2?cells/g) following 5?hr preenrichment at 35°C. The LAMP technique, with a preenrichment step for 5 and 16?hr, was shown to give 100% specificity with food samples compared to the reference culture method in which 67 out of 90 food samples gave positive results. Different food matrixes did not interfere with LAMP detection which employed a simple boiling method for DNA template preparation. The results indicate that the LAMP method, targeting the stn gene, has great potential for detection of Salmonella in food samples with both high specificity and high sensitivity. PMID:26543859

  12. Development of a Rapid Detection Method for Potato virus X by Reverse Transcription Loop-Mediated Isothermal Amplification

    PubMed Central

    Jeong, Joojin; Cho, Sang-Yun; Lee, Wang-Hyu; Lee, Kui-jae; Ju, Ho-Jong

    2015-01-01

    The primary step for efficient control of viral diseases is the development of simple, rapid, and sensitive virus detection. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been used to detect viral RNA molecules because of its simplicity and high sensitivity for a number of viruses. RT-LAMP for the detection of Potato virus X (PVX) was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR) to demonstrate its advantages over RT-PCR. RT-LAMP reactions were conducted with or without a set of loop primers since one out of six primers showed PVX specificity. Based on real-time monitoring, RT-LAMP detected PVX around 30 min, compared to 120 min for RT-PCR. By adding a fluorescent reagent during the reaction, the extra step of visualization by gel electrophoresis was not necessary. RT-LAMP was conducted using simple inexpensive instruments and a regular incubator to evaluate whether RNA could be amplified at a constant temperature instead of using an expensive thermal cycler. This study shows the potential of RT-LAMP for the diagnosis of viral diseases and PVX epidemiology because of its simplicity and rapidness compared to RT-PCR. PMID:26361470

  13. Development and application of loop-mediated isothermal amplification for detecting the highly benzimidazole-resistant isolates in Sclerotinia sclerotiorum

    PubMed Central

    Duan, Ya Bing; Yang, Ying; Wang, Jian Xin; Liu, Cong Chao; He, Ling Ling; Zhou, Ming Guo

    2015-01-01

    Resistance of benzimidazole fungicides is related to the point mutation of the ?-tubulin gene in Sclerotinia sclerotiorum. The point mutation at codon 198 (GAG???GCG, E198A) occurs in more than 90% of field resistant populations in China. Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient. To establish a suitable and rapid detection of benzimidazole-resistant mutants of S. sclerotiorum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). Eight sets of LAMP primers were designed and four sets were optimized to specially distinguish benzimidazole-resistant mutants of S. sclerotiorum. With the optimal LAMP primers, the concentration of LAMP components was optimized and the reaction conditions were set as 60–64?°C for 60?min. This method had a good specificity, sensitivity, stability and repeatability. In the 1491 sclerotia, 614 (41.18%) were positive by LAMP, and 629 (42.19%) positive by MIC. Therefore, the LAMP assay is more feasible to detect benzimidazole-resistant mutants of S. sclerotiorum than traditional detection methods. PMID:26606972

  14. Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane

    PubMed Central

    Zhou, Dinggang; Guo, Jinlong; Xu, Liping; Gao, Shiwu; Lin, Qingliang; Wu, Qibin; Wu, Luguang; Que, Youxiong

    2014-01-01

    To meet the demand for detection of foreign genes in genetically modified (GM) sugarcane necessary for regulation of gene technology, an efficient method with high specificity and rapidity was developed for the cry1Ac gene, based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed using the sequence of cry1Ac along with optimized reaction conditions: 5.25?mM of Mg2+, 4:1 ratio of inner primer to outer primer, 2.0?U of Bst DNA polymerase in a reaction volume of 25.0??L. Three post-LAMP detection methods (precipitation, calcein (0.60?mM) with Mn2+ (0.05?mM) complex and SYBR Green I visualization), were shown to be effective. The sensitivity of the LAMP method was tenfold higher than that of conventional PCR when using templates of the recombinant cry1Ac plasmid or genomic DNA from cry1Ac transgenic sugarcane plants. More importantly, this system allowed detection of the foreign gene on-site when screening GM sugarcane without complex and expensive instruments, using the naked eye. This method can not only provide technological support for detection of cry1Ac, but can also further facilitate the use of this detection technique for other transgenes in GM sugarcane. PMID:24810230

  15. Comparison of a loop-mediated isothermal amplification for orf virus with quantitative real-time PCR

    PubMed Central

    2013-01-01

    Background Orf virus (ORFV) causes orf (also known as contagious ecthyma or contagious papular dermatitis), a severe infectious skin disease in goats, sheep and other ruminants. Therefore, a rapid, highly specific and accurate method for the diagnosis of ORFV infections is essential to ensure that the appropriate treatments are administered and to reduce economic losses. Methods A loop-mediated isothermal amplification (LAMP) assay based on the identification of the F1L gene was developed for the specific detection of ORFV infections. The sensitivity and specificity of the LAMP assay were evaluated, and the effectiveness of this method was compared with that of real-time PCR. Results The sensitivity of this assay was determined to be 10 copies of a standard plasmid. Furthermore, no cross-reactivity was found with either capripox virus or FMDV. The LAMP and real-time PCR assays were both able to detect intracutaneous- and cohabitation-infection samples, with a concordance of 97.83%. LAMP demonstrated a sensitivity of 89.13%. Conclusion The LAMP assay is a highly efficient and practical method for detecting ORFV infection. This LAMP method shows great potential for monitoring the prevalence of orf, and it could prove to be a powerful supplemental tool for current diagnostic methods. PMID:23634981

  16. Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

    PubMed Central

    Barkway, Christopher P.; Pocock, Rebecca L.; Vrba, Vladimir; Blake, Damer P.

    2015-01-01

    Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm’s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable. PMID:25741643

  17. Loop-mediated isothermal amplification for diagnosis of 18 World Organization for Animal Health (OIE) notifiable viral diseases of ruminants, swine and poultry.

    PubMed

    Mansour, Shimaa M G; Ali, Haytham; Chase, Christopher C L; Cepica, Arnost

    2015-12-01

    Loop-mediated isothermal amplification (LAMP) is a simple, powerful state-of-the-art gene amplification technique used for the rapid diagnosis and early detection of microbial diseases. Many LAMP assays have been developed and validated for important epizootic diseases of livestock. We review the LAMP assays that have been developed for the detection of 18 viruses deemed notifiable of ruminants, swine and poultry by the World Organization for Animal Health (OIE). LAMP provides a fast (the assay often takes less than an hour), low cost, highly sensitive, highly specific and less laborious alternative to detect infectious disease agents. The LAMP procedure can be completed under isothermal conditions so thermocyclers are not needed. The ease of use of the LAMP assay allows adaptability to field conditions and works well in developing countries with resource-limited laboratories. However, this technology is still underutilized in the field of veterinary diagnostics despite its huge capabilities. PMID:25900363

  18. Loop region-specific oligonucleotide probes for loop-mediated isothermal amplification-enzyme-linked immunosorbent assay truly minimize the instrument needed for detection process.

    PubMed

    Ravan, Hadi; Yazdanparast, Razieh

    2013-08-15

    Enteric fever represents a significant public health burden in less-developed countries. Therefore, there is a great need for developing an improved diagnostic tool adapted to the demands of poor-resource clinical laboratories in those countries. The current study has developed a reliable loop-mediated isothermal amplification (LAMP)-enzyme-linked immunosorbent assay (ELISA) for diagnosis of enteric fever with a minimal equipment dependency. The LAMP-ELISA assay involves direct incorporation of a labeled nucleotide into amplicons during the amplification of the SPA3440 gene, their hybridization to the unique tagged oligonucleotide probes during the LAMP reaction, and finally detection of labeled LAMP amplicons by immunoassay technology. Because the designed oligonucleotide probes target the single-stranded DNA segment within the LAMP amplicons, the probe hybridization stage is performed simultaneously with the amplification process. This novel probe design strategy allows both the amplification and hybridization stages to be performed simultaneously and isothermally in a water bath. Among the bacteria tested, positive results were observed only with enteric fever causative bacteria. The LAMP-ELISA assay was successfully applied to artificially contaminated blood samples with a detection limit of 10 colony-forming units (CFU)/ml, which was 100 times more sensitive than polymerase chain reaction (PCR) and turbidity assessment-based conventional LAMP methods. The new assay is considered to be an effective method for diagnosis of enteric fever. PMID:23624347

  19. Loop-Mediated Isothermal Amplification (LAMP) assay for the identification of Echinococcus multilocularis infections in canine definitive hosts

    PubMed Central

    2014-01-01

    Background Alveolar echinococcosis, caused by the metacestode larval stage of Echinococcus multilocularis, is a zoonosis of public health significance and is highly prevalent in northwest China. To effectively monitor its transmission, we developed a new rapid and cheap diagnostic assay, based on loop-mediated isothermal amplification (LAMP), to identify canine definitive hosts infected with E. multilocularis. Methods The primers used in the LAMP assay were based on the mitochondrial nad5 gene of E. multilocularis and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR assay, using DNA extracted from the feces of dogs experimentally infected with E. multilocularis, on 189 dog fecal samples collected from three E. multilocularis-endemic regions in Qinghai province, the People’s Republic of China, and 30 negative control copro-samples from dogs from an area in Gansu province that had been subjected to an intensive de-worming program. Light microscopy was also used to examine the experimentally obtained and field collected dog copro-samples for the presence of E. multilocularis eggs. Results The E. multilocularis-positivity rates obtained for the field-collected fecal samples were 16.4% and 5.3% by the LAMP and PCR assays, respectively, and all samples obtained from the control dogs were negative. The LAMP assay was able to detect E. multilocularis DNA in the feces of experimentally infected dogs at 12 days post-infection, whereas the PCR assay was positive on the 17th day and eggs were first detectable by light microscopy at day 44 post-challenge. Conclusion The earlier specific detection of an E. multilocularis infection in dog copro-samples indicates that the LAMP assay we developed is a realistic alternative method for the field surveillance of canines in echinococcosis-endemic areas. PMID:24886279

  20. Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification

    PubMed Central

    Poole, Catherine B.; Ettwiller, Laurence; Tanner, Nathan A.; Evans, Thomas C.; Wanji, Samuel; Carlow, Clotilde K. S.

    2015-01-01

    Loa loa infections have emerged as a serious public health problem in patients co-infected with Onchocerca volvulus or Wuchereria bancrofti because of severe adverse neurological reactions after treatment with ivermectin. Accurate diagnostic tests are needed for careful mapping in regions where mass drug administration is underway. Loop-mediated isothermal amplification (LAMP) has become a widely adopted screening method because of its operational simplicity, rapidity and versatility of visual detection readout options. Here, we present a multi-step bioinformatic pipeline to generate diagnostic candidates suitable for LAMP and experimentally validate this approach using one of the identified candidates to develop a species-specific LAMP assay for L. loa. The pipeline identified ~140 new L. loa specific DNA repeat families as putative biomarkers of infection. The consensus sequence of one family, repeat family 4 (RF4), was compiled from ~ 350 sequences dispersed throughout the L. loa genome and maps to a L. loa-specific region of the long terminal repeats found at the boundaries of Bel/Pao retrotransposons. PCR and LAMP primer sets targeting RF4 specifically amplified L. loa but not W. bancrofti, O. volvulus, Brugia malayi, human or mosquito DNA. RF4 LAMP detects the DNA equivalent of one microfilaria (100 pg) in 25–30 minutes and as little as 0.060 pg of L. loa DNA (~1/1600th of a microfilaria) purified from spiked blood samples in approximately 50 minutes. In summary, we have successfully employed a bioinformatic approach to mine the L. loa genome for species-specific repeat families that can serve as new DNA biomarkers for LAMP. The RF4 LAMP assay shows promise as a field tool for the implementation and management of mass drug administration programs and warrants further testing on clinical samples as the next stage in development towards this goal. PMID:26414073

  1. Clinical Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Neisseria meningitidis in Cerebrospinal Fluid

    PubMed Central

    Kilgore, Paul E.; Kim, Soon Ae; Takahashi, Hideyuki; Ohnishi, Makoto; Anh, Dang Duc; Dong, Bai Qing; Kim, Jung Soo; Tomono, Jun; Miyamoto, Shigehiko; Notomi, Tsugunori; Kim, Dong Wook; Seki, Mitsuko

    2015-01-01

    Background Neisseria meningitidis (Nm) is a leading causative agent of bacterial meningitis in humans. Traditionally, meningococcal meningitis has been diagnosed by bacterial culture. However, isolation of bacteria from patients’ cerebrospinal fluid (CSF) is time consuming and sometimes yields negative results. Recently, polymerase chain reaction (PCR)-based diagnostic methods of detecting Nm have been considered the gold standard because of their superior sensitivity and specificity compared with culture. In this study, we developed a loop-mediated isothermal amplification (LAMP) method and evaluated its ability to detect Nm in cerebrospinal fluid (CSF). Methodology/Principal Findings We developed a meningococcal LAMP assay (Nm LAMP) that targets the ctrA gene. The primer specificity was validated using 16 strains of N. meningitidis (serogroup A, B, C, D, 29-E, W-135, X, Y, and Z) and 19 non-N. meningitidis species. Within 60 min, the Nm LAMP detected down to ten copies per reaction with sensitivity 1000-fold more than that of conventional PCR. The LAMP assays were evaluated using a set of 1574 randomly selected CSF specimens from children with suspected meningitis collected between 1998 and 2002 in Vietnam, China, and Korea. The LAMP method was shown to be more sensitive than PCR methods for CSF samples (31 CSF samples were positive by LAMP vs. 25 by PCR). The detection rate of the LAMP method was substantially higher than that of the PCR method. In a comparative analysis of the PCR and LAMP assays, the clinical sensitivity, specificity, positive predictive value, and negative predictive value of the LAMP assay were 100%, 99.6%, 80.6%, and 100%, respectively. Conclusions/Significance Compared to PCR, LAMP detected Nm with higher analytical and clinical sensitivity. This sensitive and specific LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings. PMID:25853422

  2. Mixed-Dye-Based Label-Free and Sensitive Dual Fluorescence for the Product Detection of Nucleic Acid Isothermal Multiple-Self-Matching-Initiated Amplification.

    PubMed

    Ding, Xiong; Wu, Wenshuai; Zhu, Qiangyuan; Zhang, Tao; Jin, Wei; Mu, Ying

    2015-10-20

    Visual detections based on fluorescence and the color changes under natural light are two promising product detections for isothermal nucleic acid amplifications (INAAs) such as the isothermal multiple-self-matching-initiated amplification (IMSA) as point-of-care testing techniques. However, the currently used approaches have shortcomings in application. For the former, fluorescence changes recognized by naked eye may be indistinguishable because of single fluorescence emitted and strong background noise, which requires empirical preset of cutoff intensity values. For the latter, visual detection sensitivity under natural light is not comparable to that based on fluorescence. Herein, hydroxyl naphthol blue (HNB) and SYBR Green I (SG) were coupled to acquire a label-free dual fluorescence for the visual product detection of IMSA. The mixed-dye-loaded off-chip (tube-based) and on-chip (microfluidic chip-based) IMSAs for the detection of hepatitis B virus were conducted. The results demonstrated that this dual fluorescence could realize distinguishable fluorescent color changes to improve visual detection sensitivity and avoid the preset of cutoff values. Moreover, the mixed dye is stable when kept at room temperature and compatible with the IMSA's reagents without a contamination-prone step of opening tubes after amplification. Also, this coupled dye inherits the advantages of achieving color changes under natural light from HNB and real-time detection from SG. In conclusion, the mixed-dye-based dual fluorescence has a potential in the point-of-care testing application for realizing off-chip and on-chip product detection of IMSA, loop-mediated isothermal amplification (LAMP), or other INAAs. PMID:26383158

  3. Restriction Cascade Exponential Amplification (RCEA) assay with an attomolar detection limit: a novel, highly specific, isothermal alternative to qPCR

    PubMed Central

    Ghindilis, Andrey L.; Smith, Maria W.; Simon, Holly M.; Seoudi, Ihab A.; Yazvenko, Nina S.; Murray, Iain A.; Fu, Xiaoqing; Smith, Kenneth; Jen-Jacobson, Linda; Xu, Shuang-yong

    2015-01-01

    An alternative to qPCR was developed for nucleic acid assays, involving signal rather than target amplification. The new technology, Restriction Cascade Exponential Amplification (RCEA), relies on specific cleavage of probe-target hybrids by restriction endonucleases (REase). Two mutant REases for amplification (Ramp), S17C BamHI and K249C EcoRI, were conjugated to oligonucleotides, and immobilized on a solid surface. The signal generation was based on: (i) hybridization of a target DNA to a Ramp-oligonucleotide probe conjugate, followed by (ii) specific cleavage of the probe-target hybrid using a non-immobilized recognition REase. The amount of Ramp released into solution upon cleavage was proportionate to the DNA target amount. Signal amplification was achieved through catalysis, by the free Ramp, of a restriction cascade containing additional oligonucleotide-conjugated Ramp and horseradish peroxidase (HRP). Colorimetric quantification of free HRP indicated that the RCEA achieved a detection limit of 10 aM (10?17?M) target concentration, or approximately 200 molecules, comparable to the sensitivity of qPCR-based assays. The RCEA assay had high specificity, it was insensitive to non-specific binding, and detected target sequences in the presence of foreign DNA. RCEA is an inexpensive isothermal assay that allows coupling of the restriction cascade signal amplification with any DNA target of interest. PMID:25583452

  4. Development of loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Penicillium nordicum in dry-cured meat products.

    PubMed

    Ferrara, M; Perrone, G; Gallo, A; Epifani, F; Visconti, A; Susca, A

    2015-06-01

    The need of powerful diagnostic tools for rapid, simple, and cost-effective detection of food-borne fungi has become very important in the area of food safety. Currently, several isothermal nucleic acid amplification methods have been developed as an alternative to PCR-based analyses. Loop-mediated isothermal amplification (LAMP) is one of these innovative methods; it requires neither gel electrophoresis to separate and visualize the products nor expensive laboratory equipment and it has been applied already for detection of pathogenic organisms. In the current study, we developed a LAMP assay for the specific detection of Penicillium nordicum, the major causative agent of ochratoxin A contamination in protein-rich food, especially dry-cured meat products. The assay was based on targeting otapksPN gene, a key gene in the biosynthesis of ochratoxin A (OTA) in P. nordicum. Amplification of DNA during the reaction was detected directly in-tube by color transition of hydroxynaphthol blue from violet to sky blue, visible to the naked eye, avoiding further post amplification analyses. Only DNAs isolated from several P. nordicum strains led to positive results and no amplification was observed from non-target OTA and non OTA-producing strains. The assay was able to detect down to 100 fg of purified targeted genomic DNA or 10(2) conidia/reaction within 60 min. The LAMP assay for detection and identification of P. nordicum was combined with a rapid DNA extraction method set up on serially diluted conidia, providing an alternative rapid, specific and sensitive DNA-based method suitable for application directly "on-site", notably in key steps of dry-cured meat production. PMID:25771218

  5. Real-Time Sequence-Validated Loop-Mediated Isothermal Amplification Assays for Detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

    PubMed Central

    Bhadra, Sanchita; Jiang, Yu Sherry; Kumar, Mia R.; Johnson, Reed F.; Hensley, Lisa E.; Ellington, Andrew D.

    2015-01-01

    The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens. PMID:25856093

  6. Loop-Mediated Isothermal Amplification for Laboratory Confirmation of Buruli Ulcer Disease—Towards a Point-of-Care Test

    PubMed Central

    Beissner, Marcus; Phillips, Richard Odame; Battke, Florian; Bauer, Malkin; Badziklou, Kossi; Sarfo, Fred Stephen; Maman, Issaka; Rhomberg, Agata; Piten, Ebekalisai; Frimpong, Michael; Huber, Kristina Lydia; Symank, Dominik; Jansson, Moritz; Wiedemann, Franz Xaver; Banla Kere, Abiba; Herbinger, Karl-Heinz; Löscher, Thomas; Bretzel, Gisela

    2015-01-01

    Background As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents. Methodology/Principal Findings Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR), IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%; 100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%; cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays. Conclusions/Significance Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold-chains. As validation of the assays was conducted in a third-level laboratory environment, field based evaluation trials are necessary to determine the clinical performance at peripheral health care level. PMID:26566026

  7. Rapid and Sensitive Salmonella Typhi Detection in Blood and Fecal Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification.

    PubMed

    Fan, Fenxia; Yan, Meiying; Du, Pengcheng; Chen, Chen; Kan, Biao

    2015-09-01

    Typhoid fever caused by Salmonella enterica serovar Typhi remains a significant public health problem in developing countries. Although the main method for diagnosing typhoid fever is blood culture, the test is time consuming and not always able to detect infections. Thus, it is very difficult to distinguish typhoid from other infections in patients with nonspecific symptoms. A simple and sensitive laboratory detection method remains necessary. The purpose of this study is to establish and evaluate a rapid and sensitive reverse transcription-based loop-mediated isothermal amplification (RT-LAMP) method to detect Salmonella Typhi infection. In this study, a new specific gene marker, STY1607, was selected to develop a STY1607-RT-LAMP assay; this is the first report of specific RT-LAMP detection assay for typhoid. Human-simulated and clinical blood/stool samples were used to evaluate the performance of STY1607-RT-LAMP for RNA detection; this method was compared with STY1607-LAMP, reverse transcription real-time polymerase chain reaction (rRT-PCR), and bacterial culture methods for Salmonella Typhi detection. Using mRNA as the template, STY1607-RT-LAMP exhibited 50-fold greater sensitivity than STY1607-LAMP for DNA detection. The STY1607-RT-LAMP detection limit is 3 colony-forming units (CFU)/mL for both the pure Salmonella Typhi samples and Salmonella Typhi-simulated blood samples and was 30 CFU/g for the simulated stool samples, all of which were 10-fold more sensitive than the rRT-PCR method. RT-LAMP exhibited improved Salmonella Typhi detection sensitivity compared to culture methods and to rRT-PCR of clinical blood and stool specimens from suspected typhoid fever patients. Because it can be performed without sophisticated equipment or skilled personnel, RT-LAMP is a valuable tool for clinical laboratories in developing countries. This method can be applied in the clinical diagnosis and care of typhoid fever patients as well as for a quick public health response. PMID:26270463

  8. Diagnostic accuracy of loop-mediated isothermal amplification in detection of Clostridium difficile in stool samples: a meta-analysis

    PubMed Central

    Wei, Chen; Yang-Ming, Li; Shan, Luo; Yi-Ming, Zhong

    2015-01-01

    Introduction Clostridium difficile infection (CDI) remains a diagnostic challenge for clinicians. More recently, loop-mediated isothermal amplification (LAMP) has become readily available for the diagnosis of CDI, and many studies have investigated the usefulness of LAMP for rapid and accurate diagnosis of CDI. However, the overall diagnostic accuracy of LAMP for CDI remains unclear. In this meta-analysis, our aim was to establish the overall diagnostic accuracy of LAMP in detection of Clostridium difficile (CD) in stool samples. Material and methods A search was done in PubMed, MEDLINE, EMBASE and Cochrane Library databases up to February 2014 to identify published studies that evaluated the diagnostic role of LAMP for CD. Methodological quality was assessed according to the quality assessment for studies of diagnostic accuracy (QUADAS) instrument. The sensitivities (SEN), specificities (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratio (DOR) were pooled statistically using random effects models. Statistical analysis was performed by employing Meta-Disc 1.4 software. Summary receiver operating characteristic (SROC) curves were used to summarize overall test performance. Funnel plots were used to test the potential publication bias. Result A total of 9 studies met inclusion criteria for the present meta-analysis. The pooled SEN and SPE for diagnosing CD were 0.93 (95% CI: 0.91–0.95) and 0.98 (95% CI: 0.98–0.99), respectively. The PLR was 47.72 (95% CI: 15.10–150.82), NLR was 0.07 (95% CI: 0.04–0.14) and DOR was 745.19 (95% CI: 229.30?2421.72). The area under the ROC was 0.98. Meta-regression indicated that the total number of samples was a source of heterogeneity for LAMP in detection of CD. The funnel plots suggested no publication bias. Conclusions The LAMP meets the minimum desirable characteristics of a diagnostic test of SEN, SPE and other measures of accuracy in the diagnosis of CD, and it is suitable as a rapid, effective and reliable stand-alone diagnostic test for diagnosis of CDI, potentially decreasing morbidity and nosocomial spread of CD. PMID:26528332

  9. Loop mediated isothermal amplification assay using hydroxy naphthol blue, conventional polymerase chain reaction and real-time PCR in the diagnosis of intraocular tuberculosis.

    PubMed

    Balne, P K; Basu, S; Rath, S; Barik, M R; Sharma, S

    2015-01-01

    This study is a comparative evaluation (Chi-square test) of a closed tube loop mediated isothermal amplification assay using hydroxy naphthol blue dye (HNB-LAMP), real-time polymerase chain reaction (PCR) and conventional PCR in the diagnosis of intraocular tuberculosis. Considering clinical presentation as the gold standard in 33 patients, the sensitivity of HNB-LAMP assay (75.8%) was higher (not significant, P value 0.2) than conventional PCR (57.6%) and lower than real-time PCR (90.9%). Specificity was 100% by all three methods. No amplification was observed in negative controls (n = 20) by all three methods. The cost of the HNB-LAMP assay was Rs. 500.00 and it does not require thermocycler, therefore, it can be used as an alternative to conventional PCR in resource-poor settings. PMID:26470966

  10. Rapid Detection of Subtype H10N8 Influenza Virus by One-Step Reverse Transcription-Loop-Mediated Isothermal Amplification Methods.

    PubMed

    Bao, Hongmei; Feng, Xiaoxiao; Ma, Yong; Shi, Jianzhong; Zhao, Yuhui; Gu, Linlin; Wang, Xiurong; Chen, Hualan

    2015-12-01

    We developed hemagglutinin- and neuraminidase-specific one-step reverse transcription-loop-mediated isothermal amplification assays for detecting the H10N8 virus. The detection limit of the assays was 10 copies of H10N8 virus, and the assays did not amplify nonspecific RNA. The assays can detect H10N8 virus from chicken samples with high sensitivity and specificity, and they can serve as an effective tool for detecting and monitoring H10N8 virus in live poultry markets. PMID:26378283

  11. Rapid detection of Bombyx mori nucleopolyhedrovirus (BmNPV) by loop-mediated isothermal amplification assay combined with a lateral flow dipstick method.

    PubMed

    Zhou, Yang; Wu, Jiege; Lin, Feng; Chen, Naifu; Yuan, Shaofei; Ding, Lina; Gao, Li; Hang, Bangxing

    2015-12-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) is a principal pathogen of the domestic silkworm. The disease often breaks out in sericultural countries and due to its high infectivity; it is difficult to control, resulting in heavy economic loss. In order to develop a rapid, sensitive visual detection and simple-to-use novel technology for detection of BmNPV, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) method was described. In this study, a set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the BmNPV gp41 gene, and the LAMP for the detection of BmNPV was developed by isothermal amplification at 61 °C for 45 min, followed by hybridization with an FITC-labeled DNA probe for 5 min and detected by LFD within 5 min. The detection limit of LAMP-LFD was 0.2 pg DNA extracted from silkworm infected with BmNPV and was 100 times more sensitive than conventional PCR. No product was generated from silkworm infected with other viruses. Furthermore, we applied the technique to detect BmNPV in the hemolymph and feces at different intervals post infection (pi). In conclusion, the novel LAMP-LFD setup presented here is simple, rapid, reliable, and has the potential for future use in the detection of BmNPV. PMID:26365226

  12. One simple DNA extraction device and its combination with modified visual loop-mediated isothermal amplification for rapid on-field detection of genetically modified organisms.

    PubMed

    Zhang, Miao; Liu, Yinan; Chen, Lili; Quan, Sheng; Jiang, Shimeng; Zhang, Dabing; Yang, Litao

    2013-01-01

    Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs. PMID:23181490

  13. Use of loop-mediated isothermal amplification of the IS900 sequence for rapid detection of cultured Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Enosawa, M; Kageyama, S; Sawai, K; Watanabe, K; Notomi, T; Onoe, S; Mori, Y; Yokomizo, Y

    2003-09-01

    We evaluated the usefulness of loop-mediated isothermal amplification (LAMP) in detecting specific gene sequences of Mycobacterium avium subsp. paratuberculosis (MAP). A total of 102 primer sets for LAMP was designed to amplify the IS900, HspX, and F57 gene sequences of MAP. Using each of two primer sets (P-1 and P-2) derived from the IS900 fragment, it was possible to detect MAP in a manner similar to that used with nested PCR. The sensitivity of LAMP with P-1 was 0.5 pg/tube, which was more sensitive than nested PCR. When P-2 was used, 5 pg/tube could be detected, which was the same level of sensitivity as that for nested PCR. LAMP with P-1 was specific. Although only 2 Mycobacterium scrofulaceum strains out of 43 non-MAP mycobacterial strains were amplified, the amplification reaction for these strains was less efficient than for MAP strains, and their products could be distinguished from MAP products by restriction digestion. LAMP with P-2 resulted in very specific amplification only from MAP, the same result obtained with nested PCR. Our LAMP method was highly specific, and the white turbidity of magnesium pyrophosphate, a by-product of the LAMP reaction, allowed simple visual detection. Our method is rapid, taking only 2 h, compared with 4 h for nested PCR. In addition, the LAMP method is performed under isothermal conditions and no special apparatus is needed, which makes it more economical and practical than nested PCR or real-time PCR. These results indicate that LAMP can provide a rapid yet simple test for the detection of MAP. PMID:12958269

  14. Development of a loop-mediated isothermal amplification method for the rapid detection of the dioxin-degrading bacterium Ochrobactrum anthropi in soil.

    PubMed

    Chen, Hsi-Jien; Lai, Jun-Yu; Lee, Meng-Shiou

    2015-09-01

    In this study, loop-mediated isothermal amplification (LAMP) and real-time LAMP assays were developed to detect the dioxin-degrading bacterium Ochrobactrum anthropi strain BD-1 in soil. Four primers were designed to use ITS gene amplification for the strain O. anthropi BD-1. The real-time LAMP assay was found to accomplish the reaction by 1 pg of genomic DNA load when used for nucleic acid amplification. This assay was then applied to detect O. anthropi BD-1 in eight soil samples collected from a dioxin-contaminated site. The results demonstrated that these newly developed LAMP and real-time LAMP assays will not only be useful and efficient tools for detecting the target gene, but also be used as molecular tools for monitoring the growth of dioxin-degrading O. anthropi in the soil. This is the first report to demonstrate the use of LAMP assays to monitor the presence of O. anthropi in dioxin-contaminated soil. The application of this method should improve the biomonitoring of dioxin contamination. PMID:26144562

  15. Variability of Potato virus Y in Tomato Crops in Poland and Development of a Reverse-Transcription Loop-Mediated Isothermal Amplification Method for Virus Detection.

    PubMed

    Hasiów-Jaroszewska, Beata; Stachecka, Joanna; Minicka, Julia; Sowi?ski, Mateusz; Borodynko, Natasza

    2015-09-01

    A collection of 147 Potato virus Y (PVY) isolates from tomato, originating from several commercial fields and greenhouses in different regions of Poland, was tested for the presence of PVY by reverse-transcription polymerase chain reaction. However, in some cases, the results obtained were ambiguous. Therefore, a sensitive reverse-transcription loop-mediated isothermal amplification method was developed for rapid detection of PVY isolates. Phylogenetic and recombination analyses were performed based on sequences of the coat protein gene. In comparison with results obtained in 2008, the presence of other strains besides PVY(N)Wi-P was confirmed. A novel recombinant between PVY(NTN) and PVY(N)Wi-P strains was detected. Our results indicate an increasing distribution and variability of the PVY population on tomato in Poland. PMID:25961337

  16. Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of common genetically modified organisms (GMOs).

    PubMed

    Feng, Jiawang; Tang, Shiming; Liu, Lideng; Kuang, Xiaoshan; Wang, Xiaoyu; Hu, Songnan; You, Shuzhu

    2015-03-01

    Here, we developed a loop-mediated isothermal amplification (LAMP) assay for 11 common transgenic target DNA in GMOs. Six sets of LAMP primer candidates for each target were designed and their specificity, sensitivity, and reproductivity were evaluated. With the optimized LAMP primers, this LAMP assay was simply run within 45-60 min to detect all these targets in GMOs tested. The sensitivity, specificity, and reproductivity of the LAMP assay were further analyzed in comparison with those of Real-Time PCR. In consistent with real-time PCR, detection of 0.5% GMOs in equivalent background DNA was possible using this LAMP assay for all targets. In comparison with real-time PCR, the LAMP assay showed the same results with simple instruments. Hence, the LAMP assay developed can provide a rapid and simple approach for routine screening as well as specific events detection of many GMOs. PMID:25582179

  17. Visual and Real-Time Event-Specific Loop-Mediated Isothermal Amplification Based Detection Assays for Bt Cotton Events MON531 and MON15985.

    PubMed

    Randhawa, Gurinder Jit; Chhabra, Rashmi; Bhoge, Rajesh K; Singh, Monika

    2015-01-01

    Bt cotton events MON531 and MON15985 are authorized for commercial cultivation in more than 18 countries. In India, four Bt cotton events have been commercialized; more than 95% of total area under genetically modified (GM) cotton cultivation comprises events MON531 and MON15985. The present study reports on the development of efficient event-specific visual and real-time loop-mediated isothermal amplification (LAMP) assays for detection and identification of cotton events MON531 and MON15985. Efficiency of LAMP assays was compared with conventional and real-time PCR assays. Real-time LAMP assay was found time-efficient and most sensitive, detecting up to two target copies within 35 min. The developed real-time LAMP assays, when combined with efficient DNA extraction kit/protocol, may facilitate onsite GM detection to check authenticity of Bt cotton seeds. PMID:26525238

  18. Specific detection of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella from single vegetative cells by a loop-mediated isothermal amplification method.

    PubMed

    Nagai, Satoshi; Itakura, Shigeru

    2012-09-01

    In this study, we succeeded in developing a loop-mediated isothermal amplification (LAMP) method that enables sensitive and specific detection of the toxic marine dinoflagellates Alexandrium tamarense and Alexandrium catenella from single cells of both laboratory cultures and naturally blooming cells within 25 min, by monitoring the turbidimeter from the start of the LAMP reaction. The fluorescence intensity was strong enough to allow discrimination between positive and negative results by naked eye under a UV lamp, even in amplified samples from a single cell, by using the LAMP method. Unambiguous detection by naked eye was possible even in half the volume of LAMP cocktail recommended by the manufacturer, suggesting the potential to significantly reduce the cost of Alexandrium monitoring. Therefore, we can conclude that this method is one of the most convenient, sensitive, and cost-effective molecular tools for Alexandrium monitoring. PMID:22897962

  19. Development and evaluation of a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of type II porcine reproductive and respiratory syndrome virus.

    PubMed

    Gao, Ming; Cui, Jin; Ren, Yudong; Suo, Siqingaowa; Li, Guangxing; Sun, Xuejiao; Su, Dingding; Opriessnig, Tanja; Ren, Xiaofeng

    2012-10-01

    The objective of this study was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of type II porcine reproductive and respiratory syndrome virus (PRRSV). Based on sequence alignment, four primers were designed amplifying the M gene of type II PRRSV and were subsequently utilized in an RT-LAMP assay. The RT-LAMP product had a ladder-like pattern of bands and the optimal reaction condition for this assay was determined to be 40 min at 63°C. Comparative analysis indicated that the RT-LAMP method was more sensitive than a conventional RT-PCR assay and comparable to a real-time PCR assay. In addition, the RT-LAMP assay was capable of detecting type II PRRSV in field samples and differentiating type II PRRSV from seven other porcine viruses which are all associated frequently with similar clinical symptoms. PMID:22659065

  20. Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae

    PubMed Central

    Wang, Deguo; Liu, Yanhong

    2015-01-01

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies. PMID:26016433

  1. Rapid Detection and Differentiation of Dengue Virus Serotypes by a Real-Time Reverse Transcription-Loop-Mediated Isothermal Amplification Assay

    PubMed Central

    Parida, Manmohan; Horioke, Kouhei; Ishida, Hiroyuki; Dash, Paban Kumar; Saxena, Parag; Jana, Asha Mukul; Islam, Mohammed Alimul; Inoue, Shingo; Hosaka, Norimitsu; Morita, Kouichi

    2005-01-01

    The development and validation of a one-step, real-time, and quantitative dengue virus serotype-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the 3? noncoding region for the rapid detection and differentiation of dengue virus serotypes are reported. The RT-LAMP assay is very simple and rapid, wherein the amplification can be obtained in 30 min under isothermal conditions at 63°C by employing a set of four serotype-specific primer mixtures through real-time monitoring in an inexpensive turbidimeter. The evaluation of the RT-LAMP assay for use for clinical diagnosis with a limited number of patient serum samples, confirmed to be infected with each serotype, revealed a higher sensitivity by picking up 100% samples as positive, whereas 87% and 81% of the samples were positive by reverse transcription-PCR and virus isolation, respectively. The sensitivity and specificity of the RT-LAMP assay for the detection of viral RNA in patient serum samples with reference to virus isolation were 100% and 93%, respectively. The optimal assay conditions with zero background and no cross-reaction with other closely related members of the Flavivirus family (Japanese encephalitis, West Nile, and St. Louis encephalitis viruses) as well as within the four serotypes of dengue virus were established. None of the serum samples from healthy individuals screened in this study showed any cross-reaction with the four dengue virus serotype-specific RT-LAMP assay primers. These findings demonstrate that RT-LAMP assay has the potential clinical application for detection and differentiation of dengue virus serotypes, especially in developing countries. PMID:15956414

  2. Detection of spring viraemia of carp virus (SVCV) by loop-mediated isothermal amplification (LAMP) in koi carp, Cyprinus carpio L

    USGS Publications Warehouse

    Shivappa, R.B.; Savan, R.; Kono, T.; Sakai, M.; Emmenegger, E.; Kurath, G.; Levine, J.F.

    2008-01-01

    Spring viraemia of carp virus (SVCV) is a rhabdovirus associated with systemic illness and mortality in cyprinids. Several diagnostic tests are available for detection of SVCV. However, most of these tests are time consuming and are not well adapted for field-based diagnostics. In this study, a diagnostic tool for SVCV detection based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been developed. Based on the nucleotide sequence of the glycoprotein (G) gene of SVCV North Carolina (NC) isolate, four sets (each set containing two outer and two inner) of primers were designed. Temperature and time conditions were optimized to 65 ??C and 60 min, respectively, for LAMP and RT-LAMP using one primer set. In vitro specificity was evaluated using four different strains of fish rhabdoviruses and RT-LAMP was found to be specific to SVCV. Serial dilutions of SVCV NC isolate was used to evaluate the in vitro sensitivity of RT-LAMP. Sensitivity of the assays was similar to RT-PCR and detected SVCV even at the lowest dilution of 10 1 TCID50 mL-1. The ability of RT-LAMP to detect SVCV from infected carp was also tested and the assay detected SVCV from all infected fish. The isothermal temperature requirements, high specificity and sensitivity, and short incubation time of the RT-LAMP assay make it an excellent choice as a field diagnostic test for SVCV. ?? 2008 The Authors.

  3. Development of a Rapid, Simple Method for Detecting Naegleria fowleri Visually in Water Samples by Loop-Mediated Isothermal Amplification (LAMP)

    PubMed Central

    Mahittikorn, Aongart; Mori, Hirotake; Popruk, Supaluk; Roobthaisong, Amonrattana; Sutthikornchai, Chantira; Koompapong, Khuanchai; Siri, Sukhontha; Sukthana, Yaowalark; Nacapunchai, Duangporn

    2015-01-01

    Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings. PMID:25822175

  4. A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay.

    PubMed

    Saitou, Yasumasa; Kobayashi, Yuki; Hirano, Shinji; Mochizuki, Nobuyuki; Itou, Takuya; Ito, Fumio H; Sakai, Takeo

    2010-09-01

    At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme AlwI. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RT-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RT-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field. PMID:20403387

  5. Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP(®) using reverse transcription-recombinase polymerase amplification.

    PubMed

    Zhang, Shulu; Ravelonandro, Michel; Russell, Paul; McOwen, Nathan; Briard, Pascal; Bohannon, Seven; Vrient, Albert

    2014-10-01

    Plum pox virus (PPV) causes the most destructive viral disease known as plum pox or Sharka disease in stone fruit trees. As an important regulated pathogen, detection of PPV is thus of critical importance to quarantine and eradication of the spreading disease. In this study, the innovative development of two AmplifyRP(®) tests is reported for a rapid isothermal detection of PPV using reverse transcription-recombinase polymerase amplification. In an AmplifyRP(®) test, all specific recombination and amplification reactions occur at a constant temperature without thermal cycling and the test results are either recorded in real-time with a portable fluorescence reader or displayed using a lateral flow strip contained inside an amplicon detection chamber. The major improvement of this assay is that the entire test from sample preparation to result can be completed in as little as 20min and can be performed easily both in laboratories and in the field. The results from this study demonstrated the ability of the AmplifyRP(®) technique to detect all nine PPV strains (An, C, CR, D, EA, M, Rec, T, or W). Among the economic benefits to pathogen surveys is the higher sensitivity of the AmplifyRP(®) to detect PPV when compared to the conventional ELISA and ImmunoStrip(®) assays. This is the first report describing the use of such an innovative technique to detect rapidly plant viruses affecting perennial crops. PMID:25010790

  6. A rapid molecular diagnosis of cutaneous leishmaniasis by colorimetric malachite green-loop-mediated isothermal amplification (LAMP) combined with an FTA card as a direct sampling tool.

    PubMed

    Nzelu, Chukwunonso O; Cáceres, Abraham G; Guerrero-Quincho, Silvia; Tineo-Villafuerte, Edwin; Rodriquez-Delfin, Luis; Mimori, Tatsuyuki; Uezato, Hiroshi; Katakura, Ken; Gomez, Eduardo A; Guevara, Angel G; Hashiguchi, Yoshihisa; Kato, Hirotomo

    2016-01-01

    Leishmaniasis remains one of the world's most neglected diseases, and early detection of the infectious agent, especially in developing countries, will require a simple and rapid test. In this study, we established a quick, one-step, single-tube, highly sensitive loop-mediated isothermal amplification (LAMP) assay for rapid detection of Leishmania DNA from tissue materials spotted on an FTA card. An FTA-LAMP with pre-added malachite green was performed at 64°C for 60min using a heating block and/or water bath and DNA amplification was detected immediately after incubation. The LAMP assay had high detection sensitivity down to a level of 0.01 parasites per ?l. The field- and clinic-applicability of the colorimetric FTA-LAMP assay was demonstrated with 122 clinical samples collected from patients suspected of having cutaneous leishmaniasis in Peru, from which 71 positives were detected. The LAMP assay in combination with an FTA card described here is rapid and sensitive, as well as simple to perform, and has great potential usefulness for diagnosis and surveillance of leishmaniasis in endemic areas. PMID:26516109

  7. Erwinia amylovora loop-mediated isothermal amplification (LAMP) assay for rapid pathogen detection and on-site diagnosis of fire blight.

    PubMed

    Bühlmann, Andreas; Pothier, Joël F; Rezzonico, Fabio; Smits, Theo H M; Andreou, Michael; Boonham, Neil; Duffy, Brion; Frey, Jürg E

    2013-03-01

    Several molecular methods have been developed for the detection of Erwinia amylovora, the causal agent of fire blight in pear and apple, but none are truly applicable for on-site use in the field. We developed a fast, reliable and field applicable detection method using a novel target on the E. amylovora chromosome that we identified by applying a comparative genomic pipeline. The target coding sequences (CDSs) are both uniquely specific for and all-inclusive of E. amylovora genotypes. This avoids potential false negatives that can occur with most commonly used methods based on amplification of plasmid gene targets, which can vary among strains. Loop-mediated isothermal AMPlification (LAMP) with OptiGene Genie II chemistry and instrumentation proved to be an exceptionally rapid (under 15 min) and robust method for detecting E. amylovora in orchards, as well as simple to use in the plant diagnostic laboratory. Comparative validation results using plant samples from inoculated greenhouse trials and from natural field infections (of regional and temporal diverse origin) showed that our LAMP had an equivalent or greater performance regarding sensitivity, specificity, speed and simplicity than real-time PCR (TaqMan), other LAMP assays, immunoassays and plating, demonstrating its utility for routine testing. PMID:23275135

  8. Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum.

    PubMed

    Duan, Yabing; Zhang, Xiaoke; Ge, Changyan; Wang, Yong; Cao, Junhong; Jia, Xiaojing; Wang, Jianxin; Zhou, Mingguo

    2014-01-01

    Resistance of Fusarium graminearum to carbendazim is caused by point mutations in the ?2-tubulin gene. The point mutation at codon 167 (TTT ? TAT, F167Y) occurs in more than 90% of field resistant isolates in China. To establish a suitable method for rapid detection of the F167Y mutation in F. graminearum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed and optimized to specially distinguish the F167Y mutation genotype. The LAMP reaction was optimal at 63 °C for 60 min. When hydroxynaphthol blue dye (HNB) was added prior to amplification, samples with DNA of the F167Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. The developed LAMP had good specificity, stability and repeatability and was suitable for monitoring carbendazim-resistance populations of F. graminearum in agricultural production. PMID:25403277

  9. Use of loop-mediated isothermal amplification for detection of Ophiostoma clavatum, the primary blue stain fungus associated with Ips acuminatus.

    PubMed

    Villari, Caterina; Tomlinson, Jennifer A; Battisti, Andrea; Boonham, Neil; Capretti, Paolo; Faccoli, Massimo

    2013-04-01

    Loop-mediated isothermal amplification (LAMP) is an alternative amplification technology which is highly sensitive and less time-consuming than conventional PCR-based methods. Three LAMP assays were developed, two for detection of species of symbiotic blue stain fungi associated with Ips acuminatus, a bark beetle infesting Scots pine (Pinus sylvestris), and an additional assay specific to I. acuminatus itself for use as a control. In common with most bark beetles, I. acuminatus is associated with phytopathogenic blue stain fungi involved in the process of exhausting tree defenses, which is a necessary step for the colonization of the plant by the insect. However, the identity of the main blue stain fungus vectored by I. acuminatus was still uncertain, as well as its frequency of association with I. acuminatus under outbreak and non-outbreak conditions. In this study, we employed LAMP technology to survey six populations of I. acuminatus sampled from the Southern Alps. Ophiostoma clavatum was detected at all sampling sites, while Ophiostoma brunneo-ciliatum, reported in part of the literature as the main blue stain fungus associated with I. acuminatus, was not detected on any of the samples. These results are consistent with the hypothesis that O. clavatum is the main blue stain fungus associated with I. acuminatus in the Southern Alps. The method developed in the course of this work provides a molecular tool by which it will be easy to screen populations and derive important data regarding the ecology of the species involved. PMID:23396326

  10. Use of Loop-Mediated Isothermal Amplification for Detection of Ophiostoma clavatum, the Primary Blue Stain Fungus Associated with Ips acuminatus

    PubMed Central

    Tomlinson, Jennifer A.; Battisti, Andrea; Boonham, Neil; Capretti, Paolo

    2013-01-01

    Loop-mediated isothermal amplification (LAMP) is an alternative amplification technology which is highly sensitive and less time-consuming than conventional PCR-based methods. Three LAMP assays were developed, two for detection of species of symbiotic blue stain fungi associated with Ips acuminatus, a bark beetle infesting Scots pine (Pinus sylvestris), and an additional assay specific to I. acuminatus itself for use as a control. In common with most bark beetles, I. acuminatus is associated with phytopathogenic blue stain fungi involved in the process of exhausting tree defenses, which is a necessary step for the colonization of the plant by the insect. However, the identity of the main blue stain fungus vectored by I. acuminatus was still uncertain, as well as its frequency of association with I. acuminatus under outbreak and non-outbreak conditions. In this study, we employed LAMP technology to survey six populations of I. acuminatus sampled from the Southern Alps. Ophiostoma clavatum was detected at all sampling sites, while Ophiostoma brunneo-ciliatum, reported in part of the literature as the main blue stain fungus associated with I. acuminatus, was not detected on any of the samples. These results are consistent with the hypothesis that O. clavatum is the main blue stain fungus associated with I. acuminatus in the Southern Alps. The method developed in the course of this work provides a molecular tool by which it will be easy to screen populations and derive important data regarding the ecology of the species involved. PMID:23396326

  11. Loop-mediated isothermal amplification assay targeting the blaCTX-M9 gene for detection of extended spectrum ?-lactamase-producing Escherichia coli and Klebsiella pneumoniae.

    PubMed

    Thirapanmethee, Krit; Pothisamutyothin, Kanokporn; Nathisuwan, Surakit; Chomnawang, Mullika T; Wiwat, Chanpen

    2014-12-01

    Extended-spectrum ?-lactamases (ESBLs) produced by Enterobacteriaceae are one of the resistance mechanisms to most ?-lactam antibiotics. ESBLs are currently a major problem in both hospitals and community settings worldwide. Rapid and reliable means of detecting ESBL-producing bacteria is necessary for identification, prevention and treatment. Loop-mediated isothermal amplification (LAMP) is a technique that rapidly amplifies DNA with high specificity and sensitivity under isothermal conditions. This study was aimed to develop a convenient, accurate and inexpensive method for detecting ESBL-producing bacteria by a LAMP technique. ESBLs-producing Escherichia coli and Klebsiella pneumoniae were isolated from a tertiary hospital in Bangkok, Thailand and reconfirmed by double-disk synergy test. A set of four specific oligonucleotide primers of LAMP for detection of bla(CTX-M9) gene was designed based on bla(CTX-M9) from E. coli (GenBank Accession No. AJ416345). The LAMP reaction was amplified under isothermal temperature at 63°C for 60?min. Ladder-like patterns of band sizes from 226 bp of the bla(CTX-M9) DNA target was observed. The LAMP product was further analyzed by restriction digestion with MboI and TaqI endonucleases. The fragments generated were approximately 168, 177 and 250 bp in size for MboI digestion and 165, 193, 229, 281 and 314 bp for TaqI digestion, which is in agreement with the predicted sizes. The sensitivity of the LAMP technique to bla(CTX-M9) was greater than that of the PCR method by at least 10,000-fold. These results showed that the LAMP primers specifically amplified only the bla(CTX-M9) gene. Moreover, the presence of LAMP amplicon was simply determined by adding SYBR Green I in the reaction. In conclusion, this technique for detection of ESBLs is convenient, reliable and easy to perform routinely in hospitals or laboratory units in developing countries. PMID:25284314

  12. Highly sensitive fluorescence assay of DNA methyltransferase activity by methylation-sensitive cleavage-based primer generation exponential isothermal amplification-induced G-quadruplex formation.

    PubMed

    Xue, Qingwang; Lv, Yanqin; Xu, Shuling; Zhang, Yuanfu; Wang, Lei; Li, Rui; Yue, Qiaoli; Li, Haibo; Gu, Xiaohong; Zhang, Shuqiu; Liu, Jifeng

    2015-04-15

    Site-specific identification of DNA methylation and assay of MTase activity are imperative for determining specific cancer types, provide insights into the mechanism of gene repression, and develop novel drugs to treat methylation-related diseases. Herein, we developed a highly sensitive fluorescence assay of DNA methyltransferase by methylation-sensitive cleavage-based primer generation exponential isothermal amplification (PG-EXPA) coupled with supramolecular fluorescent Zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex. In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The cleaved hairpin probe then functions as a signal primer to initiate the exponential isothermal amplification reaction (EXPAR) by hybridizing with a unimolecular DNA containing three functional domains as the amplification template, producing a large number of G-quadruplex nanostructures by utilizing polymerases and nicking enzymes as mechanical activators. The G-quadruplex nanostructures act as host for ZnPPIX that lead to supramolecular complexes ZnPPIX/G-quadruplex, which provides optical labels for amplified fluorescence detection of Dam MTase. While in the absence of Dam MTase, neither methylation/cleavage nor PG-EXPA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a wide dynamic range from 0.0002 to 20U/mL and an extremely low detection limit of 8.6×10(-5)U/mL, which is superior to most conventional approaches for the MTase assay. Owing to the specific site recognition of MTase toward its substrate, the proposed sensing system was able to readily discriminate Dam MTase from other MTase such as M.SssI and even detect the target in a complex biological matrix. Furthermore, the application of the proposed sensing strategy for screening Dam MTase inhibitors was also demonstrated with satisfactory results. This novel method not only provides a promising platform for monitoring activity and inhibition of DNA MTases, but also shows great potentials in biological process researches, drugs discovery and clinical diagnostics. PMID:25506903

  13. Protein-coding housekeeping gene Rv2461c can be used as an amplification target in loop-mediated isothermal amplification assay for the detection of Mycobacterium tuberculosis in sputum samples

    PubMed Central

    Li, Dairong; Zhao, Jianing; Nie, Xiaoping; Wan, Tao; Xu, Wenchun; Zhao, Yong

    2014-01-01

    The study is to explore the potential of the conserved Rv2461c gene as a biomarker for Tuberculosis (TB) diagnosis. The conservation of the hypothetical genes was evaluated in this study using multiple sequence alignment and phylogenetic analysis. The conservation of Rv2461c coding gene was evaluated by polymerase chain reaction using six reference strains of M. tuberculosis complex (MTC), 156 M. tuberculosis clinical isolates, 25 species of non-tuberculosis mycobacteria (NTM), and 10 non-mycobacterial species. A total of 126 clinical sputum specimens were collected from patients with respiratory symptoms, including 79 specimens from suspected TB patients, and 47 specimens from patients with respiratory diseases other than TB. Genomic DNAs were extracted and subject to polymerase chain reaction for nucleic acid amplification test. In addition, we successfully developed loop-mediated isothermal amplification (LAMP) technology for rapid detection of M. tuberculosis in sputum specimens. The sensitivity and specificity of LAMP assay were evaluated for the detection of M. tuberculosis. Phylogenetic analysis of the clpP sequences revealed that the Mycobacterium strains were split into two major clusters: i) MTC; ii) NTM strains and M. leprae. During the evaluation of the conservation of Rv2461c coding gene, all MTC strains yielded positive results, and no false-positive results were observed in NTM or other bacterial species. LAMP analysis showed high sensitivity and specificity (84.8% and 95.7%, respectively) for the detection of M. tuberculosis from sputum. Our result indicated that Rv2461c coding gene was an efficient and promising alternative nucleic acid amplification test target for the detection of M. tuberculosis. PMID:25674236

  14. Protein-coding housekeeping gene Rv2461c can be used as an amplification target in loop-mediated isothermal amplification assay for the detection of Mycobacterium tuberculosis in sputum samples.

    PubMed

    Li, Dairong; Zhao, Jianing; Nie, Xiaoping; Wan, Tao; Xu, Wenchun; Zhao, Yong

    2014-01-01

    The study is to explore the potential of the conserved Rv2461c gene as a biomarker for Tuberculosis (TB) diagnosis. The conservation of the hypothetical genes was evaluated in this study using multiple sequence alignment and phylogenetic analysis. The conservation of Rv2461c coding gene was evaluated by polymerase chain reaction using six reference strains of M. tuberculosis complex (MTC), 156 M. tuberculosis clinical isolates, 25 species of non-tuberculosis mycobacteria (NTM), and 10 non-mycobacterial species. A total of 126 clinical sputum specimens were collected from patients with respiratory symptoms, including 79 specimens from suspected TB patients, and 47 specimens from patients with respiratory diseases other than TB. Genomic DNAs were extracted and subject to polymerase chain reaction for nucleic acid amplification test. In addition, we successfully developed loop-mediated isothermal amplification (LAMP) technology for rapid detection of M. tuberculosis in sputum specimens. The sensitivity and specificity of LAMP assay were evaluated for the detection of M. tuberculosis. Phylogenetic analysis of the clpP sequences revealed that the Mycobacterium strains were split into two major clusters: i) MTC; ii) NTM strains and M. leprae. During the evaluation of the conservation of Rv2461c coding gene, all MTC strains yielded positive results, and no false-positive results were observed in NTM or other bacterial species. LAMP analysis showed high sensitivity and specificity (84.8% and 95.7%, respectively) for the detection of M. tuberculosis from sputum. Our result indicated that Rv2461c coding gene was an efficient and promising alternative nucleic acid amplification test target for the detection of M. tuberculosis. PMID:25674236

  15. Improved Detection Limit in Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 by a Novel Reverse Transcription–Isothermal Multiple-Self-Matching-Initiated Amplification Assay

    PubMed Central

    Ding, Xiong; Nie, Kai; Shi, Lei; Zhang, Yong; Guan, Li; Zhang, Dan

    2014-01-01

    Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-foot-and-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription–isothermal multiple-self-matching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R2 values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R2 values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71 and CVA16 viruses. PMID:24648558

  16. Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for detection of Leishmania DNA in buffy coat from visceral leishmaniasis patients

    PubMed Central

    2012-01-01

    Background Visceral leishmaniasis (VL) remains as one of the most neglected tropical diseases with over 60% of the world’s total VL cases occurring in the Indian subcontinent. Due to the invasive risky procedure and technical expertise required in the classical parasitological diagnosis, the goal of the VL experts has been to develop noninvasive procedure(s) applicable in the field settings. Several serological and molecular biological approaches have been developed over the last decades, but only a few are applicable in field settings that can be performed with relative ease. Recently, loop-mediated isothermal amplification (LAMP) has emerged as a novel nucleic acid amplification method for diagnosis of VL. In this study, we have evaluated the LAMP assay using buffy coat DNA samples from VL patients in Bangladesh and compared its performance with leishmania nested PCR (Ln-PCR), an established molecular method with very high diagnostic indices. Methods Seventy five (75) parasitologically confirmed VL patients by spleen smear microcopy and 101 controls (endemic healthy controls ?25, non-endemic healthy control-26, Tuberculosis-25 and other diseases-25) were enrolled in this study. LAMP assay was carried out using a set of four primers targeting L. donovani kinetoplast minicircle DNA under isothermal (62 °C) conditions in a heat block. For Ln-PCR, we used primers targeting the parasite’s small-subunit rRNA region. Results LAMP assay was found to be positive in 68 of 75 confirmed VL cases, and revealed its diagnostic sensitivity of 90.7% (95.84-81.14, 95% CI), whereas all controls were negative by LAMP assay, indicating a specificity of 100% (100–95.43, 95% CI). The Ln-PCR yielded a sensitivity of 96% (98.96-87.97, 95% CI) and a specificity of 100% (100–95.43, 95% CI). Conclusion High diagnostic sensitivity and excellent specificity were observed in this first report of LAMP diagnostic evaluation from Bangladesh. Considering its many fold advantages over conventional PCR and potential to be used as a simple and rapid test in the VL endemic areas of the Indian subcontinent, our findings are encouraging, but further evaluation of LAMP is needed. PMID:23206441

  17. Development and Application of Loop-Mediated Isothermal Amplification Assays for Rapid Visual Detection of cry2Ab and cry3A Genes in Genetically-Modified Crops

    PubMed Central

    Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

    2014-01-01

    The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field. PMID:25167136

  18. Development and application of loop-mediated isothermal amplification assays for rapid visual detection of cry2Ab and cry3A genes in genetically-modified crops.

    PubMed

    Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

    2014-01-01

    The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field. PMID:25167136

  19. Smartphone-Imaged HIV-1 Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) on a Chip from Whole Blood

    PubMed Central

    Damhorst, Gregory L.; Duarte-Guevara, Carlos; Chen, Weili; Ghonge, Tanmay; Cunningham, Brian T.; Bashir, Rashid

    2015-01-01

    Viral load measurements are an essential tool for the long-term clinical care of hum an immunodeficiency virus (HIV)-positive individuals. The gold standards in viral load instrumentation, however, are still too limited by their size, cost, and sophisticated operation for these measurements to be ubiquitous in remote settings with poor healthcare infrastructure, including parts of the world that are disproportionately affected by HIV infection. The challenge of developing a point-of-care platform capable of making viral load more accessible has been frequently approached but no solution has yet emerged that meets the practical requirements of low cost, portability, and ease-of-use. In this paper, we perform reverse-transcription loop-mediated isothermal amplification (RT-LAMP) on minimally processed HIV-spiked whole blood samples with a microfluidic and silicon microchip platform, and perform fluorescence measurements with a consumer smartphone. Our integrated assay shows amplification from as few as three viruses in a ~ 60 nL RT-LAMP droplet, corresponding to a whole blood concentration of 670 viruses per µL of whole blood. The technology contains greater power in a digital RT-LAMP approach that could be scaled up for the determination of viral load from a finger prick of blood in the clinical care of HIV-positive individuals. We demonstrate that all aspects of this viral load approach, from a drop of blood to imaging the RT-LAMP reaction, are compatible with lab-on-a-chip components and mobile instrumentation. PMID:26705482

  20. Target-regulated proximity hybridization with three-way DNA junction for in situ enhanced electronic detection of marine biotoxin based on isothermal cycling signal amplification strategy.

    PubMed

    Liu, Bingqian; Chen, Jinfeng; Wei, Qiaohua; Zhang, Bing; Zhang, Lan; Tang, Dianping

    2015-07-15

    A new signal amplification strategy based on target-regulated DNA proximity hybridization (TRPH) reaction accompanying formation of three-way DNA junction was designed for electronic detection of Microcystin-LR (MC-LR used in this case), coupling with junction-induced isothermal cycling signal amplification. Initially, a sandwiched-type immunoreaction was carried out in a low-cost PCR tube between anti-MC-LR mAb1 antibody-labeled DNA1 (mAb1-DNA1) and anti-MC-LR mAb2-labeled DNA2 (mAb2-DNA2) in the presence of target to form a three-way DNA junction. Then, the junction could undergo an unbiased strand displacement reaction on an h-like DNA nanostructure-modified electrode (labeled with methylene blue redox tag on the short DNA strand), thereby resulting in the dissociation of methylene blue-labeled signal DNA from the electrode. The newly formed double-stranded DNA could be cleaved again by exonuclease III, and the released three-way DNA junction retriggered the strand-displacement reaction with h-like DNA nanostructures for junction recycling. During the strand-displacement reaction, numerous methylene blue-labeled DNA strands were far away from the electrode, thus decreasing the detectable electrochemical signal within the applied potentials. Under optimal conditions, the TRPH-based immunosensing system exhibited good electrochemical responses for detecting target MC-LR at a concentration as low as 1.0ngkg(-1) (1.0ppt). Additionally, the precision, reproducibility, specificity and method accuracy were also investigated with acceptable results. PMID:25747510

  1. One-step detection of Bean pod mottle virus in soybean seeds by the reverse-transcription loop-mediated isothermal amplification

    PubMed Central

    2012-01-01

    Background Bean pod mottle virus (BPMV) is a wide-spread and destructive virus that causes huge economic losses in many countries every year. A sensitive, reliable and specific method for rapid surveillance is urgently needed to prevent further spread of BPMV. Methods A degenerate reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer set was designed on the conserved region of BPMV CP gene. The reaction conditions of RT-LAMP were optimized and the feasibility, specificity and sensitivity of this method to detect BPMV were evaluated using the crude RNA rapidly extracted from soybean seeds. Results The optimized RT-LAMP parameters including 6?mM MgCl2, 0.8?M betaine and temperature at 62.5-65°C could successfully amplify the ladder-like bands from BPMV infected soybean seeds. The amplification was very specific to BPMV that no cross-reaction was observed with other soybean viruses. Inclusion of a fluorescent dye makes it easily be detected in-tube by naked eye. The sensitivity of RT-LAMP assay is higher than the conventional RT-PCR under the conditions tested, and the conventional RT-PCR couldn’t be used for detection of BPMV using crude RNA extract from soybean seeds. Conclusion A highly efficient and practical method was developed for the detection of BPMV in soybean seeds by the combination of rapid RNA extraction and RT-LAMP. This RT-LAMP method has great potential for rapid BPMV surveillance and will assist in preventing further spread of this devastating virus. PMID:22958497

  2. Direct identification and discernment of Mycobacterium avium and Mycobacterium intracellulare using a real-time RNA isothermal amplification and detection method.

    PubMed

    Cui, Zhenling; Li, Yuanyuan; Cheng, Song; Yang, Hua; Lu, Junmei; Zhu, Honglei; Hu, Zhongyi

    2015-12-01

    The purpose of this work was to establish a real-time simultaneous amplification and testing method for identification and discernment of Mycobacterium avium and Mycobacterium intracellulare (SAT-MAC assay) and to evaluate the efficiency with which this method can detect isolated strains and clinical sputum specimens. The specific 16S rRNA sequences of M. avium and M. intracellulare were used as targets to design RNA probes and a reverse transcription primer containing T7 promoter. RNA isothermal amplification and real-time fluorescence detection were performed at 42 °C. SAT-MAC assay, culture tests on Lowenstein-Jensen (L-J) culture medium and PCR-sequencing were used to test the clinical isolated strains and sputum specimens. The limit of detection (LOD) of M. avium and M. intracellulare by SAT-MAC was found to be 30 CFU/mL and 20 CFU/mL. SAT-MAC showed high specificity in 21 species of mycobacteria standard strains and 5 species of non-mycobacteria bacteria. Using PCR-sequencing as the reference method, both rates of SAT-MAC assay for identifying M. avium and M. intracellulare from clinical isolates were 100% (259/259). Consistent with the results of L-J culture combined PCR-sequencing, the coincidence rate of SAT-MAC assay in clinical sputum specimens was 100% (369/369) for M. avium and 99.19% (366/369) for Mycobacterium intracellular. The SAT-MAC assay can identify and distinguish M. avium and M. intracellulare rapidly and accurately. It may be suitable for use in clinical microbiology laboratories. PMID:26316142

  3. Rapid and Sensitive Detection of Shigella spp. and Salmonella spp. by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique

    PubMed Central

    Wang, Yi; Wang, Yan; Luo, Lijuan; Liu, Dongxin; Luo, Xia; Xu, Yanmei; Hu, Shoukui; Niu, Lina; Xu, Jianguo; Ye, Changyun

    2015-01-01

    Shigella and Salmonella are frequently isolated from various food samples and can cause human gastroenteritis. Here, a novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully established and validated for simultaneous detection of Shigella strains and Salmonella strains in only a single reaction. Two sets of MERT-LAMP primers for 2 kinds of pathogens were designed from ipaH gene of Shigella spp. and invA gene of Salmonella spp., respectively. Under the constant condition at 63°C, the positive results were yielded in as short as 12 min with the genomic DNA extracted from the 19 Shigella strains and 14 Salmonella strains, and the target pathogens present in a sample could be simultaneously identified based on distinct fluorescence curves in real-time format. Accordingly, the multiplex detection assay significantly reduced effort, materials and reagents used, and amplification and differentiation were conducted at the same time, obviating the use of postdetection procedures. The analytical sensitivity of MERT-LAMP was found to be 62.5 and 125 fg DNA/reaction with genomic templates of Shigella strains and Salmonella strains, which was consist with normal LAMP assay, and at least 10- and 100-fold more sensitive than that of qPCR and conventional PCR approaches. The limit of detection of MERT-LAMP for Shigella strains and Salmonella strains detection in artificially contaminated milk samples was 5.8 and 6.4 CFU per vessel. In conclusion, the MERT-LAMP methodology described here demonstrated a potential and valuable means for simultaneous screening of Shigella and Salmonella in a wide variety of samples. PMID:26697000

  4. Ag(I)-coordinated hairpin DNA for homogenous electronic monitoring of hepatitis C virus accompanying isothermal cycling signal amplification strategy.

    PubMed

    Lu, Minghua; Xu, Linfang; Zhang, Xiaona; Xiao, Rui; Wang, Youmei

    2015-11-15

    This work designs a new homogenous electronic monitoring platform for sensitive detection of hepatitis C virus (HCV) on an immobilization-free Ag(I)-assisted hairpin DNA through the cytosine-Ag(+)-cytosine coordination chemistry. The assay consists of target-induced Ag(+) dissociation from hairpin DNA and an isothermal circular strand-displacement polymerization (ICSDP) reaction. Upon target analyte introduction, HCV DNA initially hybridizes with hairpin DNA to disrupt the Ag(I)-coordinated hairpin probe and releases the coordinated Ag(+) ion, then the newly formed DNA duplex induces the ICSDP reaction with the aid of primer and polymerase, and then the displaced target DNA retriggers Ag(I)-coordinated hairpin DNA with target recycling, thereby resulting in formation of numerous free Ag(+) ions in the detection cell. The released Ag(+) ions can be readily captured by the negatively charged screen-printed carbon electrode, and subsequent anodic-stripping voltammetric detection of the captured Ag(+) ions are conducted to form the anodic current for the production of the electrochemical signal within the applied potential. Under optimal conditions, the ICSDP-based homogenous sensing system can be utilized for the detection of HCV DNA at a concentration as low as 2.3 pM. Intra- and inter-assay coefficients of variation with identical batches are below 9.5% and 10.5%, respectively. The analysis in 5 clinical serum specimens shows good accordance between results obtained by the developed method and commercial Cobas® Amplicor HCV Test Analyzer. PMID:26071691

  5. The detection of T-Nos, a genetic element present in GMOs, by cross-priming isothermal amplification with real-time fluorescence.

    PubMed

    Zhang, Fang; Wang, Liu; Fan, Kai; Wu, Jian; Ying, Yibin

    2014-05-01

    An isothermal cross-priming amplification (CPA) assay for Agrobacterium tumefaciens nopaline synthase terminator (T-Nos) was established and investigated in this work. A set of six specific primers, recognizing eight distinct regions on the T-Nos sequence, was designed. The CPA assay was performed at a constant temperature, 63 °C, and detected by real-time fluorescence. The results indicated that real-time fluorescent CPA had high specificity, and the limit of detection was 1.06?×?10(3) copies of rice genomic DNA, which could be detected in 40 min. Comparison of real-time fluorescent CPA and conventional polymerase chain reaction (PCR) was also performed. Results revealed that real-time fluorescent CPA had a comparable sensitivity to conventional real-time PCR and had taken a shorter time. In addition, different contents of genetically modified (GM)-contaminated rice seed powder samples were detected for practical application. The result showed real-time fluorescent CPA could detect 0.5 % GM-contaminated samples at least, and the whole reaction could be finished in 35 min. Real-time fluorescent CPA is sensitive enough to monitor labeling systems and provides an attractive method for the detection of GMO. PMID:24748469

  6. A loop-mediated isothermal amplification (LAMP) assay for Strongyloides stercoralis in stool that uses a visual detection method with SYTO-82 fluorescent dye.

    PubMed

    Watts, Matthew R; James, Gregory; Sultana, Yasmin; Ginn, Andrew N; Outhred, Alexander C; Kong, Fanrong; Verweij, Jaco J; Iredell, Jonathan R; Chen, Sharon C-A; Lee, Rogan

    2014-02-01

    An assay to detect Strongyloides stercoralis in stool specimens was developed using the loop-mediated isothermal amplification (LAMP) method. Primers were based on the 28S ribosomal subunit gene. The reaction conditions were optimized and SYTO-82 fluorescent dye was used to allow real-time and visual detection of the product. The product identity was confirmed with restriction enzyme digestion, cloning, and sequence analysis. The assay was specific when tested against DNA from bacteria, fungi and parasites, and 30 normal stool samples. Analytical sensitivity was to < 10 copies of target sequence in a plasmid and up to a 10(-2) dilution of DNA extracted from a Strongyloides ratti larva spiked into stool. Sensitivity was increased when further dilutions were made in water, indicative of reduced reaction inhibition. Twenty-seven of 28 stool samples microscopy and polymerase chain reaction positive for S. stercoralis were positive with the LAMP method. On the basis of these findings, the assay warrants further clinical validation. PMID:24323513

  7. Rapid and sensitive detection of Dasheen mosaic virus infecting elephant foot yam by reverse transcription loop mediated isothermal amplification of coat protein gene.

    PubMed

    Kamala, S; Makeshkumar, T

    2015-09-15

    Dasheen mosaic virus (DsMV), the pathogen causing mosaic disease of elephant foot yam (Amorphophallus paeoniifoilius) is disseminated mainly through vegetative propagation of the tubers. For the rapid and sensitive detection of the virus, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay based on the coat protein gene has been developed. A final concentration of 5.4 mM magnesium sulphate and 0.7 M betaine in the reaction mixture was found to be optimum for getting characteristic ladder like bands of the amplified product after gel electrophoresis. The reaction was set at 65°C for 50 min followed by reaction termination at 86°C for 5 min in a water bath. The sensitivity of the assay was found to be 100 times higher than that of RT-PCR. The virus was indexed successfully from tubers of elephant foot yam. In tube detection of the DsMV was carried out using fluorescence detection reagents. The assay was validated with field samples from various regions of Kerala state, India. PMID:26096915

  8. Reverse transcription loop-mediated isothermal amplification of RNA for sensitive and rapid detection of southern rice black-streaked dwarf virus.

    PubMed

    Zhou, Tong; Du, Linlin; Fan, Yongjian; Zhou, Yijun

    2012-03-01

    Southern rice black-streaked dwarf virus (SRBSDV) causes one of the most serious viral diseases of rice in Southeast Asia. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of SRBSDV using total RNA extracted from rice tissues and the insect pest, white-backed planthopper. The assay was based on a set of four primers matching a total of six sequences in the S9 region of SRBSDV genome. Presence of the virus could be detected in RT-LAMP reactions containing 1.2×10(-6)?g of a total RNA extract, which was ten times more sensitive than a classical RT-PCR assay. The SRBSDV could be distinguished from the closely related rice black-streaked dwarf virus (RBSDV) by this method, indicating a high degree of specificity. This simple and sensitive RT-LAMP assay shows potential for detection of SRBSDV in field samples of hosts or vectors at a relatively low cost. PMID:22227615

  9. Field Evaluation of Malaria Microscopy, Rapid Malaria Tests and Loop-Mediated Isothermal Amplification in a Rural Hospital in South Western Ethiopia

    PubMed Central

    Prieto-Pérez, Laura; Martin-Martin, Ines; Berzosa, Pedro; González, Vicenta; Tisiano, Gebre; Balcha, Seble; Ramos, José Manuel; Górgolas, Miguel

    2015-01-01

    Background In up to one third of the hospitals in some rural areas of Africa, laboratory services in malaria diagnosis are limited to microscopy by thin film, as no capability to perform thick film exists (gold standard in terms of sensitivity for malaria diagnosis). A new rapid molecular malaria diagnostic test called Loop-mediated isothermal DNA amplification (LAMP) has been recently validated in clinical trials showing exceptional sensitivity and specificity features. It could be a reliable diagnostic tool to be implemented without special equipment or training. Objective The objective of this proof of concept study was to confirm the feasibility of using LAMP technique for diagnosis of malaria in a rural Ethiopian hospital with limited resources. Methodology/Principal Findings This study was carried out in Gambo General Hospital, West Arsi Province (Ethiopia), from November 1st to December 31st 2013. A total of 162 patients with a non-focal febrile syndrome were investigated. The diagnostic capability (sensitivity, specificity, positive predictive and negative predictive values) of rapid malaria tests and microscopy by thin film was evaluated in comparison with LAMP. Eleven (6.79%) out of the 162 patients with fever and suspected malaria, tested positive for LAMP, 3 (1.85%) for rapid malaria tests and none of the eleven cases was detected by thin film microscopy. Conclusions/Significance LAMP can be performed in basic rural laboratories without the need for specialized infrastructure and it may set a reliable tool for malaria control to detect a low level parasitemia. PMID:26555068

  10. The Novel Multiple Inner Primers-Loop-Mediated Isothermal Amplification (MIP-LAMP) for Rapid Detection and Differentiation of Listeria monocytogenes.

    PubMed

    Wang, Yi; Wang, Yan; Ma, Aijing; Li, Dongxun; Luo, Lijuan; Liu, Dongxin; Hu, Shoukui; Jin, Dong; Liu, Kai; Ye, Changyun

    2015-01-01

    Here, a novel model of loop-mediated isothermal amplification (LAMP), termed multiple inner primers-LAMP (MIP-LAMP), was devised and successfully applied to detect Listeria monocytogenes. A set of 10 specific MIP-LAMP primers, which recognized 14 different regions of target gene, was designed to target a sequence in the hlyA gene. The MIP-LAMP assay efficiently amplified the target element within 35 min at 63 °C and was evaluated for sensitivity and specificity. The templates were specially amplified in the presence of the genomic DNA from L. monocytogenes. The limit of detection (LoD) of MIP-LAMP assay was 62.5 fg/reaction using purified L. monocytogenes DNA. The LoD for DNA isolated from serial dilutions of L. monocytogenes cells in buffer and in milk corresponded to 2.4 CFU and 24 CFU, respectively. The amplified products were analyzed by real-time monitoring of changes in turbidity, and visualized by adding Loop Fluorescent Detection Reagent (FD), or as a ladder-like banding pattern on gel electrophoresis. A total of 48 pork samples were investigated for L. monocytogenes by the novel MIP-LAMP method, and the diagnostic accuracy was shown to be 100% when compared to the culture-biotechnical method. In conclusion, the MIP-LAMP methodology was demonstrated to be a reliable, sensitive and specific tool for rapid detection of L. monocytogenes strains. PMID:26633345

  11. Visual Detection of Canine Parvovirus Based on Loop-Mediated Isothermal Amplification Combined with Enzyme-Linked Immunosorbent Assay and with Lateral Flow Dipstick

    PubMed Central

    SUN, Yu-Ling; YEN, Chon-Ho; TU, Ching-Fu

    2013-01-01

    ABSTRACT Loop-mediated isothermal amplification (LAMP) combined with enzyme-linked immunosorbent assay (LAMP–ELISA) and with lateral flow dipstick (LAMP–LFD) are rapid, sensitive and specific methods for the visual detection of clinical pathogens. In this study, LAMP–ELISA and LAMP–LFD were developed for the visual detection of canine parvovirus (CPV). For LAMP, a set of four primers (biotin-labeled forward inner primers) was designed to specifically amplify a region of the VP2 gene of CPV. The optimum time and temperature for LAMP were 60 min and 65°C, respectively. The specific capture oligonucleotide probes, biotin-labeled CPV probe for LAMP–ELISA and fluorescein isothiocyanate-labeled CPV probe for LAMP–LFD were also designed for hybridization with LAMP amplicons on streptavidin-coated wells and LFD strips, respectively. For the comparison of detection sensitivity, conventional PCR and LAMP for CPV detection were also performed. The CPV detection limits by PCR, PCR–ELISA, LAMP, LAMP–ELISA and LAMP–LFD were 102, 102, 10?1, 10?1 and 10?1 TCID50/ml, respectively. In tests using artificially contaminated dog fecal samples, the samples with CPV inoculation levels of ?1 TCID50/ml gave positive results by both LAMP–ELISA and LAMP–LFD. Our data indicated that both LAMP–ELISA and LAMP–LFD are promising as rapid, sensitive and specific methods for an efficient diagnosis of CPV infection. PMID:24334855

  12. Evaluation of three enzyme immunoassays and a loop-mediated isothermal amplification test for the laboratory diagnosis of Clostridium difficile infection.

    PubMed

    Bruins, M J; Verbeek, E; Wallinga, J A; Bruijnesteijn van Coppenraet, L E S; Kuijper, E J; Bloembergen, P

    2012-11-01

    The laboratory diagnosis of Clostridium difficile infection (CDI) consists of the detection of toxigenic Clostridium difficile, and/or its toxins A or B in stool preferably in a two-step algorithm. In a prospective study, we compared the performance of three toxin enzyme immunoassays (EIAs)-ImmunoCard Toxins A & B, Premier Toxins A & B and C. diff Quik Chek Complete, which combines a toxins test and a glutamate dehydrogenase (GDH) antigen EIA in one device -and the loop-mediated isothermal amplification assay Illumigene C. difficile. In total 986 stool samples were analyzed. Compared with toxigenic culture as the gold standard, sensitivities, specificities, PPV and NPV values of the toxin EIAs were 41.1-54.8 %, 98.9-100 %, 75.0-100 % and 95.5-96.5 % respectively, of the Illumigene assay 93.3 %, 99.7 %, 95.8 % and 99.5 %. Illumigene assays performed significantly better for non-014/020 PCR-ribotypes than for C. difficile isolates belonging to 014/020. Discrepant analysis of three culture-negative, but Illumigene-positive samples, revealed the presence of toxin genes using real-time PCRs. In addition to the GDH EIA (NPV of 99.8 %), the performance of Illumigene allows this test to be introduced as a first screening test for CDI- or as a confirmation test for GDH -positive samples, although the initial invalid Illumigene result of 4.4 % is a point of concern. PMID:22706512

  13. Rapid and Sensitive Detection of Vibrio alginolyticus by Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick Targeted to the rpoX Gene.

    PubMed

    Plaon, Saranya; Longyant, Siwaporn; Sithigorngul, Paisarn; Chaivisuthangkura, Parin

    2015-09-01

    Vibrio alginolyticus is a major bacterial pathogen causing disease in marine animals. The present study aimed to develop a loop-mediated isothermal amplification (LAMP) coupled with a lateral flow dipstick (LFD) for rapid and simple visual detection of V. alginolyticus-specific amplicons. The biotin-labeled LAMP amplicons from the targeted portion of a gene encoding rpoS-like sigma factor (rpoX) were generated at 60°C for 1 h and then hybridized with a fluorescein isothiocyanate-labeled probe for 5 min for visual detection with LFD. In pure cultures, the detection limit of the LAMP-LFD technique for V. alginolyticus was 1.8 × 10(2) CFU/mL while that of PCR was 1.8 × 10(3) CFU/mL. In spiked whiteleg shrimp samples Penaeus vannamei, the sensitivity for V. alginolyticus detection was 2 × 10(3) CFU/g (equivalent to 4 CFU per reaction) while PCR was 10 times less sensitive. The LAMP-LFD method for V. alginolyticus correctly identified 21 isolates of V. alginolyticus but did not recognize 23 non-V. alginolyticus Vibrio isolates and 15 non-Vibrio isolates. In summary, this LAMP-LFD method targeted to the rpoX gene is a convenient assay for specific identification of V. alginolyticus with high sensitivity. PMID:26220075

  14. Establishment of reverse transcription loop-mediated isothermal amplification for rapid detection and differentiation of canine distemper virus infected and vaccinated animals.

    PubMed

    Liu, Da-Fei; Liu, Chun-Guo; Tian, Jin; Jiang, Yi-Tong; Zhang, Xiao-Zhan; Chai, Hong-Liang; Yang, Tian-Kuo; Yin, Xiu-Chen; Zhang, Hong-Ying; Liu, Ming; Hua, Yu-Ping; Qu, Lian-Dong

    2015-06-01

    Although widespread vaccination against canine distemper virus (CDV) has been conducted for many decades, several canine distemper outbreaks in vaccinated animals have been reported frequently. In order to detect and differentiate the wild-type and vaccine strains of the CDV from the vaccinated animals, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed. A set of four primers-two internal and two external-were designed to target the H gene for the specific detection of wild-type CDV variants. The CDV-H RT-LAMP assay rapidly amplified the target gene, within 60 min, using a water bath held at a constant temperature of 65°C. The assay was 100-fold more sensitive than conventional RT-PCR, with a detection limit of 10(-1)TCID50ml(-1). The system showed a preference for wild-type CDV, and exhibited less sensitivity to canine parvovirus, canine adenovirus type 1 and type 2, canine coronavirus, and canine parainfluenza virus. The assay was validated using 102 clinical samples obtained from vaccinated dog farms, and the results were comparable to a multiplex nested RT-PCR assay. The specific CDV-H RT-LAMP assay provides a simple, rapid, and sensitive tool for the detection of canines infected with wild-type CDV from canines vaccinated with attenuated vaccine. PMID:25769803

  15. Rapid and sensitive detection of type II porcine reproductive and respiratory syndrome virus by reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip.

    PubMed

    Gou, Hongchao; Deng, Jieru; Pei, Jingjing; Wang, Jiaying; Liu, Wenjun; Zhao, Mingqiu; Chen, Jinding

    2014-12-01

    Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was combined with a vertical flow (VF) nucleic acid detection strip to develop a universal assay for the detection of type II porcine reproductive and respiratory syndrome virus (PRRSV). The loop primers were labeled separately with biotin and fluorescein isothiocyanate (FITC) in this assay. Using optimized parameters, the whole reaction could be completed in <50 min in a completely enclosed environment. The detection limit of this assay was found to be 1 pg RNA, 30 tissue culture infective dose 50 (TCID50) virus, or 230 copies of recombinant plasmid DNA, which is relatively higher than that of RT-LAMP analyzed by agarose gel, RT-LAMP visualized by calcein, and the conventional RT-polymerase chain reaction (PCR). No false-positive results were obtained in the specificity assay. The efficiency of the RT-LAMP method was tested by analyzing 43 clinical samples, and the results were compared with those obtained by RT-PCR analysis, with the respective positive rates of 32.56% and 27.91%. This result confirmed that the method described is a rapid, accurate, and sensitive method for universal type II PRRSV detection. Also, this method can be used for the rapid detection of type II PRRSV during the early phase of an outbreak, especially for rapid veterinary diagnosis on the spot and in rural areas. PMID:25241142

  16. The Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Salmonella enterica serovar Typhi

    PubMed Central

    Fan, Fenxia; Du, Pengcheng; Kan, Biao; Yan, Meiying

    2015-01-01

    Typhoid fever remains a public health threat in many countries. A positive result in traditional culture is a gold-standard for typhoid diagnosis, but this method is time consuming and not sensitive enough for detection of samples containing a low copy number of the target organism. The availability of the loop-mediated isothermal amplification (LAMP) assay, which offers high speed and simplicity in detection of specific targets, has vastly improved the diagnosis of numerous infectious diseases. However, little research efforts have been made on utilizing this approach for diagnosis of Salmonella enterica serovar Typhi by targeting a single and specific gene. In this study, a LAMP assay for rapid detection of S. Typhi based on a novel marker gene, termed STY2879-LAMP, was established and evaluated with real-time PCR (RT-PCR). The specificity tests showed that STY2879 could be amplified in all S. Typhi strains isolated in different years and regions in China, whereas no amplification was observable in non-typhoidal strains covering 34 Salmonella serotypes and other pathogens causing febrile illness. The detection limit of STY2879-LAMP for S. Typhi was 15 copies/reaction in reference plasmids, 200 CFU/g with simple heat-treatment of DNA extracted from simulated stool samples and 20 CFU/ml with DNA extracted from simulated blood samples, which was 10 fold more sensitive than the parallel RT-PCR control experiment. Furthermore, the sensitivity of STY2879-LAMP and RT-PCR combining the traditional culture enrichment method for simulated stool and blood spiked with lower S. Typhi count during the 10 h enrichment time was also determined. In comparison with LAMP, the positive reaction time for RT-PCR required additional 2-3 h enrichment time for either simulated stool or blood specimens. Therefore, STY2879-LAMP is of practical value in the clinical settings and has a good potential for application in developing regions due to its easy-to-use protocol. PMID:25910059

  17. Molecular Evidence of Infections with Babesia gibsoni Parasites in Japan and Evaluation of the Diagnostic Potential of a Loop-Mediated Isothermal Amplification Method

    PubMed Central

    Ikadai, Hiromi; Tanaka, Hiroko; Shibahara, Nona; Matsuu, Aya; Uechi, Masami; Itoh, Naoyuki; Oshiro, Sugao; Kudo, Noboru; Igarashi, Ikuo; Oyamada, Takashi

    2004-01-01

    Detection and analysis of Babesia gibsoni infection were performed with whole-blood samples collected between July 2002 and July 2003 from 945 and 137 dogs from the Aomori and Okinawa Prefectures of Japan, respectively, by PCR and loop-mediated isothermal amplification (LAMP). On the basis of the criterion for positivity by PCR, 3.9% (37 of 945) and 10.9% (15 of 137) of the dogs had B. gibsoni DNA. All 37 positive animals from Aomori Prefecture were male Tosa dogs (Japanese mastiff). The 15 dogs from Okinawa Prefecture with positive PCR assay results were of various breeds, ages, and sexes. The 18S ribosomal DNA (18S rDNA) sequences from all samples showed 100% homology to each other and to published B. gibsoni sequences. The limits of detection of B. gibsoni parasitemia by the PCR and LAMP methods with an 18S rDNA-based primer set were 0.0005% each. A comparison of the PCR and LAMP methods with microscopic examination for the detection of B. gibsoni infections in blood samples from 945 field dogs in Aomori Prefecture and 137 field dogs in Okinawa Prefecture showed that 37 and 15 dogs, respectively, were positive by the PCR and LAMP methods and that 16 and 12 dogs, respectively, were positive by light microscopic examination. All samples found to be positive by microscopic examination were also positive by the PCR and LAMP methods. The results of the PCR and LAMP methods agreed for samples with positive results by either method. Moreover, nonspecific reactions were not observed by the LAMP method. These results suggest that the LAMP method provides a useful tool for the detection of B. gibsoni infections in dogs. PMID:15184421

  18. Rapid and Sensitive Detection of Bartonella bacilliformis in Experimentally Infected Sand Flies by Loop-Mediated Isothermal Amplification (LAMP) of the Pap31 Gene

    PubMed Central

    Angkasekwinai, Nasikarn; Atkins, Erin H.; Johnson, Richard N.; Grieco, John P.; Ching, Wei Mei; Chao, Chien Chung

    2014-01-01

    Background Carrion' disease, caused by Bartonella bacilliformis, remains truly neglected due to its focal geographical nature. A wide spectrum of clinical manifestations, including asymptomatic bacteremia, and lack of a sensitive diagnostic test can potentially lead to a spread of the disease into non-endemic regions where competent sand fly vectors may be present. A reliable test capable of detecting B. bacilliformis is urgently needed. Our objective is to develop a loop-mediated isothermal amplification (LAMP) assay targeting the pap31 gene to detect B. bacilliformis. Methods and Findings The sensitivity of the LAMP was evaluated in comparison to qPCR using plasmid DNA containing the target gene and genomic DNA in the absence and presence of human or sand fly DNA. The detection limit of LAMP was 1 to 10 copies/µL, depending on the sample metrics. No cross-reaction was observed when testing against a panel of various closely related bacteria. The utility of the LAMP was further compared to qPCR by the examination of 74 Lutzomyia longipalpis sand flies artificially fed on blood spiked with B. bacilliformis and harvested at days (D) 1, 3, 5, 7 and 9 post feeding. Only 86% of sand flies at D1 and 63% of flies at D3 were positive by qPCR. LAMP was able to detect B. bacilliformis in all those flies confirmed positive by qPCR. However, none of the flies after D3 were positive by either LAMP or qPCR. In addition to demonstrating the sensitivity of the LAMP assay, these results suggest that B. bacilliformis cannot propagate in artificially fed L. longipalpis. Conclusions The LAMP assay is as sensitive as qPCR for the detection of B. bacilliformis and could be useful to support diagnosis of patients in low-resource settings and also to identify B. bacilliformis in the sand fly vector. PMID:25522230

  19. Use of a combination of brushing technique and the loop-mediated isothermal amplification method as a novel, rapid, and safe system for detection of Helicobacter pylori.

    PubMed

    Minami, Masaaki; Ohta, Michio; Ohkura, Teruko; Ando, Takafumi; Torii, Keizo; Hasegawa, Tadao; Goto, Hidemi

    2006-11-01

    Gastric mucosal biopsy is widely used in the detection of Helicobacter pylori but is associated with a number of problems, including false-negative results due to sampling error and massive bleeding after biopsy. Given the extended period required to culture H. pylori, detection would be further improved by the use of rapid detection methods such as PCR. Here, we developed a rapid, safe, and convenient method for collecting H. pylori which combines endoscopic brushing with the loop-mediated isothermal amplification (LAMP) method. The specificity and sensitivity of LAMP were examined using nine urease-generating non-H. pylori bacterial species, Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Helicobacter hepaticus, and 51 H. pylori strains. Results showed that H. pylori-specific LAMP primers amplified H. pylori DNA only and that the lowest detection limit of the LAMP reaction was 10(2) CFU. Brushing and biopsy samples taken from 200 patients with peptic ulcer at Nagoya University Hospital and a regional health care center were subjected to both LAMP and culturing. No adverse effects such as severe bleeding or penetration occurred during the procedure. By LAMP assay, 123 patients were confirmed as H. pylori positive when brushing technique samples were assayed, whereas only 100 were positive when biopsy samples were assayed. Culture assay detected H. pylori in 117 patients when it was combined with the brushing technique and in 96 when it was combined with biopsy. Combination of the endoscopic brushing technique with LAMP is considered a useful and safe system for identifying H. pylori infection. PMID:17088368

  20. Use of a Combination of Brushing Technique and the Loop-Mediated Isothermal Amplification Method as a Novel, Rapid, and Safe System for Detection of Helicobacter pylori

    PubMed Central

    Minami, Masaaki; Ohta, Michio; Ohkura, Teruko; Ando, Takafumi; Torii, Keizo; Hasegawa, Tadao; Goto, Hidemi

    2006-01-01

    Gastric mucosal biopsy is widely used in the detection of Helicobacter pylori but is associated with a number of problems, including false-negative results due to sampling error and massive bleeding after biopsy. Given the extended period required to culture H. pylori, detection would be further improved by the use of rapid detection methods such as PCR. Here, we developed a rapid, safe, and convenient method for collecting H. pylori which combines endoscopic brushing with the loop-mediated isothermal amplification (LAMP) method. The specificity and sensitivity of LAMP were examined using nine urease-generating non-H. pylori bacterial species, Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Helicobacter hepaticus, and 51 H. pylori strains. Results showed that H. pylori-specific LAMP primers amplified H. pylori DNA only and that the lowest detection limit of the LAMP reaction was 102 CFU. Brushing and biopsy samples taken from 200 patients with peptic ulcer at Nagoya University Hospital and a regional health care center were subjected to both LAMP and culturing. No adverse effects such as severe bleeding or penetration occurred during the procedure. By LAMP assay, 123 patients were confirmed as H. pylori positive when brushing technique samples were assayed, whereas only 100 were positive when biopsy samples were assayed. Culture assay detected H. pylori in 117 patients when it was combined with the brushing technique and in 96 when it was combined with biopsy. Combination of the endoscopic brushing technique with LAMP is considered a useful and safe system for identifying H. pylori infection. PMID:17088368

  1. Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira.

    PubMed

    Nurul Najian, A B; Engku Nur Syafirah, E A R; Ismail, Nabilah; Mohamed, Maizan; Yean, Chan Yean

    2016-01-15

    In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10(-1) genomic equivalent ml(-1). An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device. PMID:26709307

  2. Establishment and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the semi-quantitative detection of HIV-1 group M virus.

    PubMed

    Odari, Eddy Okoth; Maiyo, Alex; Lwembe, Raphael; Gurtler, Lutz; Eberle, Josef; Nitschko, Hans

    2015-02-01

    The past decade has witnessed a dramatic increase of anti-retroviral treatment of human immunodeficiency virus (HIV) infected patients in many African countries. Due to costs and lack of currently available commercial viral load assays, insufficient attention has been paid to therapy monitoring through measurement of plasma viral load. This challenge of patient monitoring by tests as viral load, CD4 cell count, and finally HIV drug resistance could reverse achievements already made against HIV/AIDS infection. Loop-mediated isothermal amplification (LAMP) has been shown to be simple, rapid and cost-effective, characteristics which make this assay suitable for viral load monitoring in resource limited settings. This paper describes a revised LAMP assay using primers in the HIV-1 integrase region. The assay can be used for semi-quantitative measurement of HIV-1 group M viral load. The lower limit of detection (LLOD) was determined as 1200copies/mL and lower limit of quantitation (LLOQ) at 9800copies/mL. Sensitivities of 82 and 86% (in 135 and 99 plasma samples respectively from Kenya) and 93% (in 112 plasma samples from Germany) and specificities of 99 and 100% were realized. HIV-1 group O and HIV-2 virus samples were not detected. This LAMP assay has the potential for semi-quantitation of HIV-1 group M viral load in resource limited countries. There is still a need for further improvement by refinement of primers in respect to detection of HIV-1 group M non-B virus. PMID:25445795

  3. Use of reverse transcription loop-mediated isothermal amplification combined with lateral flow dipstick for an easy and rapid detection of Jembrana disease virus.

    PubMed

    Kusumawati, Asmarani; Tampubolon, Issabellina Dwades; Hendarta, Narendra Yoga; Salasia, Siti Isrina Oktavia; Wanahari, Tenri Ashari; Mappakaya, Basofi Ashari; Hartati, Sri

    2015-09-01

    Jembrana disease virus (JDV) is a viral pathogen that causes Jembrana disease in Bali cattle (Bos javanicus) with high mortality rate. An easy and rapid diagnostic method is essential for further control this disease. We used a reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with lateral flow dipstick (LFD), based on conserved tm subunit of Jembrana disease virus env gene. The RT-LAMP conditions were optimized by varying the concentration of MgSO4, betaine, dNTP, and temperature as well as the time and duration of reaction. The primers sensitivity for JDV was confirmed. The method was able to detect env-tm gene dilution which contained 2 × 10(-15) g of template. Comparatively, the sensitivity of RT-LAMP/LFD was 100-fold more sensitive than reverse transcription-polymerase chain reaction. The primers specificity for JDV was also confirmed using positive and negative controls. This work also showed that virus detection could be done not only on total RNA extracted from blood but various organs could also be analyzed for the presence of JDV using RT-LAMP/LFD method. The whole process, including the LAMP reaction and the LFD hybridization step only lasts approximately 75 min. Results of analysis can be easily observed with naked eyes without addition of any chemical or further analysis. The combination of RT-LAMP with LFD makes the method a more suitable diagnostic tool in conditions where sophisticated and expensive equipments are not available for field investigations on Jembrana disease in Bali cattle. PMID:26396986

  4. Most-probable-number loop-mediated isothermal amplification-based procedure enhanced with K antigen-specific immunomagnetic separation for quantifying tdh(+) Vibrio parahaemolyticus in molluscan Shellfish.

    PubMed

    Tanaka, Natsuko; Iwade, Yoshito; Yamazaki, Wataru; Gondaira, Fumio; Vuddhakul, Varaporn; Nakaguchi, Yoshitsugu; Nishibuchi, Mitsuaki

    2014-07-01

    Although thermostable direct hemolysin-producing (tdh(+)) Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis, the enumeration of tdh(+) V. parahaemolyticus remains challenging due to its low densities in the environment. In this study, we developed a most-probable-number (MPN)-based procedure designated A-IS(1)-LAMP, in which an immunomagnetic separation (IMS) technique targeting as many as 69 established K antigens and a loop-mediated isothermal amplification (LAMP) assay targeting the thermostable direct hemolysin (tdh) gene were applied in an MPN format. Our IMS employed PickPen, an eight-channel intrasolution magnetic particle separation device, which enabled a straightforward microtiter plate-based IMS procedure (designated as PickPen-IMS). The ability of the procedure to quantify a wide range of tdh(+) V. parahaemolyticus levels was evaluated by testing shellfish samples in Japan and southern Thailand, where shellfish products are known to contain relatively low and high levels of total V. parahaemolyticus, respectively. The Japanese and Thai shellfish samples showed, respectively, relatively low (< 3 to 11 MPN/10 g) and considerably higher (930 to 110,000 MPN/10 g) levels of tdh(+) V. parahaemolyticus, raising concern about the safety of Thai shellfish products sold to domestic consumers at local morning markets. LAMP showed similar or higher performance than conventional PCR in the detection and quantification of a wide range of tdh(+) V. parahaemolyticus levels in shellfish products. Whereas a positive effect of PickPen-IMS was not observed in MPN determination, PickPen-IMS was able to concentrate tdh(+) V. parahaemolyticus 32-fold on average from the Japanese shellfish samples at an individual tube level, suggesting a possibility of using PickPen-IMS as an optional tool for specific shellfish samples. The A-IS(1)-LAMP procedure can be used by any health authority in the world to measure the tdh(+) V. parahaemolyticus levels in shellfish products. PMID:24988012

  5. Evaluation of a Loop-Mediated Isothermal Amplification Suite for the Rapid, Reliable, and Robust Detection of Shiga Toxin-Producing Escherichia coli in Produce

    PubMed Central

    Wang, Fei; Yang, Qianru; Qu, Yinzhi; Meng, Jianghong

    2014-01-01

    Shiga toxin-producing Escherichia coli (STEC) strains are a leading cause of produce-associated outbreaks in the United States. Rapid, reliable, and robust detection methods are needed to better ensure produce safety. We recently developed a loop-mediated isothermal amplification (LAMP) suite for STEC detection. In this study, the STEC LAMP suite was comprehensively evaluated against real-time quantitative PCR (qPCR) using a large panel of bacterial strains (n = 156) and various produce items (several varieties of lettuce, spinach, and sprouts). To simulate real-world contamination events, produce samples were surface inoculated with a low level (1.2 to 1.8 CFU/25 g) of individual STEC strains belonging to seven serogroups (O26, O45, O103, O111, O121, O145, and O157) and held at 4°C for 48 h before testing. Six DNA extraction methods were also compared using produce enrichment broths. All STEC targets and their subtypes were accurately detected by the LAMP suite. The detection limits were 1 to 20 cells per reaction in pure culture and 105 to 106 CFU per 25 g (i.e., 103 to 104 CFU per g) in produce, except for strains harboring the stx2c, eae-?, and eae-? subtypes. After 6 to 8 h of enrichment, the LAMP suite achieved accurate detection of low levels of STEC strains of various stx2 and eae subtypes in lettuce and spinach varieties but not in sprouts. A similar trend of detection was observed for qPCR. The PrepMan Ultra sample preparation reagent yielded the best results among the six DNA extraction methods. This research provided a rapid, reliable, and robust method for detecting STEC in produce during routine sampling and testing. The challenge with sprouts detection by both LAMP and qPCR calls for special attention to further analysis. PMID:24509927

  6. Determination of the prevalence of African trypanosome species in indigenous dogs of Mambwe district, eastern Zambia, by loop-mediated isothermal amplification

    PubMed Central

    2014-01-01

    Background Dogs have been implicated to serve as links for parasite exchange between livestock and humans and remain an important source of emerging and re-emerging diseases including trypanosome infections. Yet, canine African trypanosomosis (CAT), particularly in indigenous dogs (mongrel breed) remains under- reported in literature. This study evaluated the performance of loop-mediated isothermal amplification (LAMP) in detecting trypanosomes in blood from indigenous dogs of tsetse-infested Mambwe district in eastern Zambia. Methods A cross sectional survey of CAT was conducted within 5 chiefdoms (Msoro, Kakumbi, Munkanya, Nsefu, Malama) of Mambwe district, eastern Zambia, during October 2012. Blood samples from 237 indigenous hunting dogs were collected and screened by microscopy and LAMP. Results Of the 237 dogs screened for CAT, 14 tested positive by microscopy (5.9%; 95% CI: 2.9 – 8.9%), all of which also tested positive by LAMP. In addition, LAMP detected 6 additional CAT cases, bringing the total cases detected by LAMP to 20 (8.4%; 95% CI: 4.9 – 12.0%). Irrespective of the detection method used, CAT was only recorded from 3 chiefdoms (Munkanya, Nsefu, Malama) out of the 5. According to LAMP, these infections were caused by Trypanosoma congolense, Trypanosoma brucei brucei and the zoonotic Trypanosoma brucei rhodesiense. Although these CAT cases generally did not manifest clinical illness, an association was observed between infection with Trypanosoma brucei subspecies and occurrence of corneal opacity. Conclusions This communication reports for the first time the occurrence of CAT in indigenous Zambian dogs. Our study indicates that LAMP is a potential diagnostic tool for trypanosome detection in animals. LAMP was more sensitive than microscopy and was further capable of distinguishing the closely related T. b. brucei and T. b. rhodesiense. In view of the sporadic cases of re-emerging HAT being reported within the Luangwa valley, detection of the human serum resistant associated (SRA) gene in trypanosomes from mongrels is intriguing and indicative of the risk of contracting HAT by local communities and tourists in Mambwe district. Consequently, there is a need for continuous trypanosome surveillances in animals, humans and tsetse flies using sensitive and specific tests such as LAMP. PMID:24411022

  7. Novel multifunction-integrated molecular beacon for the amplification detection of DNA hybridization based on primer/template-free isothermal polymerization.

    PubMed

    Dong, Haiyan; Wu, Zai-Sheng; Xu, Jianguo; Ma, Ji; Zhang, Huijuan; Wang, Jie; Shen, Weiyu; Xie, Jingjing; Jia, Lee

    2015-10-15

    Molecular beacon (MB) is widely explored as a signaling probe in powerful biosensing systems, for example, enzyme-assisted strand displacement amplification (SDA)-based system. The existing polymerization-based amplification system is often composed of recognition element, primer, template and fluorescence reporter. To develop a new MB sensing system and simply the signal amplification design, we herein attempted to propose a multifunctional integrated MB (MI-MB) for the polymerization amplification detection of target DNA via introducing a G-rich fragment into the loop of MB without using any exogenous auxiliary oligonucleotide probe. Utilizing only one MI-MB probe, the p53 target gene could trigger the cycles of hybridization/polymerization/displacement, resulting in amplification of the target hybridization event. Thus, the p53 gene can be detected down to 5 × 10(-10)M with the linear response range from 5 × 10(-10)M to 4 × 10(-7)M. Using the MI-MB, we could readily discriminate the point mutation-contained p53 from the wild-type one. As a proof-of-concept study, owing to its simplicity and multifunction, including recognition, replication, amplification and signaling, the MI-MB exhibits the great potential for the development of different biosensors for various biomedical applications, especially, for early cancer diagnosis. PMID:25982726

  8. A programmable Y-shaped junction scaffold-mediated modular and cascade amplification strategy for the one-step, isothermal and ultrasensitive detection of target DNA.

    PubMed

    Liu, Shufeng; Gong, Hongwei; Sun, Xinya; Liu, Tao; Wang, Li

    2015-12-28

    The programmable DNA polymerization across the two branches of the assembled Y-shaped junction was ingeniously manipulated for modular target recycling and cascade lambda exonuclease cleavage, which afforded the one-pot, isothermal and ultrasensitive detection of target DNA. A low detection limit of 28.2 fM of target DNA with an excellent selectivity could be obtained. PMID:26492526

  9. Increased robustness of single-molecule counting with microfluidics, digital isothermal amplification, and a mobile phone versus real-time kinetic measurements.

    PubMed

    Selck, David A; Karymov, Mikhail A; Sun, Bing; Ismagilov, Rustem F

    2013-11-19

    Quantitative bioanalytical measurements are commonly performed in a kinetic format and are known to not be robust to perturbation that affects the kinetics itself or the measurement of kinetics. We hypothesized that the same measurements performed in a "digital" (single-molecule) format would show increased robustness to such perturbations. Here, we investigated the robustness of an amplification reaction (reverse-transcription loop-mediated amplification, RT-LAMP) in the context of fluctuations in temperature and time when this reaction is used for quantitative measurements of HIV-1 RNA molecules under limited-resource settings (LRS). The digital format that counts molecules using dRT-LAMP chemistry detected a 2-fold change in concentration of HIV-1 RNA despite a 6 °C temperature variation (p-value = 6.7 × 10(-7)), whereas the traditional kinetic (real-time) format did not (p-value = 0.25). Digital analysis was also robust to a 20 min change in reaction time, to poor imaging conditions obtained with a consumer cell-phone camera, and to automated cloud-based processing of these images (R(2) = 0.9997 vs true counts over a 100-fold dynamic range). Fluorescent output of multiplexed PCR amplification could also be imaged with the cell phone camera using flash as the excitation source. Many nonlinear amplification schemes based on organic, inorganic, and biochemical reactions have been developed, but their robustness is not well understood. This work implies that these chemistries may be significantly more robust in the digital, rather than kinetic, format. It also calls for theoretical studies to predict robustness of these chemistries and, more generally, to design robust reaction architectures. The SlipChip that we used here and other digital microfluidic technologies already exist to enable testing of these predictions. Such work may lead to identification or creation of robust amplification chemistries that enable rapid and precise quantitative molecular measurements under LRS. Furthermore, it may provide more general principles describing robustness of chemical and biological networks in digital formats. PMID:24199852

  10. SPR-DNA array for detection of methicillin-resistant Staphylococcus aureus (MRSA) in combination with loop-mediated isothermal amplification.

    PubMed

    Nawattanapaiboon, Kawin; Kiatpathomchai, Wansika; Santanirand, Pitak; Vongsakulyanon, Apirom; Amarit, Ratthasart; Somboonkaew, Armote; Sutapun, Boonsong; Srikhirin, Toemsak

    2015-12-15

    In this study, we evaluated surface plasmon resonance imaging (SPR imaging) as a DNA biosensor for the detection of methicillin-resistant Staphylococcus aureus (MRSA) which is one of the most common causes of nosocomial infections. The DNA sample were collected from clinical specimens, including sputum and blood hemoculture were undergone LAMP amplification for 0.18 kbp and 0.23 kbp DNA fragments of femB and mecA genes, respectively. The self-assembled monolayer surface (SAMs) was used for immobilized streptavidin-biotinylated probes on the sensor surface for the detection of LAMP amplicons from MRSA. Both LAMP amplicons were simultaneously hybridized with ssDNA probes immobilized onto a bio-functionalized surface to detect specific targets in the multiplex DNA array platform. In addition, the sensor surface could be regenerated allowing at least five cycles of use with a shortened assay time. The detection limit of LAMP-SPR sensing was 10 copies/µl and LAMP-SPR sensing system showed a good selectivity toward the MRSA. PMID:26159153

  11. Molecular Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) Positive Bacteria from Environmental and Drinking Water Samples by Loop Mediated Isothermal Amplification of bla NDM-1.

    PubMed

    Rathinasabapathi, P; Hiremath, Deepak S; Arunraj, Rex; Parani, M

    2015-12-01

    New Delhi metallo-?-lactamase-1 gene (bla NDM-1 ) codes for New Delhi metallo-beta-lactamase-1 (NDM-1) enzyme that cleaves the amide bond of ?-lactam ring, and provides resistance against major classes of ?-lactam antibiotics. Dissemination of the plasmid borne bla NDM-1 through horizontal gene transfer is a potential threat to the society. In this study, a rapid non-culture method for detecting NDM-1 positive bacteria was developed by Loop Mediated Isothermal Amplification (LAMP) of bla NDM-1 . Sensitivity of this method was found to be one femtogram of plasmid DNA, which translates into 2.6-25.8 copies depending on the size of the plasmid DNA. This method was applied to detect NDM-1 positive bacteria in 81 water samples that were collected from environmental and drinking water sources. NDM-1 positive bacteria were detected in three drinking water samples by LAMP but not by PCR. These three samples were collected from the water sources that were treated with chlorine for decontamination before public distribution. NDM-1 positive bacteria were not detected in lake water samples or in the samples that were collected from the water sources that were purified by reverse osmosis before public distribution. Detection of NDM-1 positive bacteria using LAMP was found to be safe, sensitive and rapid for screening large number of samples from diverse sources. This method could be developed as on-field detection kit by using fluorescent dyes to visualize the amplified bla NDM-1 gene. PMID:26543265

  12. Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens.

    PubMed

    Rodriguez, Natalia M; Linnes, Jacqueline C; Fan, Andy; Ellenson, Courtney K; Pollock, Nira R; Klapperich, Catherine M

    2015-08-01

    The 2009 Influenza A (H1N1) pandemic disproportionately affected the developing world and highlighted the key inadequacies of traditional diagnostic methods that make them unsuitable for use in resource-limited settings, from expensive equipment and infrastructure requirements to unacceptably long turnaround times. While rapid immunoassay diagnostic tests were much less costly and more context-appropriate, they suffered from drastically low sensitivities and high false negative rates. An accurate, sensitive, and specific molecular diagnostic that is also rapid, low-cost, and independent of laboratory infrastructure is needed for effective point-of-care detection and epidemiological control in these developing regions. We developed a paper-based assay that allows for the extraction and purification of RNA directly from human clinical nasopharyngeal specimens through a poly(ether sulfone) paper matrix, H1N1-specific in situ isothermal amplification directly within the same paper matrix, and immediate visual detection on lateral flow strips. The complete sample-to-answer assay can be performed at the point-of-care in just 45 min, without the need for expensive equipment or laboratory infrastructure, and it has a clinically relevant viral load detection limit of 10(6) copies/mL, offering a 10-fold improvement over current rapid immunoassays. PMID:26125635

  13. Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Hyderabad, India.

    PubMed

    Neeraja, M; Lakshmi, V; Lavanya, Vanjari; Priyanka, E N; Parida, M M; Dash, P K; Sharma, Shashi; Rao, P V Lakshmana; Reddy, Gopal

    2015-01-01

    Early and rapid detection of dengue virus (DENV) infection during the acute phase of illness is crucial for proper patient management and prevention of the spread of the infection. In the present study, the standardization and validation of a one step, four tube reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for rapid detection and serotyping of the DENV targeting NS1 gene using the Genie® II flourometer was carried out. The performance of the RT-LAMP was compared to RT-PCR, CDC 1-4 Real time PCR and the NS1 antigen ELISA, IgM and IgG anti DENV antibodies. Acute DENV infection was confirmed in 250/300 patients suspected clinically of DENV infection. RT- LAMP and CDC 1-4 Real time PCR assay was positive in 148/250 patients, while 92/250 patients were positive for anti- Dengue IgM and IgG antibodies. The RT-LAMP assay and the CDC real-time RT-PCR assay showed high concordance (k=1.0). The detection rate of acute DENV infection improved to 96% (240/250) when the results of RT-LAMP were combined with NS1 Ag, IgM and IgG ELISA. The RT-LAMP had a detection limit of 100 copies for DEN-1 and DEN-2, 10 copies for DEN-3 and DEN-4 compared to 1000 copies for DEN-1 and DEN-2, 100 copies for DEN-3 and DEN-4 by the conventional RT-PCR. The assay showed 100% specificity. The RT-LAMP assay developed in this study has potential use for early clinical diagnosis, serotyping and surveillance of DENV infection in endemic countries such as India. PMID:25455901

  14. Application of loop-mediated isothermal amplification assays for direct identification of pure cultures of Aspergillus flavus, A. nomius, and A. caelatus and for their rapid detection in shelled Brazil nuts.

    PubMed

    Luo, Jie; Taniwaki, Marta H; Iamanaka, Beatriz T; Vogel, Rudi F; Niessen, Ludwig

    2014-02-17

    Brazil nuts have a high nutritional content and are a very important trade commodity for some Latin American countries. Aflatoxins are carcinogenic fungal secondary metabolites. In Brazil nuts they are produced predominantly by Aspergillus (A.) nomius and A. flavus. In the present study we applied and evaluated two sets of primers previously published for the specific detection of the two species using loop-mediated isothermal amplification (LAMP) technology. Moreover, a primer set specific for A. caelatus as a frequently occurring non-aflatoxigenic member of Aspergillus section Flavi in Brazil nuts was newly developed. LAMP assays were combined with a simplified DNA release method and used for rapid identification of pure cultures and rapid detection of A. nomius and A. flavus from samples of shelled Brazil nuts. An analysis of pure cultures of 68 isolates representing the major Aspergillus species occurring on Brazil nuts showed that the three LAMP assays had individual accuracies of 61.5%, 84.4%, and 93.3% for A. flavus, A. nomius, and A. caelatus, respectively when morphological identification was used as a reference. The detection limits for conidia added directly to the individual LAMP reactions were found to be 10? conidia per reaction with the primer set ID9 for A. nomius and 10? conidia per reaction with the primer set ID58 for A. flavus. Sensitivity was increased to 10¹ and 10² conidia per reaction for A. nomius and A. flavus, respectively, when sample preparation included a spore disruption step. The results of LAMP assays obtained during the analysis of 32 Brazil nut samples from different regions of Brazil and from different steps in the production process of the commodity were compared with results obtained from mycological analysis and aflatoxin analysis of corresponding samples. Compared with mycological analysis of the samples, the Negative Predictive Values of LAMP assays were 42.1% and 12.5% while the Positive Predictive Values were 61.5% and 66.7% for A. nomius and A. flavus, respectively. When LAMP results were compared with the presence of aflatoxins in corresponding samples, the Negative Predictive Values were 22.2% and 44.4% and the Positive Predictive Values were 52.2% and 78.3% for aflatoxins produced by A. nomius and A. flavus, respectively. The LAMP assays described in this study have been demonstrated to be a specific, sensitive and easy to use tool for the survey of Brazil nuts for contaminations with potential aflatoxin-producing A. nomius and A. flavus in low tech environments where resources may be limited. PMID:24361827

  15. Electrical and Electrochemical Monitoring of Nucleic Acid Amplification

    PubMed Central

    Goda, Tatsuro; Tabata, Miyuki; Miyahara, Yuji

    2015-01-01

    Nucleic acid amplification is a gold standard technique for analyzing a tiny amount of nucleotides in molecular biology, clinical diagnostics, food safety, and environmental testing. Electrical and electrochemical monitoring of the amplification process draws attention over conventional optical methods because of the amenability toward point-of-care applications as there is a growing demand for nucleic acid sensing in situations outside the laboratory. A number of electrical and electrochemical techniques coupled with various amplification methods including isothermal amplification have been reported in the last 10?years. In this review, we highlight recent developments in the electrical and electrochemical monitoring of nucleic acid amplification. PMID:25798440

  16. Isothermal separation processes

    NASA Technical Reports Server (NTRS)

    England, C.

    1982-01-01

    The isothermal processes of membrane separation, supercritical extraction and chromatography were examined using availability analysis. The general approach was to derive equations that identified where energy is consumed in these processes and how they compare with conventional separation methods. These separation methods are characterized by pure work inputs, chiefly in the form of a pressure drop which supplies the required energy. Equations were derived for the energy requirement in terms of regular solution theory. This approach is believed to accurately predict the work of separation in terms of the heat of solution and the entropy of mixing. It can form the basis of a convenient calculation method for optimizing membrane and solvent properties for particular applications. Calculations were made on the energy requirements for a membrane process separating air into its components.

  17. Gravitational Microlensing 1 Amplification

    E-print Network

    Murayama, Hitoshi

    Gravitational Microlensing 1 Amplification Once the deflection angle is known (see the optional the lens. To figure out the amplification due to the gravitational lensing, we con- sider the finite the line of sight within the Einstein radius. We estimate the frequency and duration of gravitational

  18. Centrifugal LabTube platform for fully automated DNA purification and LAMP amplification based on an integrated, low-cost heating system

    E-print Network

    Hoehl, Melanie M.

    This paper introduces a disposable battery-driven heating system for loop-mediated isothermal DNA amplification (LAMP) inside a centrifugally-driven DNA purification platform (LabTube). We demonstrate LabTube-based fully ...

  19. Non-instrumented nucleic acid amplification assay

    NASA Astrophysics Data System (ADS)

    Weigl, Bernhard H.; Domingo, Gonzalo; Gerlach, Jay; Tang, Dennis; Harvey, Darrel; Talwar, Nick; Fichtenholz, Alex; van Lew, Bill; LaBarre, Paul

    2008-02-01

    We have developed components of a diagnostic disposable platform that has the dual purpose of providing molecular diagnostics at the point of care (POC) as well as stabilizing specimens for further analysis via a centralized surveillance system. This diagnostic is targeted for use in low-resource settings by minimally trained health workers. The disposable device does not require any additional instrumentation and will be almost as rapid and simple to use as a lateral flow strip test - yet will offer the sensitivity and specificity of nucleic acid amplification tests (NAATs). The low-cost integrated device is composed of three functional components: (1) a sample-processing subunit that generates clean and stabilized DNA from raw samples containing nucleic acids, (2) a NA amplification subunit, and (3) visual amplicon detection sub-unit. The device integrates chemical exothermic heating, temperature stabilization using phase-change materials, and isothermal nucleic acid amplification. The aim of developing this system is to provide pathogen detection with NAAT-level sensitivity in low-resource settings where there is no access to instrumentation. If a disease occurs, patients would be tested with the disposable in the field. A nucleic acid sample would be preserved within the spent disposable which could be sent to a central laboratory facility for further analysis if needed.

  20. Early amplification options.

    PubMed

    Gabbard, Sandra Abbott; Schryer, Jennifer

    2003-01-01

    Children with permanent hearing loss have been remediated with hearing amplification devices for decades. The influx of young infants identified with hearing loss through successful newborn hearing screening programs has established a need for amplification resources for infants within the first six months of life. For the approximately two of every 1000 infants born who are identified with bilateral hearing loss [Mehl and Thomson, 1998, Pediatrics 101, p. e4], the use of amplification is commonly the first step in treating the sequella of their loss. The use of hearing aids, combined with early intervention, has been shown to significantly improve the speech and language skills of young children with hearing loss [Yoshinaga-Itano, 2000, Seminars in Hearing 21, p. 309]. Speech and language delays have contributed to compromised academic performance of school aged children with hearing loss [Johnson et al., 1997, Educational Audiology Handbook, Singular Publishing, San Diego]. Most hard-of-hearing and deaf children use hearing aids and other assistive listening devices every day throughout their lifetime and the life expectancy of a hearing aid is only five to eight years. The current challenge for pediatric audiologists is selecting and evaluating the available amplification to provide the best options for children and their families. Amplification technology has seen an explosion in growth the past few years and the options continue to expand rapidly. This article examines currently available amplification technology and reviews the selection criteria that may be used for infants and young children. Issues such as style, type, amplification features, signal processing strategies, and verification and validation tools are also discussed. PMID:14648816

  1. Cavity optoelectromechanical regenerative amplification

    E-print Network

    Taylor, Michael A; Knittel, Joachim; Lee, Kwan H; McRae, Terry G; Bowen, Warwick P

    2011-01-01

    Regenerative amplification is demonstrated in a cavity optoelectromechanical system using electrical gradient forces and optomechanical transduction. Mechanical linewidth narrowing to $6.6 \\pm 1.4$ mHz was observed at a frequency of 27.3 MHz, corresponding to an effective mechanical quality factor of $4 \\times 10^9$. A theoretical model of the system was formulated, showing that the delay in electrical feedback allows additional linewidth narrowing compared to purely optomechanical regenerative amplification. The linewidth was confirmed experimentally to scale inversely with the mechanical energy as predicted by the model.

  2. Controlled Microwave Heating Accelerates Rolling Circle Amplification

    PubMed Central

    Yoshimura, Takeo; Suzuki, Takamasa; Mineki, Shigeru; Ohuchi, Shokichi

    2015-01-01

    Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same. PMID:26348227

  3. Flux amplification in SSPX

    NASA Astrophysics Data System (ADS)

    Lodestro, Lynda; Hooper, E. B.; Jayakumar, R. J.; Pearlstein, L. D.; Wood, R. D.; McLean, H. S.

    2007-11-01

    Flux amplification---the ratio of poloidal flux enclosed between the magnetic and geometric axes to that between the separatrix and the geometric axis---is a key measure of efficiency for edge-current-driven spheromaks. With the new, modular capacitor bank, permitting flexible programming of the gun current, studies of flux amplification under various drive scenarios can be performed. Analysis of recent results of pulsed operation with the new bank finds an efficiency ˜ 0.2, in selected shots, of the conversion of gun energy to confined magnetic energy during the pulses, and suggests a route toward sustained efficiency at 0.2. Results of experiments, a model calculation of field build-up, and NIMROD simulations exploring this newly suggested scenario will be presented.

  4. Gravitomagnetic amplification in cosmology

    SciTech Connect

    Tsagas, Christos G.

    2010-02-15

    Magnetic fields interact with gravitational waves in various ways. We consider the coupling between the Weyl and the Maxwell fields in cosmology and study the effects of the former on the latter. The approach is fully analytical and the results are gauge invariant. We show that the nature and the outcome of the gravitomagnetic interaction depends on the electric properties of the cosmic medium. When the conductivity is high, gravitational waves reduce the standard (adiabatic) decay rate of the B field, leading to its superadiabatic amplification. In poorly conductive environments, on the other hand, Weyl-curvature distortions can result into the resonant amplification of large-scale cosmological magnetic fields. Driven by the gravitational waves, these B fields oscillate with an amplitude that is found to diverge when the wavelengths of the two sources coincide. We present technical and physical aspects of the gravitomagnetic interaction and discuss its potential implications.

  5. Cavity optoelectromechanical regenerative amplification

    E-print Network

    Michael A. Taylor; Alex Szorkovszky; Joachim Knittel; Kwan H. Lee; Terry G. McRae; Warwick P. Bowen

    2012-06-29

    Cavity optoelectromechanical regenerative amplification is demonstrated. An optical cavity enhances mechanical transduction, allowing sensitive measurement even for heavy oscillators. A 27.3 MHz mechanical mode of a microtoroid was linewidth narrowed to 6.6\\pm1.4 mHz, 30 times smaller than previously achieved with radiation pressure driving in such a system. These results may have applications in areas such as ultrasensitive optomechanical mass spectroscopy.

  6. Coherent white light amplification

    DOEpatents

    Jovanovic, Igor; Barty, Christopher P.

    2004-05-25

    A system for coherent simultaneous amplification of a broad spectral range of light that includes an optical parametric amplifier and a source of a seed pulse is described. A first angular dispersive element is operatively connected to the source of a seed pulse. A first imaging telescope is operatively connected to the first angular dispersive element and operatively connected to the optical parametric amplifier. A source of a pump pulse is operatively connected to the optical parametric amplifier. A second imaging telescope is operatively connected to the optical parametric amplifier and a second angular dispersive element is operatively connected to the second imaging telescope.

  7. Electricity-Free Amplification and Detection for Molecular Point-of-Care Diagnosis of HIV-1

    PubMed Central

    Singleton, Jered; Osborn, Jennifer L.; Lillis, Lorraine; Hawkins, Kenneth; Guelig, Dylan; Price, Will; Johns, Rachel; Ebels, Kelly; Boyle, David; Weigl, Bernhard; LaBarre, Paul

    2014-01-01

    In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation <0.5°C at operating temperature, a cost of approximately US$.06 per test for heater reaction materials, and an ambient temperature operating range from 16°C to 30°C. We also pair the heater with nucleic acid lateral flow (NALF)-detection for a visual readout. To further illustrate the utility of the electricity-free heater and NALF-detection platform, we demonstrate sensitive and repeatable detection of HIV-1 with a ß-actin positive internal amplification control from processed sample to result in less than 80 minutes. Together, these elements are building blocks for an electricity-free platform capable of isothermal amplification and detection of a variety of pathogens. PMID:25426953

  8. Isothermal compressors for process gases

    SciTech Connect

    Wiederuh, E.; Meinhart, D. )

    1992-09-01

    This paper reports on isothermal compressors which are more efficient for all gases. The study of several representative gases considered stage efficiencies, pressure ratios and pressure losses of the intercoolers. Generally there are two ways to reduce power consumption of a gas compression process: minimize losses of the compressor or improve the thermodynamics of the process. But there are some new ways to reduce losses of turbocompressors. Losses of the impeller labyrinth seals and the balance piston labyrinth seal can be reduced by optimizing the labyrinth geometry and minimizing labyrinth clearances. Therefore, conventional labyrinth seals are still being studied and will be improved.

  9. Rapid Identification of Black Grain Eumycetoma Causative Agents Using Rolling Circle Amplification

    PubMed Central

    Ahmed, Sarah A.; van den Ende, Bert H. G. Gerrits; Fahal, Ahmed H.; van de Sande, Wendy W. J.; de Hoog, G. S.

    2014-01-01

    Accurate identification of mycetoma causative agent is a priority for treatment. However, current identification tools are far from being satisfactory for both reliable diagnosis and epidemiological investigations. A rapid, simple, and highly efficient molecular based method for identification of agents of black grain eumycetoma is introduced, aiming to improve diagnostic in endemic areas. Rolling Circle Amplification (RCA) uses species-specific padlock probes and isothermal DNA amplification. The tests were based on ITS sequences and developed for Falciformispora senegalensis, F. tompkinsii, Madurella fahalii, M. mycetomatis, M. pseudomycetomatis, M. tropicana, Medicopsis romeroi, and Trematosphaeria grisea. With the isothermal RCA assay, 62 isolates were successfully identified with 100% specificity and no cross reactivity or false results. The main advantage of this technique is the low-cost, high specificity, and simplicity. In addition, it is highly reproducible and can be performed within a single day. PMID:25474355

  10. Rapid identification of black grain eumycetoma causative agents using rolling circle amplification.

    PubMed

    Ahmed, Sarah A; van den Ende, Bert H G Gerrits; Fahal, Ahmed H; van de Sande, Wendy W J; de Hoog, G S

    2014-12-01

    Accurate identification of mycetoma causative agent is a priority for treatment. However, current identification tools are far from being satisfactory for both reliable diagnosis and epidemiological investigations. A rapid, simple, and highly efficient molecular based method for identification of agents of black grain eumycetoma is introduced, aiming to improve diagnostic in endemic areas. Rolling Circle Amplification (RCA) uses species-specific padlock probes and isothermal DNA amplification. The tests were based on ITS sequences and developed for Falciformispora senegalensis, F. tompkinsii, Madurella fahalii, M. mycetomatis, M. pseudomycetomatis, M. tropicana, Medicopsis romeroi, and Trematosphaeria grisea. With the isothermal RCA assay, 62 isolates were successfully identified with 100% specificity and no cross reactivity or false results. The main advantage of this technique is the low-cost, high specificity, and simplicity. In addition, it is highly reproducible and can be performed within a single day. PMID:25474355

  11. CARBON ADSORPTION ISOTHERMS FOR TOXIC ORGANICS

    EPA Science Inventory

    An experimental protocol for measuring the activated carbon adsorption isotherm was developed and applied to a wide range of organic compounds. Methods for treatment of the isotherm data and a standard format for presentation of results are shown. In the early phase of the study ...

  12. Isothermal Circumstellar Dust Shell Model for Teaching

    ERIC Educational Resources Information Center

    Robinson, G.; Towers, I. N.; Jovanoski, Z.

    2009-01-01

    We introduce a model of radiative transfer in circumstellar dust shells. By assuming that the shell is both isothermal and its thickness is small compared to its radius, the model is simple enough for students to grasp and yet still provides a quantitative description of the relevant physical features. The isothermal model can be used in a…

  13. The Complexity of Hardness Amplification and Derandomization

    E-print Network

    Viola, Emanuele

    The Complexity of Hardness Amplification and Derandomization A thesis presented by Emanuele Viola Emanuele Viola The Complexity of Hardness Amplification and Derandomization Abstract This thesis studies of hardness amplification and deran- domization. Hardness amplification is the task of taking a function

  14. Laser amplification using axicon reflectors

    SciTech Connect

    Kuhnle, G.; Marowsky, G.; Reider, G.

    1988-07-01

    The amplification behavior of a laser pumped cylindrical dye cell located within an axicon reflector has been studied. Compared with a conventional, transversely pumped rectangular amplifying dye cuvette, the axicon shows similar high gain and enhanced beam quality as a result of its radially symmetric excitation geometry. This latter property was experimentally confirmed by a 30% reduction of the divergence of a seed beam after amplification and by increased second harmonic generation conversion efficiency.

  15. ISOTHERMAL AIR INGRESS VALIDATION EXPERIMENTS

    SciTech Connect

    Chang H Oh; Eung S Kim

    2011-09-01

    Idaho National Laboratory carried out air ingress experiments as part of validating computational fluid dynamics (CFD) calculations. An isothermal test loop was designed and set to understand the stratified-flow phenomenon, which is important as the initial air flow into the lower plenum of the very high temperature gas cooled reactor (VHTR) when a large break loss-of-coolant accident occurs. The unique flow characteristics were focused on the VHTR air-ingress accident, in particular, the flow visualization of the stratified flow in the inlet pipe to the vessel lower plenum of the General Atomic’s Gas Turbine-Modular Helium Reactor (GT-MHR). Brine and sucrose were used as heavy fluids, and water was used to represent a light fluid, which mimics a counter current flow due to the density difference between the stimulant fluids. The density ratios were changed between 0.87 and 0.98. This experiment clearly showed that a stratified flow between simulant fluids was established even for very small density differences. The CFD calculations were compared with experimental data. A grid sensitivity study on CFD models was also performed using the Richardson extrapolation and the grid convergence index method for the numerical accuracy of CFD calculations . As a result, the calculated current speed showed very good agreement with the experimental data, indicating that the current CFD methods are suitable for predicting density gradient stratified flow phenomena in the air-ingress accident.

  16. ISOFIT - A PROGRAM FOR FITTING SORPTION ISOTHERMS TO EXPERIMENTAL DATA

    EPA Science Inventory

    Isotherm expressions are important for describing the partitioning of contaminants in environmental systems. ISOFIT (ISOtherm FItting Tool) is a software program that fits isotherm parameters to experimental data via the minimization of a weighted sum of squared error (WSSE) obje...

  17. Innate Reverse Transcriptase Activity of DNA Polymerase for Isothermal RNA Direct Detection.

    PubMed

    Shi, Chao; Shen, Xiaotong; Niu, Shuyan; Ma, Cuiping

    2015-11-01

    RNA detection has become one of the most robust parts in molecular biology, medical diagnostics and drug discovery. Conventional RNA detection methods involve an extra reverse transcription step, which limits their further application for RNA rapid detection. We herein report a novel finding that Bst and Klenow DNA polymerases possess innate reverse transcriptase activities, so that the reverse transcription step and next amplification reaction can be combined to one step in isothermal RNA detection. We have demonstrated that Bst and Klenow DNA polymerases could be successfully used to reverse transcribe RNA within 125-nt length by real time RT-PCR and polyacrylamide gel electrophoresis (PAGE). Our findings will spur the development of a myriad of simple and easy to use RNA detection technologies for isothermal RNA direct detection. This will just meet the future needs of bioanalysis and clinical diagnosis to RNA rapid detection in POC settings and inspection and quarantine. PMID:26474356

  18. Development of loop-mediated isothermal amplification for detection of Leifsonia xyli subsp. xyli in sugarcane

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ratoon stunt, caused by the xylem-limited coryneform bacterium Leifsonia xyli subsp. xyli (Lxx), is prevalent in most sugarcane-producing countries. Because the disease does not cause characteristic external symptoms, a laboratory-based technique is needed for accurate diagnosis. We developed a diag...

  19. Loop-Mediated Isothermal Amplification for the Mapping of Microbes: An Anti-Capitalist Approach

    E-print Network

    Tonak, Ali Bekta?

    2015-01-01

    on  GMO-­?Contaminated   Honey.  Eur.  J.  Risk  Regul.  GMO  Deception:  What  You  Need  to  Know  about   the  Food,  Corporations,  and  Government  Agencies  Putting  Our  Families   and  Our   Environment  at  Risk  (

  20. Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-

    E-print Network

    Cai, Long

    , the US Food and Drug Administration has required the drug industry to publicly provide SNP data examined is reasonably fast, but to shorten the time required and simplify the detection, it is ideal to develop a one

  1. Rapid detection of porcine kobuvirus in feces by reverse transcription loop-mediated isothermal amplification

    PubMed Central

    2014-01-01

    Background PKV is a new emerging pathogen detected in diarrhea pigs. At present, no more detection methods were reported except RT-PCR method. this study was to develop a fast diagnostic method based on the LAMP reaction for rapid detection of PKV nucleic acid in fecal samples. Findings Two pairs of primers were designed to amplify the conservative 3D gene of PKV genome. The PKV RT-LAMP method possessed well specificity and had 100 times higher sensitivity than common reverse transcription PCR (RT-PCR), which could detect up to 10 RNA copies of the target gene. Conclusions The results showed that the optimal reaction condition for RT-LAMP was achieved at 64°C for 50 min. Furthermore, the RT-LAMP procedure does not demand special equipment and is time-saving. PMID:24755372

  2. Adsorption Isotherms and Surface Reaction Kinetics

    ERIC Educational Resources Information Center

    Lobo, L. S.; Bernardo, C. A.

    1974-01-01

    Explains an error that occurs in calculating the conditions for a maximum value of a rate expression for a bimolecular reaction. The rate expression is derived using the Langmuir adsorption isotherm to relate gas pressures and corresponding surface coverages. (GS)

  3. Chromosomal destabilization during gene amplification.

    PubMed Central

    Ruiz, J C; Wahl, G M

    1990-01-01

    Acentric extrachromosomal elements, such as submicroscopic autonomously replicating circular molecules (episomes) and double minute chromosomes, are common early, and in some cases initial, intermediates of gene amplification in many drug-resistant and tumor cell lines. In order to gain a more complete understanding of the amplification process, we investigated the molecular mechanisms by which such extrachromosomal elements are generated and we traced the fate of these amplification intermediates over time. The model system consists of a Chinese hamster cell line (L46) created by gene transfer in which the initial amplification product was shown previously to be an unstable extrachromosomal element containing an inverted duplication spanning more than 160 kilobases (J. C. Ruiz and G. M. Wahl, Mol. Cell. Biol. 8:4302-4313, 1988). In this study, we show that these molecules were formed by a process involving chromosomal deletion. Fluorescence in situ hybridization was performed at multiple time points on cells with amplified sequences. These studies reveal that the extrachromosomal molecules rapidly integrate into chromosomes, often near or at telomeres, and once integrated, the amplified sequences are themselves unstable. These data provide a molecular and cytogenetic chronology for gene amplification in this model system; an early event involves deletion to generate extrachromosomal elements, and subsequent integration of these elements precipitates a cascade of chromosome instability. Images PMID:2188107

  4. Unified water isotherms for clayey porous materials

    NASA Astrophysics Data System (ADS)

    Revil, A.; Lu, N.

    2013-09-01

    We provide a unified model for the soil-water retention function, including the effect of bound and capillary waters for all types of soils, including clayey media. The model combines a CEC-normalized isotherm describing the sorption of the bound water (and the filling of the trapped porosity) and the van Genuchten model to describe the capillary water sorption retention but ignore capillary condensation. For the CEC-normalized isotherm, we tested both the BET and Freundlich isotherms, and we found that the Freundlich is more suitable than the BET isotherm in fitting the data. It is also easier to combine the Freundlich isotherm with the van Genuchten model. The new model accounts for (1) the different types of clay minerals, (2) the different types of ions sorbed in the Stern layer and on the basal planes of 2:1 clays, and (3) the pore size distribution. The model is validated with different data sets, including mixtures of kaolinite and bentonite. The model parameters include two exponents (the pore size exponent of the van Genuchten model and the exponent of the Freundlich isotherm), the capillary entry pressure, and two critical water contents. The first critical water content is the water content at saturation (porosity), and the second is the maximum water content associated with adsorption forces, including the trapped nonbound water.

  5. Sequence independent amplification of DNA

    DOEpatents

    Bohlander, S.K.

    1998-03-24

    The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example, the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei. 25 figs.

  6. Sequence independent amplification of DNA

    DOEpatents

    Bohlander, Stefan K. (Chicago, IL)

    1998-01-01

    The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei.

  7. Optical chirped beam amplification and propagation

    DOEpatents

    Barty, Christopher P.

    2004-10-12

    A short pulse laser system uses dispersive optics in a chirped-beam amplification architecture to produce high peak power pulses and high peak intensities without the potential for intensity dependent damage to downstream optical components after amplification.

  8. Chronic centrosome amplification without tumorigenesis

    PubMed Central

    Vitre, Benjamin; Holland, Andrew J.; Kulukian, Anita; Shoshani, Ofer; Hirai, Maretoshi; Wang, Yin; Maldonado, Marcus; Cho, Thomas; Boubaker, Jihane; Swing, Deborah A.; Tessarollo, Lino; Evans, Sylvia M.; Fuchs, Elaine; Cleveland, Don W.

    2015-01-01

    Centrosomes are microtubule-organizing centers that facilitate bipolar mitotic spindle assembly and chromosome segregation. Recognizing that centrosome amplification is a common feature of aneuploid cancer cells, we tested whether supernumerary centrosomes are sufficient to drive tumor development. To do this, we constructed and analyzed mice in which centrosome amplification can be induced by a Cre-recombinase–mediated increase in expression of Polo-like kinase 4 (Plk4). Elevated Plk4 in mouse fibroblasts produced supernumerary centrosomes and enhanced the expected mitotic errors, but proliferation continued only after inactivation of the p53 tumor suppressor. Increasing Plk4 levels in mice with functional p53 produced centrosome amplification in liver and skin, but this did not promote spontaneous tumor development in these tissues or enhance the growth of chemically induced skin tumors. In the absence of p53, Plk4 overexpression generated widespread centrosome amplification, but did not drive additional tumors or affect development of the fatal thymic lymphomas that arise in animals lacking p53. We conclude that, independent of p53 status, supernumerary centrosomes are not sufficient to drive tumor formation. PMID:26578792

  9. Double regenerative amplification of picosecond pulses

    NASA Astrophysics Data System (ADS)

    Bai, Zhen-ao; Chen, Li-yuan; Bai, Zhen-xu; Chen, Meng; Li, Gang

    2012-04-01

    An double Nd:YAG regenerative amplification picosecond pulse laser is demonstrated under the semiconductor saturable absorption mirror(SESAM) mode-locking technology and regenerative amplification technology, using BBO crystal as PC electro-optic crystal. The laser obtained is 20.71ps pulse width at 10 KHz repetition rate, and the energy power is up to 4W which is much larger than the system without pre-amplification. This result will lay a foundation for the following amplification.

  10. Evaporation Induced Isothermal Crystallization of Silicate Melt

    NASA Astrophysics Data System (ADS)

    Nagahara, H.

    1996-03-01

    In order to investigate and role of evaporation and crystallization kinetics for silicate melt, isothermal vacuum experiments were carried out in the system MgO-SiO2. Due to successive evaporation, melt crystallized olivine at a fixed temperature. The evaporation rates and bulk chemical composition of residues varied with time, and reached a steady state. The pressure-composition phase diagram for the system at a fixed temperature well explains the experimental results. The results suggest a possibility of isothermal formation of chondrules (and some CAIs) at low pressures where evaporation takes place continuously.

  11. Equipment-Free Incubation of Recombinase Polymerase Amplification Reactions Using Body Heat

    PubMed Central

    Richards-Kortum, Rebecca

    2014-01-01

    The development of isothermal amplification platforms for nucleic acid detection has the potential to increase access to molecular diagnostics in low resource settings; however, simple, low-cost methods for heating samples are required to perform reactions. In this study, we demonstrated that human body heat may be harnessed to incubate recombinase polymerase amplification (RPA) reactions for isothermal amplification of HIV-1 DNA. After measuring the temperature of mock reactions at 4 body locations, the axilla was chosen as the ideal site for comfortable, convenient incubation. Using commonly available materials, 3 methods for securing RPA reactions to the body were characterized. Finally, RPA reactions were incubated using body heat while control RPA reactions were incubated in a heat block. At room temperature, all reactions with 10 copies of HIV-1 DNA and 90% of reactions with 100 copies of HIV-1 DNA tested positive when incubated with body heat. In a cold room with an ambient temperature of 10 degrees Celsius, all reactions containing 10 copies or 100 copies of HIV-1 DNA tested positive when incubated with body heat. These results suggest that human body heat may provide an extremely low-cost solution for incubating RPA reactions in low resource settings. PMID:25372030

  12. Hybrid chirped pulse amplification system

    DOEpatents

    Barty, Christopher P.; Jovanovic, Igor

    2005-03-29

    A hybrid chirped pulse amplification system wherein a short-pulse oscillator generates an oscillator pulse. The oscillator pulse is stretched to produce a stretched oscillator seed pulse. A pump laser generates a pump laser pulse. The stretched oscillator seed pulse and the pump laser pulse are directed into an optical parametric amplifier producing an optical parametric amplifier output amplified signal pulse and an optical parametric amplifier output unconverted pump pulse. The optical parametric amplifier output amplified signal pulse and the optical parametric amplifier output laser pulse are directed into a laser amplifier producing a laser amplifier output pulse. The laser amplifier output pulse is compressed to produce a recompressed hybrid chirped pulse amplification pulse.

  13. Frequency domain optical parametric amplification

    PubMed Central

    Schmidt, Bruno E.; Thiré, Nicolas; Boivin, Maxime; Laramée, Antoine; Poitras, François; Lebrun, Guy; Ozaki, Tsuneyuki; Ibrahim, Heide; Légaré, François

    2014-01-01

    Today’s ultrafast lasers operate at the physical limits of optical materials to reach extreme performances. Amplification of single-cycle laser pulses with their corresponding octave-spanning spectra still remains a formidable challenge since the universal dilemma of gain narrowing sets limits for both real level pumped amplifiers as well as parametric amplifiers. We demonstrate that employing parametric amplification in the frequency domain rather than in time domain opens up new design opportunities for ultrafast laser science, with the potential to generate single-cycle multi-terawatt pulses. Fundamental restrictions arising from phase mismatch and damage threshold of nonlinear laser crystals are not only circumvented but also exploited to produce a synergy between increased seed spectrum and increased pump energy. This concept was successfully demonstrated by generating carrier envelope phase stable, 1.43?mJ two-cycle pulses at 1.8??m wavelength. PMID:24805968

  14. Stochastic Cascade Amplification of Fluctuations

    E-print Network

    Michael Wilkinson; Robin Guichardaz; Marc Pradas; Alain Pumir

    2015-02-20

    We consider a dynamical system which has a stable attractor and which is perturbed by an additive noise. Under some quite typical conditions, the fluctuations from the attractor are intermittent and have a probability distribution with power-law tails. We show that this results from a stochastic cascade of amplification of fluctuations due to transient periods of instability. The exponent of the power-law is interpreted as a negative fractal dimension.

  15. Isothermal Titration Calorimetry in the Student Laboratory

    ERIC Educational Resources Information Center

    Wadso, Lars; Li, Yujing; Li, Xi

    2011-01-01

    Isothermal titration calorimetry (ITC) is the measurement of the heat produced by the stepwise addition of one substance to another. It is a common experimental technique, for example, in pharmaceutical science, to measure equilibrium constants and reaction enthalpies. We describe a stirring device and an injection pump that can be used with a…

  16. Apparatus to Measure Adiabatic and Isothermal Processes.

    ERIC Educational Resources Information Center

    Lamb, D. W.; White, G. M.

    1996-01-01

    Describes a simple manual apparatus designed to serve as an effective demonstration of the differences between isothermal and adiabatic processes for the general or elementary physics student. Enables students to verify Boyle's law for slow processes and identify the departure from this law for rapid processes and can also be used to give a clear…

  17. Performance Characteristics of an Isothermal Freeze Valve

    SciTech Connect

    Hailey, A.E.

    2001-08-22

    This document discusses performance characteristics of an isothermal freeze valve. A freeze valve has been specified for draining the DWPF melter at the end of its lifetime. Two freeze valve designs have been evaluated on the Small Cylindrical Melter-2 (SCM-2). In order to size the DWPF freeze valve, the basic principles governing freeze valve behavior need to be identified and understood.

  18. Non-Isothermal Steam Mixing Motivation & Objectives

    E-print Network

    Psaltis, Demetri

    Non-Isothermal Steam Mixing Motivation & Objectives In modern combined cycle power plants, every gas turbine is connected to a heat recovery steam generator (HRSG), which together feed a single steam turbine. When running multiple gas turbines at different loads, the HRSGs will produce steam streams

  19. USING ISOTHERMS TO PREDICT GAC'S CAPACITY FOR SYNTHETIC ORGANICS

    EPA Science Inventory

    This investigation involved operating a pilot granular activated carbon (GAC) plant to obtain capacity data under typical field conditions, determining isotherms for selected synthetic organic chemicals, and comparing the capacity predicted by the isotherm data with the pilot-pla...

  20. Instrument-free nucleic acid amplification assays for global health settings

    PubMed Central

    LaBarre, Paul; Boyle, David; Hawkins, Kenneth; Weigl, Bernhard

    2014-01-01

    Many infectious diseases that affect global health are most accurately diagnosed through nucleic acid amplification and detection. However, existing nucleic acid amplification tests are too expensive and complex for most low-resource settings. The small numbers of centralized laboratories that exist in developing countries tend to be in urban areas and primarily cater to the affluent. In contrast, rural area health care facilities commonly have only basic equipment and health workers have limited training and little ability to maintain equipment and handle reagents.1 Reliable electric power is a common infrastructure shortfall. In this paper, we discuss a practical approach to the design and development of non-instrumented molecular diagnostic tests that exploit the benefits of isothermal amplification strategies. We identify modular instrument-free technologies for sample collection, sample preparation, amplification, heating, and detection. By appropriately selecting and integrating these instrument-free modules, we envision development of an easy to use, infrastructure independent diagnostic test that will enable increased use of highly accurate molecular diagnostics at the point of care in low-resource settings. PMID:25089171

  1. Adsorption-induced auto-amplification of enantiomeric excess on an achiral surface

    NASA Astrophysics Data System (ADS)

    Yun, Yongju; Gellman, Andrew J.

    2015-06-01

    The homochirality of biomolecules is a signature of life on Earth and has significant implications in, for example, the production of pharmaceutical compounds. It has been suggested that biomolecular homochirality may have arisen from the amplification of a spontaneously formed small enantiomeric excess (e.e.). Many minerals exhibit naturally chiral surfaces and so adsorption has been proposed as one possible mechanism for such an amplification of e.e. Here we show that when gas-phase mixtures of D- and L-aspartic acid are exposed to an achiral Cu(111) surface, a small e.e. in the gas phase, e.e.g, leads to an amplification of the e.e. on the surface, e.e.s, under equilibrium conditions. Adsorption-induced amplification of e.e. does not require a chiral surface. The dependence of e.e.s on e.e.g has been modelled successfully using a Langmuir-like adsorption isotherm that incorporates the formation of homochiral adsorbate clusters on the surface.

  2. Non-isothermal Crystallization Kinetics of Linear Metallocene Polyethylenes

    E-print Network

    Hussein, Ibnelwaleed A.

    Non-isothermal Crystallization Kinetics of Linear Metallocene Polyethylenes Ibnelwaleed A. Hussein ABSTRACT The effect of Mw on the non-isothermal crystallization kinetics of metallocene linear polyethylene, metallocene polyethylene, non- isothermal crystallization, modulated DSC Email: ihussein@kfupm.edu.sa; Tel

  3. Resonant primordial gravitational waves amplification

    NASA Astrophysics Data System (ADS)

    Lin, Chunshan; Sasaki, Misao

    2016-01-01

    We propose a mechanism to evade the Lyth bound in models of inflation. We minimally extend the conventional single-field inflation model in general relativity (GR) to a theory with non-vanishing graviton mass in the very early universe. The modification primarily affects the tensor perturbation, while the scalar and vector perturbations are the same as the ones in GR with a single scalar field at least at the level of linear perturbation theory. During the reheating stage, the graviton mass oscillates coherently and leads to resonant amplification of the primordial tensor perturbation. After reheating the graviton mass vanishes and we recover GR.

  4. Sequences of Smooth Global Isothermic Immersions

    E-print Network

    Rivière, Tristan

    2012-01-01

    In the present work we study the behavior of sequences of smooth global isothermic immersions of a given closed surface and having a uniformly bounded total curvature. We prove that, if the conformal class of this sequence is bounded in the Moduli space of the surface, it weakly converges in W^{2,2} away from finitely many points, modulo extraction of a subsequence, to a possibly branched weak isothermic immersion of this surface. Moreover, if this limit happens to be smooth away from the branched points, we give an optimal description of the possible loss of strong compactness of such a subsequence by proving that, beside possibly finitely many atomic concentrations, the defect measure associated to the L^2 norm of the second fundamental form is "transported" along exceptional directions given by some holomorphic quadratic forms associated the limiting surface. We give examples where these losses of compactness, invariant along such exceptional directions, eventually happen.

  5. Dynamics and control of DNA sequence amplification

    NASA Astrophysics Data System (ADS)

    Marimuthu, Karthikeyan; Chakrabarti, Raj

    2014-10-01

    DNA amplification is the process of replication of a specified DNA sequence in vitro through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reaction are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal temperature profile. Strategies for the optimal synthesis of the DNA amplification control trajectory are proposed. Analogous methods can be used to formulate control problems for more advanced amplification objectives corresponding to the design of new types of DNA amplification reactions.

  6. Single Cell Transcriptome Amplification with MALBAC

    PubMed Central

    Tan, Longzhi; Tang, Fuchou; Xie, X. Sunney

    2015-01-01

    Recently, Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) has been developed for whole genome amplification of an individual cell, relying on quasilinear instead of exponential amplification to achieve high coverage. Here we adapt MALBAC for single-cell transcriptome amplification, which gives consistently high detection efficiency, accuracy and reproducibility. With this newly developed technique, we successfully amplified and sequenced single cells from 3 germ layers from mouse embryos in the early gastrulation stage, and examined the epithelial-mesenchymal transition (EMT) program among cells in the mesoderm layer on a single-cell level. PMID:25822772

  7. An evaluation of direct PCR amplification

    PubMed Central

    Hall, Daniel E.; Roy, Reena

    2014-01-01

    Aim To generate complete DNA profiles from blood and saliva samples deposited on FTA® and non-FTA® paper substrates following a direct amplification protocol. Methods Saliva samples from living donors and blood samples from deceased individuals were deposited on ten different FTA® and non-FTA® substrates. These ten paper substrates containing body fluids were kept at room temperature for varying lengths of time ranging from one day to approximately one year. For all assays in this research, 1.2 mm punches were collected from each substrate containing one type of body fluid and amplified with reagents provided in the nine commercial polymerase chain reaction (PCR) amplification kits. The substrates were not subjected to purification reagent or extraction buffer prior to amplification. Results Success rates were calculated for all nine amplification kits and all ten substrates based on their ability to yield complete DNA profiles following a direct amplification protocol. Six out of the nine amplification kits, and four out of the ten paper substrates had the highest success rates overall. Conclusion The data show that it is possible to generate complete DNA profiles following a direct amplification protocol using both standard (non-direct) and direct PCR amplification kits. The generation of complete DNA profiles appears to depend more on the success of the amplification kit rather than the than the FTA®- or non-FTA®-based substrates. PMID:25559837

  8. Dynamics and control of DNA sequence amplification

    SciTech Connect

    Marimuthu, Karthikeyan; Chakrabarti, Raj E-mail: rajc@andrew.cmu.edu

    2014-10-28

    DNA amplification is the process of replication of a specified DNA sequence in vitro through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reaction are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal temperature profile. Strategies for the optimal synthesis of the DNA amplification control trajectory are proposed. Analogous methods can be used to formulate control problems for more advanced amplification objectives corresponding to the design of new types of DNA amplification reactions.

  9. On the Isothermality of Solar Plasmas

    NASA Technical Reports Server (NTRS)

    Landi, E.; Klimchuk, J. A.

    2010-01-01

    Recent measurements have shown that the quiet unstructured solar corona observed at the solar limb is close to isothermal, at a temperature that does not appear to change over wide areas or with time. Some in dividual active loop structures have also been found to be nearly iso thermal both along their axis and across their cross-section. Even a complex active region observed at the solar limb has been found to be composed of three distinct isothermal plasmas. If confirmed, these r esults would pose formidable challenges to the current theoretical understanding of the thermal structure and heating of the solar corona. For example, no current theoretical model can explain the excess dens ities and lifetimes of many observed loops if the loops are in fact i sothermal. All of these measurements are based on the so-called emiss ion measure (EM) diagnostic technique that is applied to a set of opt ically thin lines under the assumption of isothermal plasma. It provi des simultaneous measurement of both the temperature and EM. However, no study has ever been carried out to quantify the uncertainties in the technique and to rigorously assess its ability to discriminate bet ween isothermal and multithermal plasmas. Such a study is the topic o f the present work. We define a formal measure of the uncertainty in the EM diagnostic technique that can easily be applied to real data. We here apply it to synthetic data based on a variety of assumed plas ma thermal distributions, and develop a method to quantitatively asse ss the degree of multithermality of a plasma.

  10. Predicting anthocyanins' isothermal and non-isothermal degradation with the endpoints method.

    PubMed

    Peleg, Micha; Kim, Amy D; Normand, Mark D

    2015-11-15

    The thermal degradation of anthocyanins in a variety of media and over a large temperature range is known to follow first-order kinetics, and the temperature-dependence of the exponential rate constant a two-parameter model. These parameters can be estimated from the initial and final concentrations of only two isothermal or non-isothermal heat treatments by numerically solving a pair of simultaneous equations of which they are the two unknowns. Once calculated they can be used to reconstruct the entire degradation curves and predict those of other heat treatments in a pertinent temperature range. Commercial mathematical software can do the calculations, as demonstrated with computer simulations and published data on the isothermal and non-isothermal degradation of anthocyanins. The endpoints method's predictions were confirmed by comparison to the reported experimentally determined final concentrations. Where applicable, the method will eliminate the need to record sets of whole isothermal degradation curves in studies of the kinetics of anthocyanins' degradation. PMID:25977061

  11. Input Locality and Hardness Amplification Andrej Bogdanov

    E-print Network

    Bogdanov, Andrej

    Input Locality and Hardness Amplification Andrej Bogdanov Alon Rosen Abstract We establish new hardness amplification results for one-way functions in which each input bit influences only a small number is injective then it is equally hard to invert f on a (1 - )-fraction of inputs. · If f is regular

  12. Hardness Amplification Proofs Require Majority Ronen Shaltiel

    E-print Network

    Viola, Emanuele

    Hardness Amplification Proofs Require Majority Ronen Shaltiel Emanuele Viola April 28, 2010 Abstract Hardness amplification is the fundamental task of converting a -hard function f : {0, 1}n {0, 1} into a (1/2 - )-hard function Amp(f), where f is -hard if small circuits fail to compute f on at least

  13. Non-isothermal and isothermal hydrogen desorption kinetics of zirconium hydride

    NASA Astrophysics Data System (ADS)

    Ma, Mingwang; Xiang, Wei; Tang, Binghua; Liang, Li; Wang, Lei; Tan, Xiaohua

    2015-12-01

    Thermal desorption behaviors of zirconium hydride powder under non-isothermal and isothermal heat treatment conditions were studied using simultaneous TG-TDS. The phase transformation sequences were established by correlating the observed peaks of H2 release and mass loss. The origins of the peaks or shoulders in the TDS spectra were described as the equilibrium hydrogen pressures of a number of consecutive phase regions that decomposition reaction passed through. Effect of held temperature on the isothermal desorption behavior was taken into consideration, which was shown to be essential for the phase transformation sequence during ZrH2 decomposition. The zirconium monohydride ?ZrH was observed at ambient conditions, which has been supposed to be metastable for a long time.

  14. Tsunami Amplification due to Focusing

    NASA Astrophysics Data System (ADS)

    Moore, C. W.; Kanoglu, U.; Titov, V. V.; Aydin, B.; Spillane, M. C.; Synolakis, C. E.

    2012-12-01

    Tsunami runup measurements over the periphery of the Pacific Ocean after the devastating Great Japan tsunami of 11 March 2011 showed considerable variation in far-field and near-field impact. This variation of tsunami impact have been attributed to either directivity of the source or by local topographic effects. Directivity arguments alone, however, cannot explain the complexity of the radiated patterns in oceans with trenches and seamounts. Berry (2007, Proc. R. Soc. Lond. A 463, 3055-3071) discovered how such underwater features may concentrate tsunamis into cusped caustics and thus cause large local amplifications at specific focal points. Here, we examine focusing and local amplification, not by considering the effects of underwater diffractive lenses, but by considering the details of the dipole nature of the initial profile, and propose that certain regions of coastline are more at-risk, not simply because of directivity but because typical tsunami deformations create focal regions where abnormal tsunami wave height can be registered (Marchuk and Titov, 1989, Proc. IUGG/IOC International Tsunami Symposium, Novosibirsk, USSR). In this work, we present a new general analytical solution of the linear shallow-water wave equation for the propagation of a finite-crest-length source over a constant depth without any restriction on the initial profile. Unlike the analytical solution of Carrier and Yeh (2005, Comp. Mod. Eng. & Sci. 10(2), 113-121) which was restricted to initial conditions with Gaussian profiles and involved approximation, our solution is not only exact, but also general and allows the use of realistic initial waveform such as N-waves as defined by Tadepalli and Synolakis (1994, Proc. R. Soc. Lond. A 445, 99-112). We then verify our analytical solution for several typical wave profiles, both with the NOAA tsunami forecast model MOST (Titov and Synolakis, 1998, J. Waterw. Port Coast. Ocean Eng. 124(4), 157-171) which is validated and verified through (Synolakis et al., 2008, Pure Appl. Geophys. 165(11-12), 2197-2228), and with a Boussinesq model, to illustrate the role focusing can play for different initial conditions, and to show the robust nature of focusing with respect to dispersion. We also show how the focusing effect might have played a role in the 17 July 1998 Papua New Guinea and 17 July 2006 Java events, and also the 11 March 2011 Great Japan earthquake and tsunami. Our results strongly imply that focusing increases the shoreline amplification of the tsunami.; Schematic of focusing; initial displacement (upper left), wave evolution (upper right, lower left), maximum wave amplitude with focusing (lower right).

  15. Amplification of Cellular Oncogenes in Solid Tumors

    PubMed Central

    Bagci, Ozkan; Kurtgöz, Serkan

    2015-01-01

    The term gene amplification refers to an increase in copy number of a gene. Upregulation of gene expression through amplification is a general mechanism to increase gene dosage. Oncogene amplifications have been shown in solid human cancers and they are often associated with progression of cancer. Defining oncogene amplification is useful since it is used as a prognostic marker in clinical oncology nowadays, especially v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (HER2) targeted agents are used in breast cancer patients with high level of HER2 overexpression as a therapeutic approach. However, patients without HER2 overexpression do not appear to benefit from these agents. We concluded that determination of oncogene amplification in solid tumors is an important factor in treatment of human cancers with many unknowns. We have referred to PubMed and some databases to prepare this article. PMID:26417556

  16. The equivalence of isothermal and non-isothermal power law distributions with temperature duality

    NASA Astrophysics Data System (ADS)

    Zheng, Yahui; Du, Jiulin

    2015-06-01

    The concept of temperature duality states that the physical temperature and Lagrange temperature both have physical sense in the nonextensive system. By use of this concept, the isothermal power law distribution and the non-isothermal power law distribution are equivalent to each other when the detailed balance is satisfied. Also, the polytropic equation in stellar system and self-gravitating gaseous system can be deduced from both of these two distributions. This indicates that the polytropic system exhibits some 'equilibrium' configuration which, in the stellar system, is probably the result of so called 'violent relaxation'.

  17. Post-Fragmentation Whole Genome Amplification-Based Method

    NASA Technical Reports Server (NTRS)

    Benardini, James; LaDuc, Myron T.; Langmore, John

    2011-01-01

    This innovation is derived from a proprietary amplification scheme that is based upon random fragmentation of the genome into a series of short, overlapping templates. The resulting shorter DNA strands (<400 bp) constitute a library of DNA fragments with defined 3 and 5 termini. Specific primers to these termini are then used to isothermally amplify this library into potentially unlimited quantities that can be used immediately for multiple downstream applications including gel eletrophoresis, quantitative polymerase chain reaction (QPCR), comparative genomic hybridization microarray, SNP analysis, and sequencing. The standard reaction can be performed with minimal hands-on time, and can produce amplified DNA in as little as three hours. Post-fragmentation whole genome amplification-based technology provides a robust and accurate method of amplifying femtogram levels of starting material into microgram yields with no detectable allele bias. The amplified DNA also facilitates the preservation of samples (spacecraft samples) by amplifying scarce amounts of template DNA into microgram concentrations in just a few hours. Based on further optimization of this technology, this could be a feasible technology to use in sample preservation for potential future sample return missions. The research and technology development described here can be pivotal in dealing with backward/forward biological contamination from planetary missions. Such efforts rely heavily on an increasing understanding of the burden and diversity of microorganisms present on spacecraft surfaces throughout assembly and testing. The development and implementation of these technologies could significantly improve the comprehensiveness and resolving power of spacecraft-associated microbial population censuses, and are important to the continued evolution and advancement of planetary protection capabilities. Current molecular procedures for assaying spacecraft-associated microbial burden and diversity have inherent sample loss issues at practically every step, particularly nucleic acid extraction. In engineering a molecular means of amplifying nucleic acids directly from single cells in their native state within the sample matrix, this innovation has circumvented entirely the need for DNA extraction regimes in the sample processing scheme.

  18. Silicate liquid immiscibility in isothermal crystallization experiments

    NASA Astrophysics Data System (ADS)

    Longhi, J.

    The role of silicate liquid immiscibility (SLI) in the petrogenesis of lunar granites was investigated in experiments in which four glasses were synthesized from reagent-grade oxides and carbonates with the compositions of two of the sets of coexisting liquids reported by Hess et al. (1975): a KREEP basalt derivative and a mare basalt derivative. Isothermal crystallization experiments showed that SLI is a stable phenomenon in residual lunar liquids saturated with plagioclase, and is likely to produce large compositional separations. The results indicate that controlled-cooling-rate experiments of Rutherford et al. (1974), and Hess et al. (1975, 1978) were substantially correct analogs of the natural process of liquid immiscibility.

  19. Temperature effects for isothermal polymer crystallization kinetics

    NASA Astrophysics Data System (ADS)

    Yang, Jiao; McCoy, Benjamin J.; Madras, Giridhar

    2005-06-01

    We adopt the cluster size distribution model to investigate the effect of temperature on homogeneous nucleation and crystal growth for isothermal polymer crystallization. The model includes the temperature effects of interfacial energy, nucleation rate, growth and dissociation rate coefficients, and equilibrium solubility. The time dependencies of polymer concentration, number and size of crystals, and crystallinity (in Avrami plots) are presented for different temperatures. The denucleation (Ostwald ripening effect) is also investigated by comparing moment and numerical solutions of the population balance equations. Agreement between the model results and temperature-sensitive experimental measurements for different polymer systems required strong temperature dependence for the crystal-melt interfacial energy.

  20. Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection.

    PubMed

    Jacroux, Thomas; Rieck, Daniel C; Cui, Rong; Ouyang, Yexin; Dong, Wen-Ji

    2013-01-15

    A rapid assay operable under isothermal or nonisothermal conditions is described, where the sensitivity of a typical molecular beacon (MB) system is improved by using thermostable RNase H to enzymatically cleave an MB composed of a DNA stem and an RNA loop (R/D-MB). On hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7× above background) due to an opening of the probe and a concomitant reduction in the Förster resonance energy transfer efficiency. The addition of thermostable RNase H resulted in the cleavage of the RNA loop, which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9× above background), resulting in an approximately 2- to 2.8-fold increase in the signal-to-noise ratio observed isothermally at 50 °C following the addition of RNase H. The probe was also used to monitor real-time polymerase chain reactions by measuring enhancement of donor fluorescence on R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB-RNase H scheme can be applied to a broad range of nucleic acid amplification methods. PMID:23000602

  1. Real-Time Duplex Applications of Loop-Mediated AMPlification (LAMP) by Assimilating Probes

    PubMed Central

    Kubota, Ryo; Jenkins, Daniel M.

    2015-01-01

    Isothermal nucleic-acid amplification methods such as Loop-Mediated isothermal AMPlification (LAMP) are increasingly appealing alternatives to PCR for use in portable diagnostic system due to the low cost, weight, and power requirements of the instrumentation. As such, interest in developing new probes and other functionality based on the LAMP reaction has been intense. Here, we report on the development of duplexed LAMP assays for pathogen detection using spectrally unique Assimilating Probes. As proof of principle, we used a reaction for Salmonella enterica as a model coupled with a reaction for ?-phage DNA as an internal control, as well as a duplexed assay to sub-type specific quarantine strains of the bacterial wilt pathogen Ralstonia solanacearum. Detection limits for bacterial DNA analyzed in individual reactions was less than 100 genomic equivalents in all cases, and increased by one to two orders of magnitude when reactions were coupled in duplexed formats. Even so, due to the more robust activity of newly available strand-displacing polymerases, the duplexed assays reported here were more powerful than analogous individual reactions reported only a few years ago, and represent a significant advance for incorporation of internal controls to validate assay results in the field. PMID:25741765

  2. Detection of Yersinia enterocolitica in milk powders by cross-priming amplification combined with immunoblotting analysis.

    PubMed

    Zhang, Hongwei; Feng, Shaolong; Zhao, Yulong; Wang, Shuo; Lu, Xiaonan

    2015-12-01

    Yersinia enterocolitica (Y. enterocolitica) is frequently isolated from a wide variety of foods and can cause human yersiniosis. Biochemical and culture-based assays are common detection methods, but require a long incubation time and easily misidentify Y. enterocolitica as other non-pathogenic Yersinia species. Alternatively, cross-priming amplification (CPA) under isothermal conditions combined with immunoblotting analysis enables a more sensitive detection in a relatively short time period. A set of specific displacement primers, cross primers and testing primers was designed on the basis of six specific sequences in Y. enterocolitica 16S-23S rDNA internal transcribed spacer. Under isothermal condition, amplification and hybridization were conducted simultaneously at 63°C for 60min. The specificity of CPA was tested for 96 different bacterial strains and 165 commercial milk powder samples. Two red lines were developed on BioHelix Express strip for all of the Y. enterocolitica strains, and one red line was shown for non-Y. enterocolitica strains. The limit of detection of CPA was 10(0)fg for genomic DNA (1000 times more sensitive than PCR assay), 10(1)CFU/ml for pure bacterial culture, and 10(0)CFU per 100g milk powder with pre-enrichment at 37°C for 24h. CPA combined with immunoblotting analysis can achieve highly specific and sensitive detection of Y. enterocolitica in milk powder in 90min after pre-enrichment. PMID:26253307

  3. [Rapid and simple detection of Legionella species by LAMP, a mew DNA amplification method].

    PubMed

    Annaka, Toshimitsu

    2003-01-01

    Legionella is a common cause of community-acquired respiratory tract infections and occasionally causes nosocomial pneumonia. Rapid and accurate detection of legionellae is important for diagnosis and treatment of patients. In order to detect legionellae, a new DNA amplification method was designed and evaluated. Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high specificity, sensitivity, and rapidity under isothermal conditions at 65 degrees C. This method employs a DNA polymerase with strand displacement activity and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. The primers targeting 16S rRNA gene were designed in order to detect a wide range of Legionella species. We could specifically detect Legionella species including Legionella pneumophila, Legionella anisa, Legionella bozemanii, Legionella dumoffii, Legionella erythra, Legionella feeleii, Legionella gormanii, Legionella longbeachae, Legionella micdadei, Legionella oakridgensis, and Legionella sainthelensi. The detection limit of the assay was 6 cfu per test of L. pneumophila strain. Furthermore, all of the positive LAMP results could be obtained within 50 minutes. The LAMP method was able to detect a wide range of Legionella species with high specificity, sensitivity, rapidity, and a simple procedure. PMID:14984304

  4. Numerical Model for Isothermal and Non-Isothermal Crystallization of Liquids and Glasses

    NASA Technical Reports Server (NTRS)

    Kelton, K. F.

    1993-01-01

    A new numerical model of isothermal and non-isothermal first order phase transformations, such as the crystallization of liquids and glasses, is presented. This model computes directly the volume fraction transformed, taking into account time-dependent nucleation rates and cluster-size-dependent growth velocities. The model is applied to the crystallization of lithium disilicate glass, using the appropriate kinetic and thermodynamic parameters. The model is used (1) to determine the validity of common methods for computing the volume fraction transformed as a function of time in isothermal experiments when a time-dependent nucleation rate is expected, (2) to simulate non-isothermal differential scanning calorimetry (DSC) studies of glass devitrification as a function of scan rate, and (3) to compute the effect of preannealing on the DSC peak parameters. A novel behavior of the nucleation rate with scan rate is predicted, arising because the relaxation of the cluster distribution cannot be described by a single relaxation time. Comparisons of the calculations with experimental data on this glass demonstrate the validity of the model.

  5. Pressure-composition-isotherms of palladium alloys

    SciTech Connect

    Flanagan, T.B.

    1996-11-01

    About one year ago a summary report was submitted covering the previous three years of the contract. This earlier report should be consulted as a useful survey and evaluation of the research carried out by the authors. Because of difficulties during the current contract period arising from the anomalous nature of the melt-spun alloys received from LANL, it is not possible to contribute much beyond that given in last year's summary with regard to the overall picture of the behavior of Pd-rich alloys towards hydrogen and its isotopes. In this contract year deuterium was employed instead of hydrogen and instead of using cycled alloys, the alloys employed for each isotherm measurement were in their virgin condition. Because of the anomalous behavior of the melt-spun alloys, it was not feasible or worthwhile in some cases, e.g., when the alloy behaved anomalously, to carry out all of the originally proposed work. Nonetheless considering these obstacles, some useful data were obtained. For example, the obtaining of deuterium isotherms for the Pd-Rh alloys down to {minus}40 C using internally oxidized melt-spun alloys may prove to be useful.

  6. Computer Modeling of Non-Isothermal Crystallization

    NASA Technical Reports Server (NTRS)

    Kelton, K. F.; Narayan, K. Lakshmi; Levine, L. E.; Cull, T. C.; Ray, C. S.

    1996-01-01

    A realistic computer model for simulating isothermal and non-isothermal phase transformations proceeding by homogeneous and heterogeneous nucleation and interface-limited growth is presented. A new treatment for particle size effects on the crystallization kinetics is developed and is incorporated into the numerical model. Time-dependent nucleation rates, size-dependent growth rates, and surface crystallization are also included. Model predictions are compared with experimental measurements of DSC/DTA peak parameters for the crystallization of lithium disilicate glass as a function of particle size, Pt doping levels, and water content. The quantitative agreement that is demonstrated indicates that the numerical model can be used to extract key kinetic data from easily obtained calorimetric data. The model can also be used to probe nucleation and growth behavior in regimes that are otherwise inaccessible. Based on a fit to data, an earlier prediction that the time-dependent nucleation rate in a DSC/DTA scan can rise above the steady-state value at a temperature higher than the peak in the steady-state rate is demonstrated.

  7. Statistical Design in Isothermal Aging of Polyimide Resins

    NASA Technical Reports Server (NTRS)

    Sutter, James K.; Jobe, Marcus; Crane, Elizabeth A.

    1995-01-01

    Recent developments in research on polyimides for high temperature applications have led to the synthesis of many new polymers. Among the criteria that determines their thermal oxidative stability, isothermal aging is one of the most important. Isothermal aging studies require that many experimental factors are controlled to provide accurate results. In this article we describe a statistical plan that compares the isothermal stability of several polyimide resins, while minimizing the variations inherent in high-temperature aging studies.

  8. Amplification-control element ACE-3 is important but not essential for autosomal chorion gene amplification.

    PubMed Central

    Swimmer, C; Delidakis, C; Kafatos, F C

    1989-01-01

    We have further characterized the cis-acting elements that control the amplification of the third chromosomal cluster of chorion genes in Drosophila melanogaster; these include the amplification-control element ACE-3 and four amplification-enhancing regions (AER-a to -d). We have used two types of deletions in the chorion cluster: the first was in vitro generated deletions of the ACE-3 region that were subsequently introduced into the germ line, and the second was deletions induced in vivo within a transposon at a preexisting chromosomal location, thus avoiding the complication of position effects. Some of the lines bearing deletions of either type showed amplification, albeit at drastically reduced levels. These unexpected results indicate that, despite its importance, ACE-3 is not essential for low-level amplification and that cis-acting amplification elements are functionally redundant within the autosomal chorion replicon. Images PMID:2554333

  9. Incorporating nonlinear isotherms into robust multilayer sorptive barrier design

    NASA Astrophysics Data System (ADS)

    Matott, L. Shawn; Bandilla, Karl; Rabideau, Alan J.

    2009-11-01

    Sorptive barrier technologies have emerged as useful tools for addressing a wide range of remediation problems. When simulating barrier performance, numerous isotherm expressions are available for relating aqueous and sorbed concentrations. However, isotherm selection is non-trivial because alternative expressions may yield comparable fits to experimental data. Additionally, concentration data collected for parameter fitting is often outside the range of concentrations relevant to simulating barrier performance. This incompatibility necessitates extrapolation of isotherm behavior during simulation-optimization. Consequently, equally plausible isotherms may predict significantly different barrier performance. Numerical experiments involving organic contaminants were performed to examine the influence of isotherm selection and extrapolation on optimal barrier design. Ten isotherms were calibrated to existing experimental data and evaluated using information-theoretic selection criteria. When incorporated into simulation-optimization, extrapolation effects were clearly evident and optimal designs varied according to the chosen isotherm. To ensure robust barrier design in the presence of such variability, a simple methodology is proposed that utilizes a piecewise-minimum isotherm concept. By favoring plausible isotherms that predict the least amount of sorption, the methodology encourages conservative barrier design while respecting available data.

  10. Advanced backward Raman amplification seeding

    NASA Astrophysics Data System (ADS)

    Malkin, Vladimir; Fisch, Nathaniel

    2010-11-01

    Next generations of ultrapowerful laser pulses, reaching exawatt and zetawatt powers within reasonably compact facilities, might be based on the backward Raman amplification (BRA) in plasmas. Amplified pulse intensities hundreds times higher than the pump intensity are already observed experimentally. More advanced BRA stages should produce even higher intensities. The largest nonfocused intensity, limited primarily by instabilities associated with the relativistic electron nonlinearity of the amplified laser pulse, is, roughly speaking, 0.1 of the fully relativistic value. It corresponds to the amplified pulse final (and shortest) duration be about the electron plasma wave period. The needed seed pulse should be at least that short then to stay ahead of the amplified pulse, rather than be shadowed by it (which would much reduce the seeding efficiency). However, at earlier BRA stages, when the amplified pulse is longer, the optimal duration of the seed pulse is also longer. This work proposes the use of self-contracting seed pulses for further optimizing the advanced BRA.

  11. AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES - Book Chapter

    EPA Science Inventory

    This book chapter contains the following headings and subheadings: Introduction; Experimental Approach - Precautions, Template, Primers, Reaction Conditions, Enhancers, Post Amplification; Procedures - Template DNA, Basic PCR, Thermal Cycle Parameters, Enzyme Addition, Agarose Ge...

  12. Mechanisms of Gene Duplication and Amplification

    E-print Network

    Roth, John R.

    Mechanisms of Gene Duplication and Amplification Andrew B. Reams1 and John R. Roth2 1 Department of mutation types (Anderson and Roth 1977; Reams and Neidle 2004; Lynch et al. 2008; Lipinski et al. 2011

  13. Sequence-independent amplification coupled with DNA microarray analysis for detection and genotyping of noroviruses.

    PubMed

    Hu, Yuan; Yan, Huijun; Mammel, Mark; Chen, Haifeng

    2015-12-01

    Noroviruses (NoVs) have high levels of genetic sequence diversities, which lead to difficulties in designing robust universal primers to efficiently amplify specific viral genomes for molecular analysis. We here described the practicality of sequence-independent amplification combined with DNA microarray analysis for simultaneous detection and genotyping of human NoVs in fecal specimens. We showed that single primer isothermal linear amplification (Ribo-SPIA) of genogroup I (GI) and genogroup II (GII) NoVs could be run through the same amplification protocol without the need to design and use any virus-specific primers. Related virus could be subtyped by the unique pattern of hybridization with the amplified product to the microarray. By testing 22 clinical fecal specimens obtained from acute gastroenteritis cases as blinded samples, 2 were GI positive and 18 were GII positive as well as 2 negative for NoVs. A NoV GII positive specimen was also identified as having co-occurrence of hepatitis A virus. The study showed that there was 100 % concordance for positive NoV detection at genogroup level between the results of Ribo-SPIA/microarray and the phylogenetic analysis of viral sequences of the capsid gene. In addition, 85 % genotype agreement was observed for the new assay compared to the results of phylogenetic analysis. PMID:26556029

  14. Isothermal aging of three polyurethane elastomers

    SciTech Connect

    Guess, T.R.

    1996-05-01

    Two polyurethane systems, EN-7 and L-100, have a long history as encapsulants and coatings in Sandia programs. These materials contain significant amounts of toluene diisocyanate (TDI), a suspect human carcinogen. As part of efforts to reduce the use of hazardous materials in the workplace, PET-90A, a polyurethane with less than 0.1% free TDI, was identified as a candidate for new applications and as a replacement for the more hazardous polyurethanes in selected programs. This report documents the results of a two-year accelerated aging study of PET-90A, EN-7, and L-100 polyurethane elastomers to characterize the effect of 135{degrees}F isothermal aging on selected physical, electrical, mechanical and thermal properties. In general, there was very little change in properties over the two year period for the three elastomers. The largest changes occurred in EN-7, which is the polyurethane with the longest service history in Sandia applications.

  15. Mathematical Formalism for Isothermal Linear Irreversibility

    E-print Network

    Hong Qian

    2001-06-27

    We prove the equivalence among symmetricity, time reversibility, and zero entropy production of the stationary solutions of linear stochastic differential equations. A sufficient and necessary reversibility condition expressed in terms of the coefficients of the equations is given. The existence of a linear stationary irreversible process is established. Concerning reversibility, we show that there is a contradistinction between any 1-dimensional stationary Gaussian process and stationary Gaussian process of dimension $n>1$. A concrete criterion for differentiating stationarity and sweeping behavior is also obtained. The mathematical result is a natural generalization of Einstein's fluctuation-dissipation relation, and provides a rigorous basis for the isothermal irreversibility in a linear regime which is the basis for applying Onsager's theory to macromolecules in aqueous solution.

  16. Isothermal dendritic growth: A low gravity experiment

    NASA Technical Reports Server (NTRS)

    Glicksman, M. E.; Hahn, R. C.; Lograsso, T. A.; Rubinstein, E. R.; Selleck, M. E.; Winsa, E.

    1988-01-01

    The Isothermal Dendritic Growth Experiment is an active crystal growth experiment designed to test dendritic growth theory at low undercoolings where convection prohibits such studies at 1 g. The experiment will be essentially autonomous, though limited in-flight interaction through a computer interface is planned. One of the key components of the apparatus will be a crystal growth chamber capable of achieving oriented single crystal dendritic growth. Recent work indicates that seeding the chamber with a crystal of the proper orientation will not, in and of itself, be sufficient to meet this requirement. Additional flight hardware and software required for the STS flight experiment are currently being developed at NASA Lewis Research Center and at Rensselaer Polytechnic Institute.

  17. Isothermal decomposition of ?-irradiated erbium acetate

    NASA Astrophysics Data System (ADS)

    Mahfouz, R. M.; Al-Shehri, S. M.; Monshi, M. A. S.; Alhaizan, A. I.; El-Salam, N. M. Abd

    Isothermal decomposition of un-irradiated and pre-?-irradiated erbium acetate has been investigated at different temperatures between 583 and 603 K. Irradiation was observed to enhance the rate of decomposition without modifying the mechanism of the thermal decomposition. Thermal decomposition of erbium acetate has been shown to proceed by a nucleation and growth mechanism (Erofe'ev model) both for un-irradiated and pre-?-irradiated samples. The enhancement of the decomposition was found to increase with an increase in the ?-ray dose applied to the sample and may be attributed to an increase in point defects and formation of additional nucleation centers generated in the host lattice. Thermodynamic values of the main decomposition process were calculated and evaluated.

  18. Isothermal decomposition of ?-irradiated samarium acetate

    NASA Astrophysics Data System (ADS)

    Mahfouz, R. M.; Monshi, M. A. S.; Alshehri, S. M.; Abd El-Salam, N. M.

    2000-10-01

    Isothermal decomposition of un-irradiated and pre-?-irradiated samarium acetate has been investigated at different temperatures between 613 and 633 K. Irradiation was observed to enhance the rate of decomposition without modifying the mechanism of thermal decomposition. Thermal decomposition of samarium acetate has been shown to proceed by two-dimensional phase-boundary reaction both for un-irradiated and pre-?-irradiated samples. The enhancement of the decomposition was found to increase with an increase in the ?-ray dose applied to the sample and may be attributed to an increase in point defects and formation of additional nucleation centers generated in the host lattice. Thermodynamic values of the main decomposition process were calculated and evaluated.

  19. Isothermal Decomposition of Hydrogen Peroxide Dihydrate

    NASA Technical Reports Server (NTRS)

    Loeffler, M. J.; Baragiola, R. A.

    2011-01-01

    We present a new method of growing pure solid hydrogen peroxide in an ultra high vacuum environment and apply it to determine thermal stability of the dihydrate compound that forms when water and hydrogen peroxide are mixed at low temperatures. Using infrared spectroscopy and thermogravimetric analysis, we quantified the isothermal decomposition of the metastable dihydrate at 151.6 K. This decomposition occurs by fractional distillation through the preferential sublimation of water, which leads to the formation of pure hydrogen peroxide. The results imply that in an astronomical environment where condensed mixtures of H2O2 and H2O are shielded from radiolytic decomposition and warmed to temperatures where sublimation is significant, highly concentrated or even pure hydrogen peroxide may form.

  20. A family of lowered isothermal models

    NASA Astrophysics Data System (ADS)

    Gieles, Mark; Zocchi, Alice

    2015-11-01

    We present a family of self-consistent, spherical, lowered isothermal models, consisting of one or more mass components, with parametrized prescriptions for the energy truncation and for the amount of radially biased pressure anisotropy. The models are particularly suited to describe the phase-space density of stars in tidally limited, mass-segregated star clusters in all stages of their life-cycle. The models extend a family of isotropic, single-mass models by Gomez-Leyton and Velazquez, of which the well-known Woolley, King and Wilson (in the non-rotating and isotropic limit) models are members. We derive analytic expressions for the density and velocity dispersion components in terms of potential and radius, and introduce a fast model solver in PYTHON (LIMEPY), that can be used for data fitting or for generating discrete samples.

  1. Rolling circle amplification of metazoan mitochondrialgenomes

    SciTech Connect

    Simison, W. Brian; Lindberg, D.R.; Boore, J.L.

    2005-07-31

    Here we report the successful use of rolling circle amplification (RCA) for the amplification of complete metazoan mt genomes to make a product that is amenable to high-throughput genome sequencing techniques. The benefits of RCA over PCR are many and with further development and refinement of RCA, the sequencing of organellar genomics will require far less time and effort than current long PCR approaches.

  2. Amplification of chromosomal DNA in situ

    DOEpatents

    Christian, Allen T. (Tracy, CA); Coleman, Matthew A. (Livermore, CA); Tucker, James D. (Livermore, CA)

    2002-01-01

    Amplification of chromosomal DNA in situ to increase the amount of DNA associated with a chromosome or chromosome region is described. The amplification of chromosomal DNA in situ provides for the synthesis of Fluorescence in situ Hybridization (FISH) painting probes from single dissected chromosome fragments, the production of cDNA libraries from low copy mRNAs and improved in Comparative Genomic Hybridization (CGH) procedures.

  3. Gravitational instability of finite isothermal spheres

    NASA Astrophysics Data System (ADS)

    Chavanis, P. H.

    2002-01-01

    We investigate the stability of bounded self-gravitating systems in the canonical ensemble by using a thermodynamical approach. Our study extends the earlier work of Padmanabhan (\\cite{pad89}) in the microcanonical ensemble. By studying the second variations of the free energy, we find that instability sets in precisely at the point of minimum temperature in agreement with the theorem of Katz (\\cite{kat78}). The perturbation that induces instability at this point is calculated explicitly; it has not a ``core-halo'' structure contrary to what happens in the microcanonical ensemble. We also study Jeans type gravitational instability of isothermal gaseous spheres described by Navier-Stokes equations. The introduction of a container and the consideration of an inhomogeneous distribution of matter avoids the Jeans swindle. We show analytically the equivalence between dynamical stability and thermodynamical stability and the fact that the stability of isothermal gas spheres does not depend on the viscosity. This confirms the findings of Semelin et al. (\\cite{sem01}) who used numerical methods or approximations. We also give a simpler derivation of the geometric hierarchy of scales inducing instability discovered by these authors. The density profiles that trigger these instabilities are calculated explicitly; high order modes of instability present numerous oscillations whose nodes also follow a geometric progression. This suggests that the system will fragment in a series of ``clumps'' and that these ``clumps'' will themselves fragment in substructures. The fact that both the domain sizes leading to instability and the ``clumps'' sizes within a domain follow a geometric progression with the same ratio suggests a fractal-like behavior. This gives further support to the interpretation of de Vega et al. (1996) concerning the fractal structure of the interstellar medium.

  4. ANALYTICAL SOLUTIONS OF SINGULAR ISOTHERMAL QUADRUPOLE LENS

    SciTech Connect

    Chu Zhe; Lin, W. P.; Yang Xiaofeng E-mail: linwp@shao.ac.cn

    2013-06-20

    Using an analytical method, we study the singular isothermal quadrupole (SIQ) lens system, which is the simplest lens model that can produce four images. In this case, the radial mass distribution is in accord with the profile of the singular isothermal sphere lens, and the tangential distribution is given by adding a quadrupole on the monopole component. The basic properties of the SIQ lens have been studied in this Letter, including the deflection potential, deflection angle, magnification, critical curve, caustic, pseudo-caustic, and transition locus. Analytical solutions of the image positions and magnifications for the source on axes are derived. We find that naked cusps will appear when the relative intensity k of quadrupole to monopole is larger than 0.6. According to the magnification invariant theory of the SIQ lens, the sum of the signed magnifications of the four images should be equal to unity, as found by Dalal. However, if a source lies in the naked cusp, the summed magnification of the left three images is smaller than the invariant 1. With this simple lens system, we study the situations where a point source infinitely approaches a cusp or a fold. The sum of the magnifications of the cusp image triplet is usually not equal to 0, and it is usually positive for major cusps while negative for minor cusps. Similarly, the sum of magnifications of the fold image pair is usually not equal to 0 either. Nevertheless, the cusp and fold relations are still equal to 0 in that the sum values are divided by infinite absolute magnifications by definition.

  5. Amplification uncertainty relation for probabilistic amplifiers

    NASA Astrophysics Data System (ADS)

    Namiki, Ryo

    2015-09-01

    Traditionally, quantum amplification limit refers to the property of inevitable noise addition on canonical variables when the field amplitude of an unknown state is linearly transformed through a quantum channel. Recent theoretical studies have determined amplification limits for cases of probabilistic quantum channels or general quantum operations by specifying a set of input states or a state ensemble. However, it remains open how much excess noise on canonical variables is unavoidable and whether there exists a fundamental trade-off relation between the canonical pair in a general amplification process. In this paper we present an uncertainty-product form of amplification limits for general quantum operations by assuming an input ensemble of Gaussian-distributed coherent states. It can be derived as a straightforward consequence of canonical uncertainty relations and retrieves basic properties of the traditional amplification limit. In addition, our amplification limit turns out to give a physical limitation on probabilistic reduction of an Einstein-Podolsky-Rosen uncertainty. In this regard, we find a condition that probabilistic amplifiers can be regarded as local filtering operations to distill entanglement. This condition establishes a clear benchmark to verify an advantage of non-Gaussian operations beyond Gaussian operations with a feasible input set of coherent states and standard homodyne measurements.

  6. Structure of magnesium alloy MA14 after multistep isothermal forging and subsequent isothermal rolling

    NASA Astrophysics Data System (ADS)

    Nugmanov, D. R.; Sitdikov, O. Sh.; Markushev, M. V.

    2015-10-01

    Optical metallography and electron microscopy have been used to analyze the structural changes in magnesium MA14 alloy subjected to processing that combines multistep isothermal forging and isothermal rolling. It has been found that forging of a bulk workpiece leads to the formation of a structure, 85-90% of which consists of recrystallized grains with an average size of less than 5 µm. Subsequent rolling results in a completely recrystallized structure with a grain size of 1-2 µm. It is shown that the resultant structural states are characterized by grain size nonuniformity inherited from the initial hot-pressed semi-finished product. The nature and features of crystallization processes that take place in the alloy during processing are discussed.

  7. The density variance-Mach number relation in isothermal and non-isothermal adiabatic turbulence

    NASA Astrophysics Data System (ADS)

    Nolan, C. A.; Federrath, C.; Sutherland, R. S.

    2015-08-01

    The density variance-Mach number relation of the turbulent interstellar medium is relevant for theoretical models of the star formation rate, efficiency, and the initial mass function of stars. Here we use high-resolution hydrodynamical simulations with grid resolutions of up to 10243 cells to model compressible turbulence in a regime similar to the observed interstellar medium. We use FYRIS ALPHA, a shock-capturing code employing a high-order Godunov scheme to track large density variations induced by shocks. We investigate the robustness of the standard relation between the logarithmic density variance (? _s^2) and the sonic Mach number M of isothermal interstellar turbulence, in the non-isothermal regime. Specifically, we test ideal gases with diatomic molecular (? = 7/5) and monatomic (? = 5/3) adiabatic indices. A periodic cube of gas is stirred with purely solenoidal forcing at low wavenumbers, leading to a fully developed turbulent medium. We find that as the gas heats in adiabatic compressions, it evolves along the relationship in the density variance-Mach number plane, but deviates significantly from the standard expression for isothermal gases. Our main result is a new density variance-Mach number relation that takes the adiabatic index into account: ? _s^2=ln (1+b^2 M^{(5? +1)/3}) and provides good fits for b M? 1. A theoretical model based on the Rankine-Hugoniot shock jump conditions is derived, ? _s^2 = ln {1 + (? +1)b^2{M}^2/[(? -1)b^2{M}^2+2]}, and provides good fits also for b M>1. We conclude that this new relation for adiabatic turbulence may introduce important corrections to the standard relation, if the gas is not isothermal (? ? 1).

  8. Heat induces gene amplification in cancer cells

    SciTech Connect

    Yan, Bin; Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 ; Ouyang, Ruoyun; Huang, Chenghui; Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 ; Liu, Franklin; Neill, Daniel; Li, Chuanyuan; Dewhirst, Mark

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and telomere functions are denatured. To our knowledge, this is the first study to provide direct evidence of hyperthermia induced gene amplification.

  9. New electrochromism isotherm in anodic iridium oxide films

    NASA Astrophysics Data System (ADS)

    Beni, G.; Shay, J. L.

    1980-01-01

    We show that there are two distinct electrochromic processes in anodic iridium oxide films. The measured isotherms for both processes have simple theoretical forms dominated by internal entropy changes of noninteracting particles. Such isotherms have never before been observed for amorphous films.

  10. Depth-varying constitutive properties observed in an isothermal glacier

    E-print Network

    Marshall, Hans-Peter

    Depth-varying constitutive properties observed in an isothermal glacier H. P. Marshall,1 J. T. Humphrey, Depth-varying constitutive properties observed in an isothermal glacier, Geophys. Res. Lett., 29 of polycrystalline glacier ice such as large crystals, widely ranging crystal sizes, and natural inhomo- geneities

  11. Resolving vibrational and structural contributions to isothermal compressibility

    E-print Network

    Resolving vibrational and structural contributions to isothermal compressibility Frank H determines T for low-temperature crystals, but both operate in the liquid phase. As a supercooled liquid, expresses the isothermal compressibility T in terms of number or density fluctuations,1 kBT T /V N N 2 / N 2

  12. A new shallow approximation for tridimensional non-isothermal viscoplastic

    E-print Network

    Saramito, Pierre

    A new shallow approximation for tridimensional non-isothermal viscoplastic lava flows Noé Bernabeu, , Pierre Saramito, and Claude Smutek Abstract ­ A new shallow reduced model for the non-isothermal tridimen, as diffusion-convection in lava, radiation and convection in air, diffusion in the substrate while the fluid

  13. Heat conductors with a stationary isothermic surface by Shigeru Sakaguchi

    E-print Network

    Magnanini, Rolando

    Heat conductors with a stationary isothermic surface by Shigeru Sakaguchi (Ehime University, Japan) We consider a bounded heat conductor and suppose that, initially, it has temperature 0 and, at all ball soup: Heat conductors with a stationary isothermic surface, submitted. [Z] L. Zalcman, Some

  14. Rapid and sensitive detection of Enterobacter sakazakii by cross-priming amplification combined with immuno-blotting analysis.

    PubMed

    Yulong, Zhao; Xia, Zhang; Hongwei, Zhang; Wei, Liu; Wenjie, Zheng; Xitai, Huang

    2010-12-01

    Enterobacter sakazakii is a widespread and life-threatening bacterium especially in polluted powdered infant milk formula. Several methods have been developed for detection of E. sakazakii such as physiological and biochemical methods, PCR and loop-mediated isothermal amplification. However, these procedures were disadvantages due to a long assay time, low sensitivity or the use of toxic reagents. Our method of cross-priming amplification (CPA) under isothermal conditions combined with immuno-blotting analysis made the whole detection procedure more sensitivity and lower time-consuming. A set of specific displacement primers, cross primers and testing primers were designed based on six specific sequences in E. sakazakii 16S-23S rDNA internal transcribed spacer. Under isothermal condition at 63 °C for 60 min, the specific amplification and hybridization steps were processed simultaneously. The specificity of the CPA was tested in panel of 54 different bacterial strains and 236 milk powder products. Two red signal lines were developed on the BioHelix Express strip in all of positive E. sakazakii strains, and only one signal line was demonstrated by non- E. sakazakii bacterial strains. The limit of decetion of CPA was 6.3 ± 2.7277 fg for the genomic DNA, 88 ± 8.7892 cfu/ml for pure bacterial culture, and 3.2 ± 2.0569 cfu per 100 g milk powder with pre-enrichment. The current study demonstrated that the assay method of CPA combined with immuno-blotting analysis was a specific and sensitive detection for the rapid detection of E. sakazakii. PMID:20850522

  15. Primase-based whole genome amplification

    PubMed Central

    Li, Ying; Kim, Hyun-Jin; Zheng, Chunyang; Chow, Wing Huen A.; Lim, Jeonghwa; Keenan, Brendan; Pan, Xiaojing; Lemieux, Bertrand; Kong, Huimin

    2008-01-01

    In vitro DNA amplification methods, such as polymerase chain reaction (PCR), rely on synthetic oligonucleotide primers for initiation of the reaction. In vivo, primers are synthesized on-template by DNA primase. The bacteriophage T7 gene 4 protein (gp4) has both primase and helicase activities. In this study, we report the development of a primase-based Whole Genome Amplification (pWGA) method, which utilizes gp4 primase to synthesize primers, eliminating the requirement of adding synthetic primers. Typical yield of pWGA from 1 ng to 10 ng of human genomic DNA input is in the microgram range, reaching over a thousand-fold amplification after 1 h of incubation at 37°C. The amplification bias on human genomic DNA is 6.3-fold among 20 loci on different chromosomes. In addition to amplifying total genomic DNA, pWGA can also be used for detection and quantification of contaminant DNA in a sample when combined with a fluorescent reporter dye. When circular DNA is used as template in pWGA, 108-fold of amplification is observed from as low as 100 copies of input. The high efficiency of pWGA in amplifying circular DNA makes it a potential tool in diagnosis and genotyping of circular human DNA viruses such as human papillomavirus (HPV). PMID:18559358

  16. Hydrodynamic simulations of propellers: Isothermal model

    NASA Astrophysics Data System (ADS)

    Seiß, M.; Hoffmann, H.; Spahn, F.

    2015-10-01

    Small moons embedded in Saturn's rings can cause S-shaped density structures, called propellers, in their close vicinity. This structures have been predicted first on base of a combined model involving gravitational scattering of test particles (creating the structure) and diffusion (smearing out the structure) [1, 2]. The propeller model was confirmed later with the help of Nbody simulations showing the additional appearance of moon wakes adjacent to the S-shaped gaps [3, 4]. It was a great success of the Cassini mission when the propeller were detected in the ISS imaging [5, 6] and UVIS occultation data [7]. Here we present an isothermal hydrodynamic simulation of a propeller as a further development of the original model [1, 2] where gravitational scattering and diffusion had to be treated separately. With this new approach we prove the correctness of the predicted scaling laws for the radial and azimuthal extent of the propeller. Furthermore, we will show a comparison between results of N-body and hydrodynamic simulations. Finally, we will present simulation results of the giant propeller Bleriot, which can not be modeled by N-body simulations in its full extent yet.

  17. Adsorption isotherm special study. Final report

    SciTech Connect

    1993-05-01

    The study was designed to identify methods to determine adsorption applicable to Uranium Mill Tailings Remedial Action (UMTRA) Project sites, and to determine how changes in aquifer conditions affect metal adsorption, resulting retardation factors, and estimated contaminant migration rates. EPA and ASTM procedures were used to estimate sediment sorption of U, As, and Mo under varying groundwater geochemical conditions. Aquifer matrix materials from three distinct locations at the DOE UMTRA Project site in Rifle, CO, were used as the adsorbents under different pH conditions; these conditions stimulated geochemical environments under the tailings, near the tailings, and downgradient from the tailings. Grain size, total surface area, bulk and clay mineralogy, and petrography of the sediments were characterized. U and Mo yielded linear isotherms, while As had nonlinear ones. U and Mo were adsorbed strongly on sediments acidified to levels similar to tailings leachate. Changes in pH had much less effect on As adsorption. Mo was adsorbed very little at pH 7-7.3, U was weakly sorbed, and As was moderately sorbed. Velocities were estimated for metal transport at different pHs. Results show that the aquifer materials must be characterized to estimate metal transport velocities in aquifers and to develop groundwater restoration strategies for the UMTRA project.

  18. Material Compatibility with Isothermal Pb-Li

    SciTech Connect

    Pint, Bruce A; Walker, Larry R; Unocic, Kinga A

    2012-01-01

    Eutectic Pb-Li is a leading candidate for current fusion blanket concepts as a coolant. However, there is very little data about the compatibility of most materials with Pb-Li above 500 C where the dissolution rate of many conventional alloys increases rapidly. Current work is beginning to assess Pb-Li compatibility from 500 to 800 C using isothermal capsule experiments. Aluminide coatings hold some promise in protecting conventional Fe-base alloys at 600-700 C. However, there is a significant initial Al loss that has not been clearly explained. Furthermore, the reaction product with coated materials is LiAlO{sub 2} rather than Al{sub 2}O{sub 3} at 600 and 700 C. Even when pre-oxidized to form {alpha}-Al{sub 2}O{sub 3}, an alumina layer on FeCrAl transformed to LiAlO{sub 2} at 700 and 800 C. At 500 C, the preformed oxide partially transformed from alumina and some Li was detected in the oxide layer.

  19. ISOTHERMAL AIR-INGRESS VALIDATION EXPERIMENTS

    SciTech Connect

    Chang H. Oh; Eung S. Kim

    2013-01-01

    Idaho National Laboratory has conducted airingress experiments as part of a campaign to validate computational fluid dynamics (CFD) calculations for very high-temperature gas-cooled reactor (VHTR) analysis. An isothermal test loop was designed to recreate exchange or stratified flow that occurs in the lower plenum of VHTR after a break in the primary loop allows helium to leak out and reactor building air to enter the reactor core. The experiment was designed to measure stratified flow in the inlet pipe connecting to the lower plenum of the General Atomics gas turbine–modular helium reactor (GT-MHR). Instead of helium and air, brine and sucrose were used as heavy fluids, and water was used as the lighter fluid to create, using scaling laws, the appropriate flow characteristics of the lower plenum immediately after depressurization. These results clearly indicate that stratified flow is established even for very small density differences. Corresponding CFD results were validated with the experimental data. A grid sensitivity study on CFD models was also performed using the Richardson extrapolation and the grid convergence index method for the numerical accuracy of CFD calculations. The calculated current speed showed very good agreement with the experimental data, indicating that current CFD methods are suitable for simulating density gradient stratified flow phenomena in an air-ingress accident.

  20. Iodine isothermal migration behaviour in titanium nitride

    NASA Astrophysics Data System (ADS)

    Gavarini, S.; Jaffrezic, H.; Martin, P.; Peaucelle, C.; Toulhoat, N.; Cardinal, S.; Moncoffre, N.; Pichon, C.; Tribet, M.

    2008-02-01

    Titanium nitride is one of the inert matrixes proposed to surround the fuel in gas cooled fast reactor (GFR) systems. These reactors will operate at high temperature and refractory materials with a high chemical stability and good mechanical properties are required. Furthermore, a total retention of the most volatile fission products, such as I, Xe or Cs, by the inert matrix is needed during the in-pile process. The isothermal migration of iodine in TiN was studied by implanting 800 keV I ++ ions in sintered samples at an ion fluence of 5 × 10 15 cm -2. Thermal treatments were performed under secondary vacuum at temperatures ranging from 1200 to 1700 °C. Iodine concentration profiles were determined by 2.5 MeV ?-particle elastic backscattering. The migration of iodine seems to be correlated with point defects created by implanted ions near the surface. The Arrhenius plot corresponding to iodine detrapping is curved with possibly two straight-line regions which could indicate either the presence of two types of traps, or a strong dependence of trap's concentration on temperature above 1500 °C. The activation energies associated with each linear region of the Arrhenius plot were found to be: Ea = 2.4 ± 0.2 eV below 1500 °C and E=11.4±0.2 eV above 1500 °C. Nitrogen evaporation from TiN surface under secondary vacuum was proposed as a contributing factor to the enhanced mobility of iodine at high temperature.

  1. Determining Kinetic Parameters for Isothermal Crystallization of Glasses

    NASA Technical Reports Server (NTRS)

    Ray, C. S.; Zhang, T.; Reis, S. T.; Brow, R. K.

    2006-01-01

    Non-isothermal crystallization techniques are frequently used to determine the kinetic parameters for crystallization in glasses. These techniques are experimentally simple and quick compared to the isothermal techniques. However, the analytical models used for non-isothermal data analysis, originally developed for describing isothermal transformation kinetics, are fundamentally flawed. The present paper describes a technique for determining the kinetic parameters for isothermal crystallization in glasses, which eliminates most of the common problems that generally make the studies of isothermal crystallization laborious and time consuming. In this technique, the volume fraction of glass that is crystallized as a function of time during an isothermal hold was determined using differential thermal analysis (DTA). The crystallization parameters for the lithium-disilicate (Li2O.2SiO2) model glass were first determined and compared to the same parameters determined by other techniques to establish the accuracy and usefulness of the present technique. This technique was then used to describe the crystallization kinetics of a complex Ca-Sr-Zn-silicate glass developed for sealing solid oxide fuel cells.

  2. Non isothermal drying process optimisation - Drying of clay tiles

    NASA Astrophysics Data System (ADS)

    Vasi?, M.; Radojevi?, Z.

    2015-11-01

    In our previous studies we have developed a model for determination of the variable effective diffusivity and identification of the exact transition points between possible drying mechanisms. The next goal was to develop a drying regime which could in advance characterize the real non isothermal process of drying clay tiles. In order to do this four isothermal experiments were recorded. Temperature and humidity were maintained at 350C / 75%; 450C / 70%; 450C / 60% and 500C / 60%; respectively in each experiment. All experimentally collected data were analyzed and the exact transition points between possible drying mechanisms were detected. Characteristic drying period (time) for each isothermal drying mechanism was also detected. The real, non-isothermal drying process was approximated by 5 segments. In each of these segments approximately isothermal drying condition were maintained. Temperature and humidity of the drying air, in the first four segments, was maintained on the same level as in recorded isothermal experiments while in the fifth segment, it were maintained at 700C / 40%. The duration of the first four segments were calculated from the diagrams Deff - t respectively for each experiment. The clay tile in experiment five was dried without cracking using the proposed non isothermal drying regime.

  3. Privacy Amplification in the Isolated Qubits Model

    E-print Network

    Yi-Kai Liu

    2015-02-11

    Isolated qubits are a special class of quantum devices, which can be used to implement tamper-resistant cryptographic hardware such as one-time memories (OTM's). Unfortunately, these OTM constructions leak some information, and standard methods for privacy amplification cannot be applied here, because the adversary has advance knowledge of the hash function that the honest parties will use. In this paper we show a stronger form of privacy amplification that solves this problem, using a fixed hash function that is secure against all possible adversaries in the isolated qubits model. This allows us to construct single-bit OTM's which only leak an exponentially small amount of information. We then study a natural generalization of the isolated qubits model, where the adversary is allowed to perform a polynomially-bounded number of entangling gates, in addition to unbounded local operations and classical communication (LOCC). We show that our technique for privacy amplification is also secure in this setting.

  4. Parametric Amplification of Scattered Atom Pairs

    SciTech Connect

    Campbell, Gretchen K.; Mun, Jongchul; Boyd, Micah; Streed, Erik W.; Ketterle, Wolfgang; Pritchard, David E.

    2006-01-20

    We have observed parametric generation and amplification of ultracold atom pairs. A {sup 87}Rb Bose-Einstein condensate was loaded into a one-dimensional optical lattice with quasimomentum k{sub 0} and spontaneously scattered into two final states with quasimomenta k{sub 1} and k{sub 2}. Furthermore, when a seed of atoms was first created with quasimomentum k{sub 1} we observed parametric amplification of scattered atoms pairs in states k{sub 1} and k{sub 2} when the phase-matching condition was fulfilled. This process is analogous to optical parametric generation and amplification of photons and could be used to efficiently create entangled pairs of atoms. Furthermore, these results explain the dynamic instability of condensates in moving lattices observed in recent experiments.

  5. Time varying arctic climate change amplification

    SciTech Connect

    Chylek, Petr; Dubey, Manvendra K; Lesins, Glen; Wang, Muyin

    2009-01-01

    During the past 130 years the global mean surface air temperature has risen by about 0.75 K. Due to feedbacks -- including the snow/ice albedo feedback -- the warming in the Arctic is expected to proceed at a faster rate than the global average. Climate model simulations suggest that this Arctic amplification produces warming that is two to three times larger than the global mean. Understanding the Arctic amplification is essential for projections of future Arctic climate including sea ice extent and melting of the Greenland ice sheet. We use the temperature records from the Arctic stations to show that (a) the Arctic amplification is larger at latitudes above 700 N compared to those within 64-70oN belt, and that, surprisingly; (b) the ratio of the Arctic to global rate of temperature change is not constant but varies on the decadal timescale. This time dependence will affect future projections of climate changes in the Arctic.

  6. Spin Amplification in an Inhomogeneous System

    NASA Astrophysics Data System (ADS)

    Endo, Suguru; Matsuzaki, Yuichiro; Munro, William J.; Koike, Tatsuhiko; Saito, Shiro

    2015-10-01

    A long-lived qubit is usually well-isolated from all other systems and the environments, and so is not easy to couple with measurement apparatus. It is sometimes difficult to implement reliable projective measurements on such a qubit. One potential solution is spin amplification with many ancillary qubits. Here, we propose a spin amplification technique, where the ancillary qubits state changes depending on the state of the target qubit. The technique works even for an inhomogeneous system. We show that fast and accurate amplification is possible even if the coupling and frequency of the ancillary qubits is inhomogeneous. Since our scheme is robust against realistic imperfections, this could provide a new mechanism for reading out a single spin that could not have been measured using the previous approaches.

  7. On the isothermal geometry of corrugated graphene sheets

    E-print Network

    Andrzej Trzesowski

    2014-12-22

    Variational geometries describing corrugated graphene sheets are proposed. The isothermal thermomechanical properties of these sheets are described by a 2-dimensional Weyl space. The equation that couples the Weyl geometry with isothermal distributions of the temperature of graphene sheets, is formulated. This material space is observed in a 3-dimensional orthogonal configurational point space as regular surfaces which are endowed with a thermal state vector field fulfilling the isothermal thermal state equation. It enables to introduce a non-topological dimensionless thermal shape parameter of non-developable graphene sheets. The properties of the congruence of lines generated by the thermal state vector field are discussed.

  8. Adsorption isotherm studies of methyl chloride on MgO

    SciTech Connect

    Sprung, Michael; Larese, J. Z.

    2000-05-15

    The wetting properties of methyl chloride (CH{sub 3}Cl) on MgO (100) surfaces have been investigated between 132 and 180 K using high-resolution adsorption isotherms. At low temperatures only one adsorption step is observed. At higher temperatures, i.e., T>158.9 K, a second isotherm step appears, signaling the presence of a layering transition. Unlike adsorption behavior on graphite, however, there is no evidence of a low-to-high-density transition in the monolayer phase. In addition to the layering properties, the isothermal compressibility and isosteric heat of adsorption of the adsorbed films are calculated. (c) 2000 The American Physical Society.

  9. Gas amplification in coaxial proportional counters

    NASA Astrophysics Data System (ADS)

    Miyahara, Hiroshi; Watanabe, Masatoshi; Watanabe, Tamaki

    1985-11-01

    We have measured gas amplification factors in three types of coaxial proportional counters for a 90% argon: 10% methane mixture (0.1-1.6 MPa) and a methane gas (0.1-0.5 MPa). The results showed that the formula by Diethorn or Williams and Sara was inadequate for the expression of gas amplification but that the Zastawny and Charles formulas could give a good fit for the given counter conditions. New values for constants of these formulas were determined from the experiments.

  10. Optical amplification enhancement in photonic crystals

    SciTech Connect

    Sapienza, R.; Leonetti, M.; Froufe-Perez, L. S.; Galisteo-Lopez, J. F.; Lopez, C.; Conti, C.

    2011-02-15

    Improving and controlling the efficiency of a gain medium is one of the most challenging problems of laser research. By measuring the gain length in an opal-based photonic crystal doped with laser dye, we demonstrate that optical amplification is more than twenty-fold enhanced along the {Gamma}-K symmetry directions of the face-centered-cubic photonic crystal. These results are theoretically explained by directional variations of the density of states, providing a quantitative connection between density of the states and light amplification.

  11. A dual amplification fluorescent strategy for sensitive detection of DNA methyltransferase activity based on strand displacement amplification and DNAzyme amplification.

    PubMed

    Cui, Wanling; Wang, Lei; Jiang, Wei

    2016-03-15

    DNA methyltransferase (MTase) plays a critical role in many biological processes and has been regarded as a predictive cancer biomarker and a therapeutic target in cancer treatment. Sensitive detection of DNA MTase activity is essential for early cancer diagnosis and therapeutics. Here, we developed a dual amplification fluorescent strategy for sensitive detection of DNA MTase activity based on strand displacement amplification (SDA) and DNAzyme amplification. A trifunctional double-stranded DNA (dsDNA) probe was designed including a methylation site for DNA MTase recognition, a complementary sequence of 8-17 DNAzyme for synthesizing DNAzyme, and a nicking site for nicking enzyme cleavage. Firstly, the trifunctional dsDNA probe was methylated by DNA MTase to form the methylated dsDNA. Subsequently, HpaII restriction endonuclease specifically cleaved the residue of unmethylated dsDNA. Next, under the action of polymerase and nicking enzyme, the methylared dsDNA initiated SDA, releasing numbers of 8-17 DNAzymes. Finally, the released 8-17 DNAzymes triggered DNAzyme amplification reaction to induce a significant fluorescence enhancement. This strategy could detect DNA MTase activity as low as 0.0082U/mL. Additionally, the strategy was successfully applied for evaluating the inhibitions of DNA MTase using two anticancer drugs, 5-azacytidine and 5-aza-2'-deoxycytidine. The results indicate the proposed strategy has a potential application in early cancer diagnosis and therapeutics. PMID:26492469

  12. New Insights into Chitosan-DNA Interactions Using Isothermal Titration Microcalorimetry

    E-print Network

    Buschmann, Michael

    New Insights into Chitosan-DNA Interactions Using Isothermal Titration Microcalorimetry Pei Lian Ma of deacetylation (DDA), and molecular weight (Mn) of chitosan, using isothermal titration microcalorimetry (ITC

  13. The Calculation of Adsorption Isotherms from Chromatographic Peak Shapes

    ERIC Educational Resources Information Center

    Neumann, M. G.

    1976-01-01

    Discusses the relationship between adsorption isotherms and elution peak shapes in gas chromatography, and describes a laboratory experiment which involves the adsorption of hexane, cyclohexane, and benzene on alumina at different temperatures. (MLH)

  14. Non-isothermal Crystallization Behaviors of HDPE/MWCNT Nanocomposites

    NASA Astrophysics Data System (ADS)

    Kim, Jihun; Seo, Youngwook P.; Seo, Yongsok; Hong, Soon Man

    2011-05-01

    Thermal properties and non-isothermal crystallization kinetics of polyolefin nanocomposites (high-density polyethylene/multi-walled carbon nanotubes) were characterized by differential scanning calorimetry and thermogravimetric analysis. In situ metallocence polymerization was used to prepare nanocomposites of multi-walled carbon nanotubes (MWCNTs) and high-density polyethylene (HDPE). This polymerization method consists of attaching a metallocene catalyst complex onto the surface of the MWCNTs followed by surface-initiated polymerization to generate polymer brushes on the surface. A kinetic equation for the non-isothermal crystallization was employed to analyze the crystallization characteristics of the nanocomposites. The Avramic exponent, n, can be reasonably well determined from the non-isothermal crystallization exotherm. The polarized optical microscopy showed that neat polyethylene possessed a well developed spherulite morphology: whereas, the nanocomposites displayed elongated entities that subsequently developed as bundle-like entities. Non-isothermal analysis implicitly provides clues about the morphological development history and HDPE molecular ordering around the carbon nanotubes.

  15. Isothermal Gas-liquid Flow Using the Lattice Boltzmann Method 

    E-print Network

    Kim, Donghoon

    2012-10-19

    tracking enable us to capture specific two-phase flow and do not require additional empirical closure relations. In this thesis, we simulate isothermal, two-dimensional bubble dynamics as a starting point toward direct simulation of the subcooled boiling...

  16. Isothermal model of ICF burn with finite alpha range treatment

    E-print Network

    Galloway, Conner Daniel (Conner Daniel Cross)

    2009-01-01

    A simple model for simulating deuterium tritium burn in inertial confinement fusion capsules is developed. The model, called the Isothermal Rarefaction Model, is zero dimensional (represented as ordinary differential ...

  17. Novel DNA Polymer for Amplification Pretargeting.

    PubMed

    Li, Xiao; Huang, Qingqing; Xiao, Jie; Liu, Guozheng; Dou, Shuping; Rusckowski, Mary; Shi, Hongcheng; Liu, Yuxia; Cheng, Dengfeng

    2015-09-10

    In this Letter, different from conventional pretargeting, an additional novel DNA polymer with multiple copies of a target was first designed to be administrated between the antitumor antibody, and the labeled effector served as an amplification pretargeting strategy. Two phosphorothioate DNA strands, a bridging and a target strand, were hybridized to form a polymer. Polymer size, as a function of molar ratios, was then monitored by size exclusion HPLC and electrophoretic mobility shift assay. Moreover, binding efficiency of polymers with the radiolabeled effector and polymer size after hybridization were measured by HPLC as well. As the polymer was expected to produce more binding sites that would be targeted by effectors, amplification pretargeting can greatly improve accumulation of effectors in tumor. This novel proof-of-concept was then well demonstrated by the in vitro test of signal amplification in antibody-binding protein L coated plate and LS174T cells. Compared to conventional pretargeting, significantly increasing radioactive signal was observed in this designed amplification pretargeting, which would serve as a useful paradigm of the potential of oligomer polymers to improve pretargeting and other related approaches. PMID:26396682

  18. Social amplification of risk: a conceptual framework

    SciTech Connect

    Kasperson, R.E.; Renn, O.; Slovic, P.; Brown, H.S.; Emel, J.; Goble, R.; Kasperson, J.X.; Ratick, S.

    1988-06-01

    One of the most perplexing problems in risk analysis is why some relatively minor risks or risk events, as assessed by technical experts, often elicit strong public concerns and result in substantial impacts upon society and economy. This article sets forth a conceptual framework that seeks to link systematically the technical assessment of risk with psychological, sociological, and cultural perspectives of risk perception and risk-related behavior. The main thesis is that hazards interact with psychological, social, institutional, and cultural processes in ways that may amplify or attenuate public responses to the risk or risk event. A structural description of the social amplification of risk is now possible. Amplification occurs at two stages: in the transfer of information about the risk, and in the response mechanisms of society. Signals about risk are processed by individual and social amplification stations, including the scientist who communicates the risk assessment, the news media, cultural groups, interpersonal networks, and others. Key steps of amplifications can be identified at each stage. The amplified risk leads to behavioral responses, which, in turn, result in secondary impacts. Models are presented that portray the elements and linkages in the proposed conceptual framework.

  19. Triggered amplification by hybridization chain reaction

    E-print Network

    Pierce, Niles A.

    Triggered amplification by hybridization chain reaction Robert M. Dirks and Niles A. Pierce chain reaction (HCR), in which stable DNA monomers assemble only upon exposure to a target DNA fragment termed hybridization chain reaction (HCR). This class of mech- anisms suggests the possibility

  20. Texts in Computational Complexity: Amplification of Hardness

    E-print Network

    Goldreich, Oded

    Texts in Computational Complexity: Amplification of Hardness Oded Goldreich Department of Computer a positive side, because hard problem can be ``put to work'' to our benefit, most notably in cryptography. One key issue that arises whenever one tries to utilize hard problem is bridging the gap between

  1. Hardness Amplification within NP against Deterministic Algorithms

    E-print Network

    Guruswami, Venkatesan

    Hardness Amplification within NP against Deterministic Algorithms Parikshit Gopalan Microsoft Carnegie Mellon University. Abstract We study the average-case hardness of the class NP against algorithms these classes, namely we ask: How hard are languages in NP on average for deterministic polynomial time

  2. Optical amplifier-powered quantum optical amplification

    E-print Network

    John Jeffers

    2011-05-16

    I show that an optical amplifier, when combined with photon subtraction, can be used for quantum state amplification, adding noise at a level below the standard minimum. The device could be used to significantly decrease the probability of incorrectly identifying coherent states chosen from a finite set.

  3. A versatile immobilization-free photoelectrochemical biosensor for ultrasensitive detection of cancer biomarker based on enzyme-free cascaded quadratic amplification strategy.

    PubMed

    Ge, Lei; Wang, Wenxiao; Hou, Ting; Li, Feng

    2016-03-15

    In this work, an ultrasensitive immobilization-free photoelectrochemical (PEC) biosensor was successfully developed for the first time based on a novel enzyme-free cascaded quadratic signal amplification strategy. This rationally designed homogeneous dual amplification strategy consists of a target-analog recycling circuit based on catalytic hairpin assembly (CHA) and a hybridization chain reaction (HCR) mediated amplification circuit. In the presence of carcinoembryonic antigen (CEA), a proof-of-concept target, target-analog is released to trigger the upstream CHA recycling circuit. The generated dsDNA complexes from CHA recycling could further induce the downstream HCR amplification, leading to the formation of numerous hemin/G-quadruplex DNAzymes. This would accordingly stimulate the biocatalytic precipitation of 4-chloro-1-naphthol, inducing a distinct decrease in the photocurrent signal due to the formed insoluble/insulating products on electrode surface. Under the optimal conditions, this PEC biosensor achieved ultrasensitive detection of CEA down to the atto-gram level. The introduction of this aptamer-based cascaded quadratic amplification strategy not only remarkably improves the selectivity and sensitivity of CEA assay, but also allows the ultrasensitive detection of other proteins by designing specific aptamers, providing a universal, isothermal and label-free PEC biosensing platform for ultrasensitive detection of different kinds of cancer biomarkers and holding a great potential for early-diagnosis of cancer. PMID:26409022

  4. Optimization of strand displacement amplification-sensitized G-quadruplex DNAzyme-based sensing system and its application in activity detection of uracil-DNA glycosylase.

    PubMed

    Du, Yi-Chen; Jiang, Hong-Xin; Huo, Yan-Fang; Han, Gui-Mei; Kong, De-Ming

    2016-03-15

    As an isothermal nucleic acid amplification technique, strand displacement amplification (SDA) reaction has been introduced in G-quadruplex DNAzyme-based sensing system to improve the sensing performance. To further provide useful information for the design of SDA-amplified G-quadruplex DNAzyme-based sensors, the effects of nicking site number in SDA template DNA were investigated. With the increase of the nicking site number from 1 to 2, enrichment of G-quadruplex DNAzyme by SDA is changed from a linear amplification to an exponential amplification, thus greatly increasing the amplification efficiency and subsequently improving the sensing performance of corresponding sensing system. The nicking site number cannot be further increased because more nicking sites might result in high background signals. However, we demonstrated that G-quadruplex DNAzyme enrichment efficiency could be further improved by introducing a cross-triggered SDA strategy, in which two templates each has two nicking sites are used. To validate the proposed cross-triggered SDA strategy, we used it to develop a sensing platform for the detection of uracil-DNA glycosylase (UDG) activity. The sensor enables sensitive detection of UDG activity in the range of 1×10(-4)-1U/mL with a detection limit of 1×10(-4)U/mL. PMID:26544872

  5. Observationally based assessment of polar amplification of global warming

    E-print Network

    Bhatt, Uma

    Observationally based assessment of polar amplification of global warming Igor V. Polyakov,1) are similar, and do not support the predicted polar amplification of global warming. The possible moderating amplification of global warming. Intrinsic arctic variability obscures long-term changes, limiting our ability

  6. Non-Instrumented Incubation of a Recombinase Polymerase Amplification Assay for the Rapid and Sensitive Detection of Proviral HIV-1 DNA

    PubMed Central

    Lillis, Lorraine; Lehman, Dara; Singhal, Mitra C.; Cantera, Jason; Singleton, Jered; Labarre, Paul; Toyama, Anthony; Piepenburg, Olaf; Parker, Mathew; Wood, Robert; Overbaugh, Julie; Boyle, David S.

    2014-01-01

    Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25–43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS. PMID:25264766

  7. Coherent amplification of ultrashort solitons in doped fibers.

    PubMed

    Mel'nikov, I V; Nabiev, R F; Nazarkin, A V

    1990-12-01

    We present the results of computer simulation of coherent amplification of solitons in doped fibers and explain the first stage of an evolution of such solitons by means of a perturbation theory applied to the inverse scattering problem in two limiting cases, noncoherent and pure coherent amplification. We show that it is possible to get adiabatic amplification of solitons. In the case of noncoherent amplification the amplitude of the solitons increases exponentially and the pulse duration decreases exponentially. In the opposite case of pure coherent amplification the amplitude of the solitons increases linearly with distance z, whereas the pulse duration displays a z(-1) dependence. PMID:19771086

  8. Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification.

    PubMed

    Silva, Gonçalo; Bömer, Moritz; Nkere, Chukwuemeka; Kumar, P Lava; Seal, Susan E

    2015-09-15

    Yam mosaic virus (YMV; genus Potyvirus) is considered to cause the most economically important viral disease of yams (Dioscorea spp.) in West Africa which is the dominant region for yam production globally. Yams are a vegetatively propagated crop and the use of virus-free planting material forms an essential component of disease control. Current serological and PCR-based diagnostic methods for YMV are time consuming involving a succession of target detection steps. In this study, a novel assay for specific YMV detection is described that is based on isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA). This test has been shown to be reproducible and able to detect as little as 14 pg/?l of purified RNA obtained from an YMV-infected plant, a sensitivity equivalent to that obtained with the reverse transcription-polymerase chain reaction (RT-PCR) in current general use. The RT-exoRPA assay has, however, several advantages over the RT-PCR; positive samples can be detected in less than 30 min, and amplification only requires a single incubation temperature (optimum 37°C). These features make the RT-exoRPA assay a promising candidate for adapting into a field test format to be used by yam breeding programmes or certification laboratories. PMID:26115609

  9. Field-Applicable Recombinase Polymerase Amplification Assay for Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae

    PubMed Central

    Yu, Mingyan; O'Brien, Elizabeth; Heller, Martin; Nepper, Julia F.; Weibel, Douglas B.; Gluecks, Ilona; Younan, Mario; Frey, Joachim; Falquet, Laurent; Jores, Joerg

    2015-01-01

    Contagious caprine pleuropneumonia (CCPP) is a highly contagious disease caused by Mycoplasma capricolum subsp. capripneumoniae that affects goats in Africa and Asia. Current available methods for the diagnosis of Mycoplasma infection, including cultivation, serological assays, and PCR, are time-consuming and require fully equipped stationary laboratories, which make them incompatible with testing in the resource-poor settings that are most relevant to this disease. We report a rapid, specific, and sensitive assay employing isothermal DNA amplification using recombinase polymerase amplification (RPA) for the detection of M. capricolum subsp. capripneumoniae. We developed the assay using a specific target sequence in M. capricolum subsp. capripneumoniae, as found in the genome sequence of the field strain ILRI181 and the type strain F38 and that was further evidenced in 10 field strains from different geographical regions. Detection limits corresponding to 5 × 103 and 5 × 104 cells/ml were obtained using genomic DNA and bacterial culture from M. capricolum subsp. capripneumoniae strain ILRI181, while no amplification was obtained from 71 related Mycoplasma isolates or from the Acholeplasma or the Pasteurella isolates, demonstrating a high degree of specificity. The assay produces a fluorescent signal within 15 to 20 min and worked well using pleural fluid obtained directly from CCPP-positive animals without prior DNA extraction. We demonstrate that the diagnosis of CCPP can be achieved, with a short sample preparation time and a simple read-out device that can be powered by a car battery, in <45 min in a simulated field setting. PMID:26085615

  10. Field-Applicable Recombinase Polymerase Amplification Assay for Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae.

    PubMed

    Liljander, Anne; Yu, Mingyan; O'Brien, Elizabeth; Heller, Martin; Nepper, Julia F; Weibel, Douglas B; Gluecks, Ilona; Younan, Mario; Frey, Joachim; Falquet, Laurent; Jores, Joerg

    2015-09-01

    Contagious caprine pleuropneumonia (CCPP) is a highly contagious disease caused by Mycoplasma capricolum subsp. capripneumoniae that affects goats in Africa and Asia. Current available methods for the diagnosis of Mycoplasma infection, including cultivation, serological assays, and PCR, are time-consuming and require fully equipped stationary laboratories, which make them incompatible with testing in the resource-poor settings that are most relevant to this disease. We report a rapid, specific, and sensitive assay employing isothermal DNA amplification using recombinase polymerase amplification (RPA) for the detection of M. capricolum subsp. capripneumoniae. We developed the assay using a specific target sequence in M. capricolum subsp. capripneumoniae, as found in the genome sequence of the field strain ILRI181 and the type strain F38 and that was further evidenced in 10 field strains from different geographical regions. Detection limits corresponding to 5 × 10(3) and 5 × 10(4) cells/ml were obtained using genomic DNA and bacterial culture from M. capricolum subsp. capripneumoniae strain ILRI181, while no amplification was obtained from 71 related Mycoplasma isolates or from the Acholeplasma or the Pasteurella isolates, demonstrating a high degree of specificity. The assay produces a fluorescent signal within 15 to 20 min and worked well using pleural fluid obtained directly from CCPP-positive animals without prior DNA extraction. We demonstrate that the diagnosis of CCPP can be achieved, with a short sample preparation time and a simple read-out device that can be powered by a car battery, in <45 min in a simulated field setting. PMID:26085615

  11. A Simple Mixture Theory for Isothermal and Non-isothermal Flows of n Newtonian and Generalized Newtonian Constituents including Interaction Effects

    E-print Network

    Hirst, Thomas Thayer

    2013-12-31

    Development of mathematical models based on conservation and balance laws including constitutive theories are presented for a saturated mixture of n homogeneous, isotropic, and incompressible constituents for isothermal and non-isothermal flows...

  12. Functional integral for optical parametric amplification

    E-print Network

    Fuyong Wang

    2015-02-28

    It is demonstrated that the nature of optical parametric amplification is a quantum phenomenon. The system Lagrangian can be constructed by the path integral of coherent state. The equations of motion for photon operators are indeed the Euler-Lagrange equations of a Lagrangian. The quantum state evolution equation can also be obtained without resorting to quantum Hamiltonian or Lagrangian. Starting with classical Newton equation, quantum transition amplitude of the system can be educed by surface integral.

  13. Amplification of trace amounts of nucleic acids

    DOEpatents

    Church, George M. (Brookline, MA); Zhang, Kun (Brighton, MA)

    2008-06-17

    Methods of reducing background during amplification of small amounts of nucleic acids employ careful analysis of sources of low level contamination. Ultraviolet light can be used to reduce nucleic acid contaminants in reagents and equipment. "Primer-dimer" background can be reduced by judicious design of primers. We have shown clean signal-to-noise with as little as starting material as one single human cell (.about.6 picogram), E. coli cell (.about.5 femtogram) or Prochlorococcus cell (.about.3 femtogram).

  14. Colossal magnetoelectric effect induced by parametric amplification

    NASA Astrophysics Data System (ADS)

    Wang, Yi; Onuta, Tiberiu-Dan; Long, Christian J.; Geng, Yunlong; Takeuchi, Ichiro

    2015-11-01

    We describe the use of parametric amplification to substantially increase the magnetoelectric (ME) coefficient of multiferroic cantilevers. Parametric amplification has been widely used in sensors and actuators based on optical, electronic, and mechanical resonators to increase transducer gain. In our system, a microfabricated mechanical cantilever with a magnetostrictive layer is driven at its fundamental resonance frequency by an AC magnetic field. The resulting actuation of the cantilever at the resonance frequency is detected by measuring the voltage across a piezoelectric layer in the same cantilever. Concurrently, the spring constant of the cantilever is modulated at twice the resonance frequency by applying an AC voltage across the piezoelectric layer. The spring constant modulation results in parametric amplification of the motion of the cantilever, yielding a gain in the ME coefficient. Using this method, the ME coefficient was amplified from 33 V/(cm Oe) to 2.0 MV/(cm Oe), an increase of over 4 orders of magnitude. This boost in the ME coefficient directly resulted in an enhancement of the magnetic field sensitivity of the device from 6.0 nT /?{Hz } to 1.0 nT /?{Hz } . The enhancement in the ME coefficient and magnetic field sensitivity demonstrated here may be beneficial for a variety actuators and sensors based on resonant multiferroic devices.

  15. Blocking variability: Arctic Amplification versus Arctic Oscillation

    NASA Astrophysics Data System (ADS)

    Hassanzadeh, Pedram; Kuang, Zhiming

    2015-10-01

    To predict future changes in blocking and the resulting weather extremes, some studies have proposed the negative phase of Arctic Oscillation (-AO) as an analogue for Arctic amplification because of similarities between their mean states: reduced midlatitude-to-pole temperature gradients and weakened, equatorward shifted jet streams. Using well-controlled modeling experiments, we show that blocking variations associated with mean state anomalies are opposite depending on whether these anomalies are driven by the internal dynamics as in AO or forced externally as in Arctic amplification. While blocking increases and its latitudinal-distribution shifts poleward in -AO, we find opposite responses when a mean state identical to the -AO mean state is externally forced. Findings suggest that the observed blocking-AO relationship is a correlation which does not imply that the -AO mean state causes increased blocking and should not be employed as a prototype for Arctic amplification. Furthermore, results urge for a careful consideration of causality before using internal variability to predict low-frequency response to external forcings.

  16. Electrochemical biosensing using amplification-by-polymerization.

    PubMed

    Wu, Yafeng; Liu, Songqin; He, Lin

    2009-08-15

    A novel signal amplification strategy for electrochemical detection of DNA and proteins based on the amplification-by-polymerization concept is described. Specifically, a controlled radical polymerization reaction is triggered after the capture of target molecules on the electrode surface. Growth of long chain polymeric materials provides numerous sites for subsequent aminoferrocene coupling, which in turn significantly enhances electrochemical signal output. Activators generated electron transfer for atom transfer radical polymerization (AGET ATRP) is used in this study for its high efficiency in polymer grafting and better tolerance toward oxygen in air. 2-Hydroxyethyl methacrylate (HEMA) and glycidyl methacrylate (GMA) are examined to provide excess hydroxyl or epoxy groups for aminoferrocene coupling. A limit of detection of 15 pM and 0.07 ng/mL is demonstrated for DNA and ovalbumin, respectively. More than 7-fold signal enhancement in ovalbumin detection has been achieved comparing to the unamplified method. In addition, a more than 5 orders of magnitude of dynamic range is achieved with a linear correlation coefficient (R(2)) of 0.997 for DNA, and a more than 3 orders of magnitude with R(2) of 0.999 for ovalbumin. Together, the results show that the coupling of amplification-by-polymerization concept with electrochemical detection offers great promises in providing a sensitive and cost-effective solution for biosensing applications. PMID:19583218

  17. Optically Controlled Signal Amplification for DNA Computation.

    PubMed

    Prokup, Alexander; Hemphill, James; Liu, Qingyang; Deiters, Alexander

    2015-10-16

    The hybridization chain reaction (HCR) and fuel-catalyst cycles have been applied to address the problem of signal amplification in DNA-based computation circuits. While they function efficiently, these signal amplifiers cannot be switched ON or OFF quickly and noninvasively. To overcome these limitations, a light-activated initiator strand for the HCR, which enabled fast optical OFF ? ON switching, was developed. Similarly, when a light-activated version of the catalyst strand or the inhibitor strand of a fuel-catalyst cycle was applied, the cycle could be optically switched from OFF ? ON or ON ? OFF, respectively. To move the capabilities of these devices beyond solution-based operations, the components were embedded in agarose gels. Irradiation with customizable light patterns and at different time points demonstrated both spatial and temporal control. The addition of a translator gate enabled a spatially activated signal to travel along a predefined path, akin to a chemical wire. Overall, the addition of small light-cleavable photocaging groups to DNA signal amplification circuits enabled conditional control as well as fast photocontrol of signal amplification. PMID:25621535

  18. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis.

    PubMed

    Santiago-Felipe, S; Tortajada-Genaro, L A; Puchades, R; Maquieira, A

    2014-02-01

    Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR-ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA-ELISA combination is proposed for amplification at a low, constant temperature (40°C) in a short time (40 min), for the hybridisation of labelled products to specific 5'-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA-ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA-ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings. PMID:24456598

  19. Electrical detection of nucleic acid amplification using an on-chip quasi-reference electrode and a PVC REFET.

    PubMed

    Salm, Eric; Zhong, Yu; Reddy, Bobby; Duarte-Guevara, Carlos; Swaminathan, Vikhram; Liu, Yi-Shao; Bashir, Rashid

    2014-07-15

    Electrical detection of nucleic acid amplification through pH changes associated with nucleotide addition enables miniaturization, greater portability of testing apparatus, and reduced costs. However, current ion-sensitive field effect transistor methods for sensing nucleic acid amplification rely on establishing the fluid gate potential with a bulky, difficult to microfabricate reference electrode that limits the potential for massively parallel reaction detection. Here we demonstrate a novel method of utilizing a microfabricated solid-state quasi-reference electrode (QRE) paired with a pH-insensitive reference field effect transistor (REFET) for detection of real-time pH changes. The end result is a 0.18 ?m, silicon-on-insulator, foundry-fabricated sensor that utilizes a platinum QRE to establish a pH-sensitive fluid gate potential and a PVC membrane REFET to enable pH detection of loop mediated isothermal amplification (LAMP). This technique is highly amendable to commercial scale-up, reduces the packaging and fabrication requirements for ISFET pH detection, and enables massively parallel droplet interrogation for applications, such as monitoring reaction progression in digital PCR. PMID:24940939

  20. Electrical Detection of Nucleic Acid Amplification Using an On-Chip Quasi-Reference Electrode and a PVC REFET

    PubMed Central

    2015-01-01

    Electrical detection of nucleic acid amplification through pH changes associated with nucleotide addition enables miniaturization, greater portability of testing apparatus, and reduced costs. However, current ion-sensitive field effect transistor methods for sensing nucleic acid amplification rely on establishing the fluid gate potential with a bulky, difficult to microfabricate reference electrode that limits the potential for massively parallel reaction detection. Here we demonstrate a novel method of utilizing a microfabricated solid-state quasi-reference electrode (QRE) paired with a pH-insensitive reference field effect transistor (REFET) for detection of real-time pH changes. The end result is a 0.18 ?m, silicon-on-insulator, foundry-fabricated sensor that utilizes a platinum QRE to establish a pH-sensitive fluid gate potential and a PVC membrane REFET to enable pH detection of loop mediated isothermal amplification (LAMP). This technique is highly amendable to commercial scale-up, reduces the packaging and fabrication requirements for ISFET pH detection, and enables massively parallel droplet interrogation for applications, such as monitoring reaction progression in digital PCR. PMID:24940939

  1. Critical dynamics of an isothermal compressible non-ideal fluid

    E-print Network

    Markus Gross; Fathollah Varnik

    2012-12-17

    A pure fluid at its critical point shows a dramatic slow-down in its dynamics, due to a divergence of the order-parameter susceptibility and the coefficient of heat transport. Under isothermal conditions, however, sound waves provide the only possible relaxation mechanism for order-parameter fluctuations. Here we study the critical dynamics of an isothermal, compressible non-ideal fluid via scaling arguments and computer simulations of the corresponding fluctuating hydrodynamics equations. We show that, below a critical dimension of 4, the order-parameter dynamics of an isothermal fluid effectively reduces to "model A," characterized by overdamped sound waves and a divergent bulk viscosity. In contrast, the shear viscosity remains finite above two dimensions. Possible applications of the model are discussed.

  2. Moisture adsorption isotherms and glass transition temperature of pectin.

    PubMed

    Basu, Santanu; Shivhare, U S; Muley, S

    2013-06-01

    The moisture adsorption isotherms of low methoxyl pectin were determined at 30-70°C and water activity ranging from 0.11 to 0.94. The moisture adsorption isotherms revealed that the equilibrium moisture content increased with water activity. Increase in temperature, in general, resulted in decreased equilibrium moisture content. However in some cases, equilibrium moisture content values increased with temperature at higher water activities. Selected sorption models (GAB, Halsey, Henderson, Oswin, modified Oswin) were tested for describing the adsorption isotherms. Parameters of each sorption models were determined by nonlinear regression analysis. Oswin model gave the best fit for pectin sorption behaviour. Isosteric heat of sorption decreased with increase in moisture content and varied between 14.607 and 0.552 kJ/mol. Glass transition temperature decreased with increase in moisture content of pectin. PMID:24425957

  3. Isothermal decomposition of gamma-irradiated palladium acetate

    NASA Astrophysics Data System (ADS)

    Mahfouz, R. M.; Alshehri, S. M.; Monshi, M. A. S.; Abd El-Salam, N. M.

    2004-06-01

    The isothermal decomposition of un-irradiated (pristine) and pre-gamma-irradiated palladium acetate was studied in the temperature range (498-508 K) and in air using the isothermal thermogravimetric technique. The data were analysed using various solid state reaction models. The results showed that the kinetics of isothermal decomposition of palladium acetate was governed by random nucleation reaction (Erofe'ev equation A(3)). The activation energies of the main decomposition process for un-irradiated and pre-gamma-irradiated samples were calculated. The change in texture and crystal structure of the investigated palladium acetate by gamma-irradiation was studied using electron microscopy and X-ray diffraction techniques.

  4. Setups for simultaneous measurement of isotherms and adsorption heats

    NASA Astrophysics Data System (ADS)

    Moreno, Juan Carlos; Giraldo, Liliana

    2005-05-01

    A unit for the simultaneous measurement of isotherms and heats of adsorption was designed and built, and some of the results obtained are presented here. For this purpose, a Calvet heat-conducting microcalorimeter was developed and connected to a gas volumetric unit built to record adsorption isotherms. The microcalorimeter was electrically calibrated to establish its sensitivity and reproducibility, obtaining K =15.12±0.32WV-1. Additionally, the equipment was tested using the heat of neutralization for the tris-(hydroxmethyl)-aminomethane-HCL system, obtaining ?H =-30.92±0.03kJmol-1. The unit was assembled to obtain adsorption isotherms and the corresponding heats of adsorption, from which Gibbs free energy was deduced.

  5. FGFR2 gene amplification and clinicopathological features in gastric cancer

    PubMed Central

    Matsumoto, K; Arao, T; Hamaguchi, T; Shimada, Y; Kato, K; Oda, I; Taniguchi, H; Koizumi, F; Yanagihara, K; Sasaki, H; Nishio, K; Yamada, Y

    2012-01-01

    Background: Frequency of FGFR2 amplification, its clinicopathological features, and the results of high-throughput screening assays in a large cohort of gastric clinical samples remain largely unclear. Methods: Drug sensitivity to a fibroblast growth factor receptor (FGFR) inhibitor was evaluated in vitro. The gene amplification of the FGFRs in formalin-fixed, paraffin-embedded (FFPE) gastric cancer tissues was determined by a real-time PCR-based copy number assay and fluorescence in situ hybridisation (FISH). Results: FGFR2 amplification confers hypersensitivity to FGFR inhibitor in gastric cancer cell lines. The copy number assay revealed that 4.1% (11 out of 267) of the gastric cancers harboured FGFR2 amplification. No amplification of the three other family members (FGFR1, 3 and 4) was detected. A FISH analysis was performed on 7 cases among 11 FGFR2-amplified cases and showed that 6 of these 7 cases were highly amplified, while the remaining 1 had a relatively low grade of amplification. Although the difference was not significant, patients with FGFR2 amplification tended to exhibit a shorter overall survival period. Conclusion: FGFR2 amplification was observed in 4.1% of gastric cancers and our established PCR-based copy number assay could be a powerful tool for detecting FGFR2 amplification using FFPE samples. Our results strongly encourage the development of FGFR-targeted therapy for gastric cancers with FGFR2 amplification. PMID:22240789

  6. A Simple, Inexpensive Device for Nucleic Acid Amplification without Electricity—Toward Instrument-Free Molecular Diagnostics in Low-Resource Settings

    PubMed Central

    LaBarre, Paul; Hawkins, Kenneth R.; Gerlach, Jay; Wilmoth, Jared; Beddoe, Andrew; Singleton, Jered; Boyle, David; Weigl, Bernhard

    2011-01-01

    Background Molecular assays targeted to nucleic acid (NA) markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS) where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR) instrumentation (another is sample preparation). Methodology/Principal Findings In this article, we investigate the hypothesis that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays. We assess the heater's equivalence to commercially available PCR instruments through the characterization of the temperature profiles produced, and a minimal method comparison. Versions of the prototype for several different isothermal techniques are presented. Conclusions/Significance We demonstrate that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays, and that the results of those assays are not significantly different from ones incubated in parallel in commercially available PCR instruments. These results clearly suggest the potential of the non-instrumented nucleic acid amplification (NINA) heater for molecular diagnostics in LRS. When combined with other innovations in development that eliminate power requirements for sample preparation, cold reagent storage, and readout, the NINA heater will comprise part of a kit that should enable electricity-free NA testing for many important analytes. PMID:21573065

  7. Acceptance of noise with monaural and binaural amplification.

    PubMed

    Freyaldenhoven, Melinda C; Plyler, Patrick N; Thelin, James W; Burchfield, Samuel B

    2006-10-01

    The present study investigated the effects of monaural and binaural amplification on speech understanding in noise and acceptance of noise for 39 listeners with hearing impairment. Results demonstrated that speech understanding in noise improved with binaural amplification; however, acceptance of noise was not dependent on monaural or binaural amplification for most listeners. These results suggest that although two hearing aids maximize speech understanding ability in noise, most individuals' acceptance of noise, which is directly related to hearing aid use, may not be affected by the use of binaural amplification. It should be noted that monaural amplification resulted in greater acceptance of noise for some listeners, indicating that binaural amplification may negatively affect some individuals' willingness to wear hearing aids. It should also be noted that interaural differences in acceptance of noise might exist for some listeners; therefore, if only one hearing aid is fitted, monaural ANLs should be measured. PMID:17039768

  8. Derivation of the Freundlich Adsorption Isotherm from Kinetics

    ERIC Educational Resources Information Center

    Skopp, Joseph

    2009-01-01

    The Freundlich adsorption isotherm is a useful description of adsorption phenomena. It is frequently presented as an empirical equation with little theoretical basis. In fact, a variety of derivations exist. Here a new derivation is presented using the concepts of fractal reaction kinetics. This derivation provides an alternative basis for…

  9. ORIGINAL ARTICLE Comparative genomics-guided loop-mediated isothermal

    E-print Network

    Hsiang, Tom

    Pseudomonas syringae pathovars and other Pseudomonas spe- cies, as well as other plant pathogenic bacteria, e and plant resources. One new tech- nology that is particularly interesting is loop-mediated isothermal pathogenicity-related secondary metabolites in the PSP genome was targeted for developing a LAMP assay

  10. Friction factor for isothermal and nonisothermal flow through porous media

    NASA Technical Reports Server (NTRS)

    Koh, J. C.; Dutton, J. L.; Benson, B. A.; Fortini, A.

    1977-01-01

    Measurements were performed to determine the pressure drops for gaseous flow through porous materials of different microstructures, porosities, and thickness under isothermal and nonisothermal conditions at various temperature levels. Results were satisfactorily correlated by a simple equation relating the friction factor to the Reynolds number and porosities.

  11. Dynamics of non-isothermal martensitic phase transitions and hysteresis

    E-print Network

    Vainchtein, Anna

    ­fast interface motion and damped temporal oscillations in both released heat and end load. We show that at higher. Two dissipation mechanisms are considered: heat conduction and the internal viscous dissipation regime to accommodate large deformations (up to 8% strain) during isothermal mechanical loading

  12. Kink Wave Propagation in Thin Isothermal Magnetic Flux Tubes

    NASA Astrophysics Data System (ADS)

    Lopin, I. P.; Nagorny, I. G.; Nippolainen, E.

    2014-08-01

    We investigated the propagation of kink waves in thin and isothermal expanding flux tubes in cylindrical geometry. By using the method of radial expansion for fluctuating variables we obtained a new kink wave equation. We show that including the radial component of the tube magnetic field leads to cutoff-free propagation of kink waves along thin flux tubes.

  13. Finite Mass Isothermal Spheres and the Structure of Globular Clusters

    E-print Network

    Jes Madsen

    1996-01-23

    Utilizing a recently derived extension of the Maxwell-Boltzmann distribution to low occupation numbers (Simons 1994) this investigation discusses the structure of stellar dynamical isothermal spheres. The resulting models, which resemble King models, constitute a new sequence of physically well-motivated spherical equilibrium configurations of finite mass, and give nice fits to pre-core collapse globular cluster data.

  14. Quantitative Detection of Hepatitis B Virus DNA by Real-Time Nucleic Acid Sequence-Based Amplification with Molecular Beacon Detection

    PubMed Central

    Yates, Sol; Penning, Maarten; Goudsmit, Jaap; Frantzen, Inge; van de Weijer, Bert; van Strijp, Dianne; van Gemen, Bob

    2001-01-01

    We have developed a hepatitis B virus (HBV) DNA detection and quantification system based on amplification with nucleic acid sequence-based amplification (NASBA) technology and real-time detection with molecular beacon technology. NASBA is normally applied to amplify single-stranded target RNA, producing RNA amplicons. In this work we show that with modifications like primer design, sample extraction method, and template denaturation, the NASBA technique can be made suitable for DNA target amplification resulting in RNA amplicons. A major advantage of our assay is the one-tube, isothermal nature of the method, which allows high-throughput applications for nucleic acid detection. The homogeneous real-time detection allows a closed-tube format of the assay, avoiding any postamplification handling of amplified material and therefore minimizing the risk of contamination of subsequent reactions. The assay has a detection range of 103 to 109 HBV DNA copies/ml of plasma or serum (6 logs), with good reproducibility and precision. Compared with other HBV DNA assays, our assay provides good sensitivity, a wide dynamic range, and high-throughput applicability, making it a viable alternative to those based on other amplification or detection methods. PMID:11574587

  15. Recombinase Polymerase Amplification (RPA) of CaMV-35S Promoter and nos Terminator for Rapid Detection of Genetically Modified Crops

    PubMed Central

    Xu, Chao; Li, Liang; Jin, Wujun; Wan, Yusong

    2014-01-01

    Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that enables the amplification of DNA within 30 min at a constant temperature of 37–42 °C by simulating in vivo DNA recombination. In this study, based on the regulatory sequence of the cauliflower mosaic virus 35S (CaMV-35S) promoter and the Agrobacterium tumefaciens nopaline synthase gene (nos) terminator, which are widely incorporated in genetically modified (GM) crops, we designed two sets of RPA primers and established a real-time RPA detection method for GM crop screening and detection. This method could reliably detect as few as 100 copies of the target molecule in a sample within 15–25 min. Furthermore, the real-time RPA detection method was successfully used to amplify and detect DNA from samples of four major GM crops (maize, rice, cotton, and soybean). With this novel amplification method, the test time was significantly shortened and the reaction process was simplified; thus, this method represents an effective approach to the rapid detection of GM crops. PMID:25310647

  16. Amplification sans bruit d'images optiques

    NASA Astrophysics Data System (ADS)

    Gigan, S.; Delaubert, V.; Lopez, L.; Treps, N.; Maitre, A.; Fabre, C.

    2004-11-01

    Nous utilisons un Oscillateur Paramétrique Optique (OPO) pompé sous le seuil dans le but d'amplifier une image multimode transverse sans dégradation du rapport signal à bruit. Le dispositif expérimental met en œuvre un OPO de type II triplement résonant et semi-confocal pour le faisceau amplifié. L'existence d'effets quantiques lors de l'amplification multimode dans un tel dispositif a été montrée expérimentalement. Plus généralement, ceci nous a amené à étudier les propriétés quantiques transverses des faisceaux lumineux amplifiés. Une telle étude peut trouver des applications non seulement en imagerie, mais également dans le traitement quantique de l'information.

  17. Mechanical entanglement via detuned parametric amplification

    NASA Astrophysics Data System (ADS)

    Szorkovszky, A.; Clerk, A. A.; Doherty, A. C.; Bowen, W. P.

    2014-06-01

    We propose two schemes to generate entanglement between a pair of mechanical oscillators using parametric amplification. In contrast to existing parametric drive-based protocols, both schemes operate in the steady-state. Using a detuned parametric drive to maintain equilibrium and to couple orthogonal quadratures, our approach can be viewed as a two-mode extension of previous proposals for parametric squeezing. We find that robust steady-state entanglement is possible for matched oscillators with well-controlled coupling. In addition, one of the proposed schemes is robust to differences in the damping rates of the two oscillators.

  18. Chirped Pulse Amplification of Femtosecond Optical Pulses

    NASA Astrophysics Data System (ADS)

    Pessot, Maurice Alfonso

    Chirped Pulse Amplification (CPA) has been instrumental in pushing forward the state of the art in ultrashort pulse amplification. As conceived however, limitations in the methods used for pulse manipulation restrict its utility to regimes in which pulse compression techniques can provide high compression ratios, limiting its use to long pulse (>=q50 psec) oscillators and compressed pulses >=q1 psec. Significantly, this also prevents its use with ultrashort sources where further compression of the pulse is not desired. In this thesis, we develop and demonstrate new methods for optical pulse manipulation enabling us to extend CPA techniques into the femtosecond regime. A generalized diffraction grating pair is shown to be a fully reversible means of expanding femtosecond pulses while providing sufficient positive group velocity dispersion to expand femtosecond pulses by factors >10^3 . A CPA system utilizing these techniques is used for the amplification of 275 fsec pulses from a modelocked dye oscillator. The 275 fsec pulses are expanded to 50 psec and amplified in a multipass regenerative amplifier utilizing the tunable solid-state material alexandrite as the gain medium. The 3 mJ pulses are then compressed to 300 fsec. An analysis of the dispersion properties of the system is shown to lead to limitations in the pulsewidth obtainable from such a system. The presence of dispersive components within the resonator cavity forces the expansion/compression system to be used in a mismatched geometry. The resulting contributions to the cubic phase shift from diffraction gratings and material elements limits the system to pulses of the order of 200 fsec. For amplification and compression of shorter pulses, simultaneous compensation of quadratic and cubic phase shifts becomes necessary. A number of methods for full and partial compensation of cubic phase shifts are examined and one method, based upon a combination of intracavity prisms and external diffraction gratings is implemented. With this and other modifications we show that bandwidths sufficient to support pulses as short as 60 fsec can be amplified to the mJ level. Partial compensation of cubic phase shifts is demonstrated, resulting in pulses of 106 fsec duration with peak powers of nearly 20 GW.

  19. Amplification of curvature perturbations in cyclic cosmology

    SciTech Connect

    Zhang Jun; Liu Zhiguo; Piao Yunsong

    2010-12-15

    We analytically and numerically show that through the cycles with nonsingular bounce, the amplitude of curvature perturbation on a large scale will be amplified and the power spectrum will redden. In some sense, this amplification will eventually destroy the homogeneity of the background, which will lead to the ultimate end of cycles of the global universe. We argue that for the model with increasing cycles, it might be possible that a fissiparous multiverse will emerge after one or several cycles, in which the cycles will continue only at corresponding local regions.

  20. Internal entanglement amplification by external interactions

    SciTech Connect

    Peskin, Uri; Huang Zhen; Kais, Sabre

    2007-07-15

    We propose a scheme to control the level of entanglement between two fixed spin-1/2 systems by interaction with a third particle. For specific designs, entanglement is shown to be 'pumped' into the system from the surroundings even when the spin-spin interaction within the system is small or nonexistent. The effect of the external particle on the system is introduced by including a dynamic spinor in the Hamiltonian. Controlled amplification of the internal entanglement to its maximum value is demonstrated. The possibility of entangling noninteracting spins in a stationary state is also demonstrated by coupling each one of them to a flying qubit in a quantum wire.

  1. Parametric amplification by coupled flux qubits

    SciTech Connect

    Rehák, M.; Neilinger, P.; Grajcar, M.; Oelsner, G.; Hübner, U.; Meyer, H.-G.; Il'ichev, E.

    2014-04-21

    We report parametric amplification of a microwave signal in a Kerr medium formed from superconducting qubits. Two mutually coupled flux qubits, embedded in the current antinode of a superconducting coplanar waveguide resonator, are used as a nonlinear element. Shared Josephson junctions provide the qubit-resonator coupling, resulting in a device with a tunable Kerr constant (up to 3?×?10{sup ?3}) and a measured gain of about 20?dB. This arrangement represents a unit cell which can be straightforwardly extended to a quasi one-dimensional quantum metamaterial with large tunable Kerr nonlinearity, providing a basis for implementation of wide-band travelling wave parametric amplifiers.

  2. Raman Amplification in Plasma: Thermal Effects

    SciTech Connect

    Farmer, John; Ersfeld, Bernhard; Jaroszynski, Dino

    2009-01-22

    The impact of thermal effects on Raman amplification in plasma is investigated theoretically. It is shown that damping and the shift in plasma resonance at finite temperature can alter the evolution of the amplified pulse and lead to pulse compression which is not predicted by the cold plasma model. Although thermal effects can lead to a reduction in the efficiency of the interaction, this can be ameliorated by using a chirped pump. In this case thermal effects can be beneficial and suppress the development of the train of pulses that develops behind the amplified pulse, as observed in the cold plasma model.

  3. Direct amplification of casework bloodstains using the Promega PowerPlex(®) 21 PCR amplification system.

    PubMed

    Gray, Kerryn; Crowle, Damian; Scott, Pam

    2014-09-01

    A significant number of evidence items submitted to Forensic Science Service Tasmania (FSST) are blood swabs or bloodstained items. Samples from these items routinely undergo phenol:chloroform:isoamyl alcohol organic extraction and quantitative Polymerase Chain Reaction (qPCR) testing prior to PowerPlex(®) 21 amplification. This multi-step process has significant cost and timeframe implications in a fiscal climate of tightening government budgets, pressure towards improved operating efficiencies, and an increasing emphasis on rapid techniques better supporting intelligence-led policing. Direct amplification of blood and buccal cells on cloth and Whatman FTA™ card with PowerPlex(®) 21 has already been successfully implemented for reference samples, eliminating the requirement for sample pre-treatment. Scope for expanding this method to include less pristine casework blood swabs and samples from bloodstained items was explored in an endeavour to eliminate lengthy DNA extraction, purification and qPCR steps for a wider subset of samples. Blood was deposited onto a range of substrates including those historically found to inhibit STR amplification. Samples were collected with micro-punch, micro-swab, or both. The potential for further fiscal savings via reduced volume amplifications was assessed by amplifying all samples at full and reduced volume (25 and 13?L). Overall success rate data showed 80% of samples yielded a complete profile at reduced volume, compared to 78% at full volume. Particularly high success rates were observed for the blood on fabric/textile category with 100% of micro-punch samples yielding complete profiles at reduced volume and 85% at full volume. Following the success of this trial, direct amplification of suitable casework blood samples has been implemented at reduced volume. Significant benefits have been experienced, most noticeably where results from crucial items have been provided to police investigators prior to interview of suspects, and a coronial identification has been successfully completed in a short timeframe to avoid delay in the release of human remains to family members. PMID:24905337

  4. Induction of gene amplification in Plasmodium falciparum

    SciTech Connect

    Rogers, P.L.

    1985-01-01

    Human erythrocytic in vitro cultures of Honduras I strain of the malaria parasite Plasmodium falciparum have been stressed stepwise with increasing concentrations of methotrexate (MTX), a folate antagonist. This selection has produced a strain that is 450 times more resistant to the drug than the original culture. Uptake of sublethal doses of radiolabeled MTX by infected red blood cells was 6-36 times greater in the resistant cultures than in the nonresistant controls. DNA isolated from all of the parasites was probed by hybridization with /sup 35/S-labeled DNA derived from a clone of the yeast thymidylate synthetase (TS) gene. This showed 50 to 100 times more increased hybridization of the TS probe to the DNA from the resistant parasites is direct evidence of gene amplification because DHFR and TS are actually one and the same bifunctional enzyme in P. falciparum. Hence, the evidence presented indicates that induced resistance of the malaria parasite to MTX in this case is due to overproduction of DHFR resulting from amplification of the DHFR-TS gene.

  5. Thermal amplification of field-correlation harvesting

    NASA Astrophysics Data System (ADS)

    Brown, Eric G.

    2013-12-01

    We study the harvesting of quantum and classical correlations from a hot scalar field in a periodic cavity by a pair of spatially separated oscillator-detectors. Specifically, we utilize nonperturbative and exact (non-numerical) techniques to solve for the evolution of the detectors-field system and then we examine how the entanglement, Gaussian quantum discord, and mutual information obtained by the detectors change with the temperature of the field. While (as expected) the harvested entanglement rapidly decays to zero as temperature is increased, we find remarkably that both the mutual information and the discord can actually be increased by multiple orders of magnitude via increasing the temperature. We go on to explain this phenomenon by a variety of means and are able to make accurate predictions of the behavior of thermal amplification. By doing this we also introduce a new perspective on harvesting in general and illustrate that the system can be represented as two dynamically decoupled systems, each with only a single detector. The thermal amplification of discord harvesting represents an exciting prospect for discord-based quantum computation, including its use in entanglement activation.

  6. Ultra-broadband amplification through nanotechnology

    NASA Astrophysics Data System (ADS)

    DiMaio, Jeffrey R.; Kokuoz, Baris; Ballato, John

    2006-10-01

    As demands for bandwidth continue to increase, telecommunication networks would greatly benefit from the development of broader-band amplifiers. The currently erbium doped fiber amplifiers are limited to amplification of approximately 100 nm bandwidth window. One method to increase the bandwidth of the fiber amplifier would be to incorporate multiple rare earths (REs) into a single fiber which exhibit emissions from ~1000-1800 nm. Unfortunately, energy transfer between rare earth ions typically results in quenching all but selected emissions negating this approach to potential ultra-broadband amplification. It would be ideal if one could take the individual spectra of an ion and place that ion into a host with no regard to other lanthanides that also are present in the host. This problem can be solved by using a composite material that utilizes nanoparticles to constrain different REs to individual particles thereby controlling or preventing energy transfer. In order to control energy transfer, RE doped LaF 3 nanocrystals were grown in an aqueous solution using a core/shell technique to constrain different rare earth into separate particles or shells within a single particle. Using these techniques, we show that energy transfer can be controlled.

  7. Weak value amplification of atomic cat states

    NASA Astrophysics Data System (ADS)

    Huang, Sumei; Agarwal, Girish S.

    2015-09-01

    We show the utility of the weak value amplification to observe the quantum interference between two close lying atomic coherent states in a post-selected atomic cat state, produced in a system of N identical two-level atoms weakly interacting with a single photon field. Through the observation of the negative parts of the Wigner distribution of the post-selected atomic cat state, we find that the post-selected atomic cat state becomes more nonclassical when the post-selected polarization state of the single photon field tends toward becoming orthogonal to its pre-selected state. We show that the small phase shift in the post-selected atomic cat state can be amplified via measuring the peak shift of its phase distribution when the post-selected state of the single photon field is nearly orthogonal to its pre-selected state. We find that the amplification factor of 15 [5] can be obtained for a sample of 10 [100] atoms. This effectively provides us with a method to discriminate between two close lying states on the Bloch sphere. We discuss possible experimental implementation of the scheme, and conclude with a discussion of the Fisher information.

  8. Space Optical Communications Using Laser Beam Amplification

    NASA Technical Reports Server (NTRS)

    Agrawal, Govind

    2015-01-01

    The Space Optical Communications Using Laser Beam Amplification (SOCLBA) project will provide a capability to amplify a laser beam that is received in a modulating retro-reflector (MRR) located in a satellite in low Earth orbit. It will also improve the pointing procedure between Earth and spacecraft terminals. The technology uses laser arrays to strengthen the reflected laser beam from the spacecraft. The results of first year's work (2014) show amplification factors of 60 times the power of the signal beam. MMRs are mirrors that reflect light beams back to the source. In space optical communications, a high-powered laser interrogator beam is directed from the ground to a satellite. Within the satellite, the beam is redirected back to ground using the MMR. In the MMR, the beam passes through modulators, which encode a data signal onto the returning beam. MMRs can be used in small spacecraft for optical communications. The SOCLBA project is significant to NASA and small spacecraft due to its application to CubeSats for optical data transmission to ground stations, as well as possible application to spacecraft for optical data transmission.

  9. Boosting riboswitch efficiency by RNA amplification

    PubMed Central

    Emadpour, Masoumeh; Karcher, Daniel; Bock, Ralph

    2015-01-01

    Riboswitches are RNA sensors that regulate gene expression in response to binding of small molecules. Although they conceptually represent simple on/off switches and, therefore, hold great promise for biotechnology and future synthetic biology applications, the induction of gene expression by natural riboswitches after ligand addition or removal is often only moderate and, consequently, the achievable expression levels are not very high. Here, we have designed an RNA amplification-based system that strongly improves the efficiency of riboswitches. We have successfully implemented the method in a biological system for which currently no efficient endogenous tools for inducible (trans)gene expression are available: the chloroplasts of higher plants. We further show that an HIV antigen whose constitutive expression from the chloroplast genome is deleterious to the plant can be inducibly expressed under the control of the RNA amplification-enhanced riboswitch (RAmpER) without causing a mutant phenotype, demonstrating the potential of the method for the production of proteins and metabolites that are toxic to the host cell. PMID:25824954

  10. Weak value amplification of atomic cat states

    E-print Network

    Sumei Huang; Girish S. Agarwal

    2015-09-09

    We show the utility of the weak value amplification to observe the quantum interference between two close lying atomic coherent states in a post-selected atomic cat state, produced in a system of $N$ identical two-level atoms weakly interacting with a single photon field. Through the observation of the negative parts of the Wigner distribution of the post-selected atomic cat state, we find that the post-selected atomic cat state becomes more nonclassical when the post-selected polarization state of the single photon field tends toward becoming orthogonal to its pre-selected state. We show that the small phase shift in the post-selected atomic cat state can be amplified via measuring the peak shift of its phase distribution when the post-selected state of the single photon field is nearly orthogonal to its pre-selected state. We find that the amplification factor of 15 [5] can be obtained for a sample of 10 [100] atoms. This effectively provides us with a method to discriminate two close lying states on the Bloch sphere. We discuss possible experimental implementation of the scheme, and conclude with a discussion of the Fisher information.

  11. A PARAMETER STUDY FOR BAROCLINIC VORTEX AMPLIFICATION

    SciTech Connect

    Raettig, Natalie; Klahr, Hubert; Lyra, Wladimir E-mail: klahr@mpia.de

    2013-03-10

    Recent studies have shown that baroclinic vortex amplification is strongly dependent on certain factors, namely, the global entropy gradient, the efficiency of thermal diffusion and/or relaxation as well as numerical resolution. We conduct a comprehensive study of a broad range and combination of various entropy gradients, thermal diffusion and thermal relaxation timescales via local shearing sheet simulations covering the parameter space relevant for protoplanetary disks. We measure the Reynolds stresses as a function of our control parameters and see that there is angular momentum transport even for entropy gradients as low as {beta} = -dln s/dln r = 1/2. Values we expect in protoplanetary disks are between {beta} = 0.5-2.0 The amplification-rate of the perturbations, {Gamma}, appears to be proportional to {beta}{sup 2} and thus proportional to the square of the Brunt-Vaeisaelae frequency ({Gamma}{proportional_to}{beta}{sup 2}{proportional_to}N {sup 2}). The saturation level of Reynolds stresses, on the other hand, seems to be proportional to {beta}{sup 1/2}. This highlights the importance of baroclinic effects even for the low entropy gradients expected in protoplanetary disks.

  12. Effects of branching characteristics and copolymer composition distribution on non-isothermal crystallization

    E-print Network

    Hussein, Ibnelwaleed A.

    ; Copolymer composition distribution (CCD); Non-isothermal crystallization; MDSC 1. IntroductionEffects of branching characteristics and copolymer composition distribution on non-isothermal crystallization kinetics of metallocene LLDPEs Mohammad Ashraful Islam a , Ibnelwaleed A. Hussein a,*, Muhammad

  13. Amplification of seismic waves by the Seattle basin, Washington State

    USGS Publications Warehouse

    Pratt, T.L.; Brocher, T.M.; Weaver, C.S.; Creager, K.C.; Snelson, C.M.; Crosson, R.S.; Miller, K.C.; Trehu, A.M.

    2003-01-01

    Recordings of the 1999 Mw 7.6 Chi-Chi (Taiwan) earthquake, two local earthquakes, and five blasts show seismic-wave amplification over a large sedimentary basin in the U.S. Pacific Northwest. For weak ground motions from the Chi-Chi earthquake, the Seattle basin amplified 0.2- to 0.8-Hz waves by factors of 8 to 16 relative to bedrock sites west of the basin. The amplification and peak frequency change during the Chi-Chi coda: the initial S-wave arrivals (0-30 sec) had maximum amplifications of 12 at 0.5-0.8 Hz, whereas later arrivals (35-65 sec) reached amplifications of 16 at 0.3-0.5 Hz. Analysis of local events in the 1.0- to 10.0-Hz frequency range show fourfold amplifications for 1.0-Hz weak ground motion over the Seattle basin. Amplifications decrease as frequencies increase above 1.0 Hz, with frequencies above 7 Hz showing lower amplitudes over the basin than at bedrock sites. Modeling shows that resonance in low-impedance deposits forming the upper 550 m of the basin beneath our profile could cause most of the observed amplification, and the larger amplification at later arrival times suggests surface waves also play a substantial role. These results emphasize the importance of shallow deposits in determining ground motions over large basins.

  14. Nucleic Acid Amplification Testing in Suspected Child Sexual Abuse

    ERIC Educational Resources Information Center

    Esernio-Jenssen, Debra; Barnes, Marilyn

    2011-01-01

    The American Academy of Pediatrics recommends that site-specific cultures be obtained, when indicated, for sexually victimized children. Nucleic acid amplification testing is a highly sensitive and specific methodology for identifying sexually transmitted infections. Nucleic acid amplification tests are also less invasive than culture, and this…

  15. Counterexamples to Hardness Amplification Beyond Negligible Yevgeniy Dodis

    E-print Network

    International Association for Cryptologic Research (IACR)

    Counterexamples to Hardness Amplification Beyond Negligible Yevgeniy Dodis Abhishek Jain Tal Moran Daniel Wichs§ January 19, 2012 Abstract If we have a problem that is mildly hard, can we create a problem that is significantly harder? A natural approach to hardness amplification is the "direct product"; instead of asking

  16. HARDNESS AMPLIFICATION VIA SPACE-EFFICIENT DIRECT PRODUCTS

    E-print Network

    Guruswami, Venkatesan

    HARDNESS AMPLIFICATION VIA SPACE-EFFICIENT DIRECT PRODUCTS Venkatesan Guruswami and Valentine this construction, we get a deterministic linear-space "worst-case to con- stant average-case" hardness- panders (Dinur 2006). Keywords. Direct products, hardness amplification, error-correcting codes, expanders

  17. Explanatory Model for Sound Amplification in a Stethoscope

    ERIC Educational Resources Information Center

    Eshach, H.; Volfson, A.

    2015-01-01

    In the present paper we suggest an original physical explanatory model that explains the mechanism of the sound amplification process in a stethoscope. We discuss the amplification of a single pulse, a continuous wave of certain frequency, and finally we address the resonant frequencies. It is our belief that this model may provide students with…

  18. Research paper Fast cochlear amplification with slow outer hair cells

    E-print Network

    Sarpeshkar, Rahul

    Research paper Fast cochlear amplification with slow outer hair cells Timothy K. Lu a,b , Serhii hair cells (OHCs) produce mechanical amplification over the entire audio-frequency range (up to 100 k reserved. Keywords: Cochlear amplifier; Outer hair cell; Receptor potential; Membrane time constant low

  19. 78 FR 66940 - Regulatory Requirements for Hearing Aid Devices and Personal Sound Amplification Products; Draft...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-07

    ...Requirements for Hearing Aid Devices and Personal Sound Amplification Products; Draft Guidance...Requirements for Hearing Aid Devices and Personal Sound Amplification Products.'' This draft...distinction between hearing aids and personal sound amplification products (PSAPs), as...

  20. Core and filament formation in magnetized, self-gravitating isothermal layers

    SciTech Connect

    Van Loo, Sven; Keto, Eric; Zhang, Qizhou

    2014-07-01

    We examine the role of the gravitational instability in an isothermal, self-gravitating layer threaded by magnetic fields on the formation of filaments and dense cores. Using a numerical simulation, we follow the non-linear evolution of a perturbed equilibrium layer. The linear evolution of such a layer is described in the analytic work of Nagai et al. We find that filaments and dense cores form simultaneously. Depending on the initial magnetic field, the resulting filaments form either a spiderweb-like network (for weak magnetic fields) or a network of parallel filaments aligned perpendicular to the magnetic field lines (for strong magnetic fields). Although the filaments are radially collapsing, the density profile of their central region (up to the thermal scale height) can be approximated by a hydrodynamical equilibrium density structure. Thus, the magnetic field does not play a significant role in setting the density distribution of the filaments. The density distribution outside of the central region deviates from the equilibrium. The radial column density distribution is then flatter than the expected power law of r {sup –4} and similar to filament profiles observed with Herschel. Our results do not explain the near constant filament width of ?0.1pc. However, our model does not include turbulent motions. It is expected that the accretion-driven amplification of these turbulent motions provides additional support within the filaments against gravitational collapse. Finally, we interpret the filamentary network of the massive star forming complex G14.225-0.506 in terms of the gravitational instability model and find that the properties of the complex are consistent with being formed out of an unstable layer threaded by a strong, parallel magnetic field.

  1. Gibbs Adsorption Isotherm Combined with Monte Carlo Sampling to See Action of Cosolutes on Protein Folding

    E-print Network

    Harries, Daniel

    Gibbs Adsorption Isotherm Combined with Monte Carlo Sampling to See Action of Cosolutes on Protein of simulation allow only fixed numbers of these solutes. By combining the analytic Gibbs adsorption isotherm the analytic expressions for the adsorption isotherm31 to evaluate the free energy change from cosolute

  2. A modified Langmuir-Freundlich isotherm model for simulating pH-dependent adsorption effects

    E-print Network

    Clement, Prabhakar

    , these isotherms are primarily useful for simulating data collected at a fixed pH value and cannot be easily isotherm uses a linear correlation between pH and affinity coefficient values. We validat- ed the proposedA modified Langmuir-Freundlich isotherm model for simulating pH-dependent adsorption effects

  3. Monitoring of an RNA Multistep Folding Pathway by Isothermal Titration Calorimetry

    E-print Network

    Perreault, Jean-Pierre

    Monitoring of an RNA Multistep Folding Pathway by Isothermal Titration Calorimetry Ce´dric Reymond Sherbrooke, Sherbrooke, Que´bec, Canada ABSTRACT Isothermal titration calorimetry was used to monitor.1 pseudoknot is the limiting step of the molecular mechanism. Last, as illustrated here, isothermal titration

  4. Signal Amplification of Bioassay Using Zinc Nanomaterials

    NASA Astrophysics Data System (ADS)

    Cowles, Chad L.

    An emerging trend in the analytical detection sciences is the employment of nanomaterials for bioassay signal transduction to identify analytes critical to public health. These nanomaterials have been specifically investigated for applications which require identification of trace levels of cells, proteins, or other molecules that can have broad ranging impacts to human health in fields such as clinical diagnostics, environmental monitoring, food and drink control, and the prevention of bioterrorism. Oftentimes these nanoparticle-based signal transduction or amplification approaches offer distinct advantages over conventional methods such as increased sensitivity, rapidity, or stability. The biological application of nanoparticles however, does suffer from drawbacks that have limited more widespread adoption of these techniques. Some of these drawbacks are, high cost and toxicity, arduous synthesis methods, functionalization and bioconjugation challenges, and laboratory disposal and environmental hazard issues, all of which have impeded the progression of this technology in some way or another. This work aims at developing novel techniques that offer solutions to a number of these hurdles through the development of new nanoparticle-based signal transduction approaches and the description of a previously undescribed nanomaterial. Zinc-based nanomaterials offer the opportunity to overcome some of the limitations that are encountered when other nanomaterials are employed for bioassay signal transduction. On the other hand, the biological application of zinc nanomaterials has been difficult because in general their fluorescence is in the blue range and the reported quantum yields are usually too low for highly sensitive applications. The advantages of using zinc nanomaterials for biological applications, such as reduced toxicity, simple synthesis, low cost, and straightforward functionalization strategies contribute to the research interest in their application as bioassay signal tranducers. To overcome the limitations associated with zinc-based nanomaterials, a novel signal transduction approach was developed that relies on zinc ion release from nanoparticle labels during an immunoassay. The development of an innovative method for zinc ion detection and the description of a previously undescribed zinc-based nanomaterial are also described in this work. There are three major contributions to science in this work: (1) The development of an original and innovative signal transduction approach for immunoassays that adopts fluorescence detection of zinc ions released from ZnS nanoparticle labels; (2) The discovery and development of dual signal amplification for immunoassay signal transduction using ion release and subsequent activation of a zinc dependent metallozyme; (3) The synthesis and characterization of a novel zinc-based nanomaterial and its biosensing application using both single and dual signal amplification strategies.

  5. Solid-state Raman image amplification

    NASA Astrophysics Data System (ADS)

    Calmes, Lonnie Kirkland

    Amplification of low-light-level optical images is important for extending the range of lidar systems that image and detect objects in the atmosphere and underwater. The use of range-gating to produce images of particular range bins is also important in minimizing the image degradation due to light that is scattered backward from aerosols, smoke, or water along the imaging path. For practical lidar systems that must be operated within sight of unprotected observers, eye safety is of the utmost importance. This dissertation describes a new type of eye-safe, range-gated lidar sensing element based on Solid-state Raman Image Amplification (SSRIA) in a solid- state optical crystal. SSRIA can amplify low-level images in the eye-safe infrared at 1.556 ?m with gains up to 106 with the addition of only quantum- limited noise. The high gains from SSRIA can compensate for low quantum efficiency detectors and can reduce the need for detector cooling. The range-gate of SSRIA is controlled by the pulsewidth of the pump laser and can be as short as 30-100 cm, using pump pulses of 2-6.7 nsec FWHM. A rate equation theoretical model is derived to help in the design of short pulsed Raman lasers. A theoretical model for the quantum noise properties of SSRIA is presented. SSRIA results in higher SNR images throughout a broad range of incident light levels, in contrast to the increasing noise factor with reduced gain in image intensified CCD's. A theoretical framework for the optical resolution of SSRIA is presented and it is shown that SSRIA can produce higher resolution than ICCD's. SSRIA is also superior in rejecting unwanted sunlight background, further increasing image SNR. Lastly, SSRIA can be combined with optical pre-filtering to perform optical image processing functions such as high-pass filtering and automatic target detection/recognition. The application of this technology to underwater imaging, called Marine Raman Image Amplification (MARIA) is also discussed. MARIA operates at a wavelength of 563 nm, which passes most efficiently through coastal ocean waters. The imaging resolution of MARIA in the marine environment can be superior to images produced by laser line scan or standard range-gated imaging systems.

  6. Isothermal dehydration of thin films of water and sugar solutions

    NASA Astrophysics Data System (ADS)

    Heyd, R.; Rampino, A.; Bellich, B.; Elisei, E.; Cesàro, A.; Saboungi, M.-L.

    2014-03-01

    The process of quasi-isothermal dehydration of thin films of pure water and aqueous sugar solutions is investigated with a dual experimental and theoretical approach. A nanoporous paper disk with a homogeneous internal structure was used as a substrate. This experimental set-up makes it possible to gather thermodynamic data under well-defined conditions, develop a numerical model, and extract needed information about the dehydration process, in particular the water activity. It is found that the temperature evolution of the pure water film is not strictly isothermal during the drying process, possibly due to the influence of water diffusion through the cellulose web of the substrate. The role of sugar is clearly detectable and its influence on the dehydration process can be identified. At the end of the drying process, trehalose molecules slow down the diffusion of water molecules through the substrate in a more pronounced way than do the glucose molecules.

  7. Isothermal dehydration of thin films of water and sugar solutions

    SciTech Connect

    Heyd, R.; Rampino, A.; Laboratory of Physical and Macromolecular Chemistry, University of Trieste, Via Giorgieri 1, 34127 Trieste ; Bellich, B.; Elisei, E.; Cesàro, A.; Elettra Sincrotrone Trieste, Area Science Park, I-34149 Trieste ; Saboungi, M.-L.; Institut de Minéralogie, de Physique des Matériaux, et de Cosmochimie , Sorbonne Univ-UPMC, Univ Paris 06, UMR CNRS 7590, Museum National d’Histoire Naturelle, IRD UMR 206, 4 Place Jussieu, F-75005 Paris

    2014-03-28

    The process of quasi-isothermal dehydration of thin films of pure water and aqueous sugar solutions is investigated with a dual experimental and theoretical approach. A nanoporous paper disk with a homogeneous internal structure was used as a substrate. This experimental set-up makes it possible to gather thermodynamic data under well-defined conditions, develop a numerical model, and extract needed information about the dehydration process, in particular the water activity. It is found that the temperature evolution of the pure water film is not strictly isothermal during the drying process, possibly due to the influence of water diffusion through the cellulose web of the substrate. The role of sugar is clearly detectable and its influence on the dehydration process can be identified. At the end of the drying process, trehalose molecules slow down the diffusion of water molecules through the substrate in a more pronounced way than do the glucose molecules.

  8. Isothermal measurement and thermal desorption using SAW devices

    NASA Astrophysics Data System (ADS)

    Martin, S. J.; Ricco, A. J.; Zipperian, T. E.

    The conventional implementation of surface acoustic wave (SAW) devices is departed from as gas sensors to describe how they may be used to measure adsorption isotherms and to perform thermal desorption spectroscopy. Adsorption isotherms are measurements, made at constant temperature, of surface coverage as a function of gas or vapor partial pressure for the species of interest. It was shown that by incorporating a heater as an integral part of a SAW device, a sensor can be constructed which is capable of measuring thermal desorption spectra. A greater spread in the temperatures at which molecules are desorbed, and thus greater discrimination between species, could be obtained by using coatings which form stronger chemical bonds rather than relying on physisorption.

  9. Chip Scale Package Integrity Assessment by Isothermal Aging

    NASA Technical Reports Server (NTRS)

    Ghaffarian, Reza

    1998-01-01

    Many aspects of chip scale package (CSP) technology, with focus on assembly reliability characteristics, are being investigated by the JPL-led consortia. Three types of test vehicles were considered for evaluation and currently two configurations have been built to optimize attachment processes. These test vehicles use numerous package types. To understand potential failure mechanisms of the packages, particularly solder ball attachment, the grid CSPs were subjected to environmental exposure. Package I/Os ranged from 40 to nearly 300. This paper presents both as assembled, up to 1, 000 hours of isothermal aging shear test results and photo micrographs, and tensile test results before and after 1,500 cycles in the range of -30/100 C for CSPs. Results will be compared to BGAs with the same the same isothermal aging environmental exposures.

  10. Non-isothermal buckling behavior of viscoplastic shell structures

    NASA Technical Reports Server (NTRS)

    Riff, Richard; Simitses, G. J.

    1988-01-01

    Described are the mathematical model and solution methodologies for analyzing the structural response of thin, metallic elasto-viscoplastic shell structures under large thermomechanical loads and their non-isothermal buckling behavior. Among the system responses associated with these loads and conditions are snap-through, buckling, thermal buckling, and creep buckling. This geometric and material nonlinearities (of high order) can be anticipated and are considered in the model and the numerical treatment.

  11. Multi-stage, isothermal CO preferential oxidation reactor

    DOEpatents

    Skala, Glenn William (Churchville, NY); Brundage, Mark A. (Pittsford, NY); Borup, Rodney Lynn (East Rochester, NY); Pettit, William Henry (Rochester, NY); Stukey, Kevin (W. Henrietta, NY); Hart-Predmore, David James (Rochester, NY); Fairchok, Joel (Alexander, NY)

    2000-01-01

    A multi-stage, isothermal, carbon monoxide preferential oxidation (PrOx) reactor comprising a plurality of serially arranged, catalyzed heat exchangers, each separated from the next by a mixing chamber for homogenizing the gases exiting one heat exchanger and entering the next. In a preferred embodiment, at least some of the air used in the PrOx reaction is injected directly into the mixing chamber between the catalyzed heat exchangers.

  12. Photochromism-induced amplification of critical current density in superconducting boron-doped diamond with an azobenzene molecular layer.

    PubMed

    Natsui, Keisuke; Yamamoto, Takashi; Akahori, Miku; Einaga, Yasuaki

    2015-01-14

    A key issue in molecular electronics is the control of electronic states by optical stimuli, which enables fast and high-density data storage and temporal-spatial control over molecular processes. In this article, we report preparation of a photoswitchable superconductor using a heavily boron-doped diamond (BDD) with a photochromic azobenzene (AZ) molecular layer. BDDs electrode properties allow for electrochemical immobilization, followed by copper(I)-catalyzed alkyne-azide cycloaddition (a "click" reaction). Superconducting properties were examined with magnetic and electrical transport measurements, such as field-dependent isothermal magnetization, temperature-dependent resistance, and the low-temperature voltage-current response. These measurements revealed reversible amplification of the critical current density by 55% upon photoisomerization. This effect is explained as the reversible photoisomerization of AZ inducing an inhomogeneous electron distribution along the BDD surface that renormalizes the surface pinning contribution to the critical current. PMID:25494096

  13. Dispersion compensation in chirped pulse amplification systems

    DOEpatents

    Bayramian, Andrew James; Molander, William A.

    2014-07-15

    A chirped pulse amplification system includes a laser source providing an input laser pulse along an optical path. The input laser pulse is characterized by a first temporal duration. The system also includes a multi-pass pulse stretcher disposed along the optical path. The multi-pass pulse stretcher includes a first set of mirrors operable to receive input light in a first plane and output light in a second plane parallel to the first plane and a first diffraction grating. The pulse stretcher also includes a second set of mirrors operable to receive light diffracted from the first diffraction grating and a second diffraction grating. The pulse stretcher further includes a reflective element operable to reflect light diffracted from the second diffraction grating. The system further includes an amplifier, a pulse compressor, and a passive dispersion compensator disposed along the optical path.

  14. Strengthening weak value amplification with recycled photons

    E-print Network

    Dressel, Justin; Jordan, Andrew N; Graham, Trent M; Kwiat, Paul G

    2013-01-01

    We consider the use of cyclic weak measurements to improve the sensitivity of weak-value amplification precision measurement schemes. Previous weak-value experiments have used only a small fraction of events, while discarding the rest through the process of "post-selection". We extend this idea by considering recycling of events which are typically unused in a weak measurement. Here we treat a sequence of polarized laser pulses effectively trapped inside an interferometer using a Pockels cell and polarization optics. In principle, all photons can be post-selected, which will improve the measurement sensitivity. We first provide a qualitative argument for the expected improvements from recycling photons, followed by the exact result for the recycling of collimated beam pulses, and numerical calculations for diverging beams. We show that beam degradation effects can be mitigated via profile flipping or Zeno reshaping. The main advantage of such a recycling scheme is an effective power increase, while maintainin...

  15. Strengthening weak value amplification with recycled photons

    E-print Network

    Justin Dressel; Kevin Lyons; Andrew N. Jordan; Trent M. Graham; Paul G. Kwiat

    2013-05-20

    We consider the use of cyclic weak measurements to improve the sensitivity of weak-value amplification precision measurement schemes. Previous weak-value experiments have used only a small fraction of events, while discarding the rest through the process of "post-selection". We extend this idea by considering recycling of events which are typically unused in a weak measurement. Here we treat a sequence of polarized laser pulses effectively trapped inside an interferometer using a Pockels cell and polarization optics. In principle, all photons can be post-selected, which will improve the measurement sensitivity. We first provide a qualitative argument for the expected improvements from recycling photons, followed by the exact result for the recycling of collimated beam pulses, and numerical calculations for diverging beams. We show that beam degradation effects can be mitigated via profile flipping or Zeno reshaping. The main advantage of such a recycling scheme is an effective power increase, while maintaining an amplified deflection.

  16. Optimizing biased semiconductor superlattices for terahertz amplification

    SciTech Connect

    Lei, Xiaoli; Wang, Dawei; Wu, Zhaoxin; Dignam, M. M.

    2014-08-11

    Over the past 15 yr or more, researchers have been trying to achieve gain for electromagnetic fields in the terahertz frequency region using biased semiconductor superlattices, but with little success. In this work, we employ our model of the excitonic states in biased GaAs/Al{sub 0.3}Ga{sub 0.7}As semiconductor superlattices to find the optimal structures for amplification of terahertz radiation. In particular, we determine the optimum well width, barrier width, and bias field for terahertz fields with frequencies ranging from 1 to 4 terahertz. We find that gain coefficients on the order of 40?cm{sup ?1} should be achievable over most of this frequency range.

  17. Superparamagnetic Nanoparticle Capture of Prions for Amplification?

    PubMed Central

    Miller, Michael B.; Supattapone, Surachai

    2011-01-01

    Prion diseases are associated with the presence of PrPSc, a disease-associated misfolded conformer of the prion protein. We report that superparamagnetic nanoparticles bind PrPSc molecules efficiently and specifically, permitting magnetic separation of prions from a sample mixture. Captured PrPSc molecules retain the activity to seed protein misfolding cyclic amplification (PMCA) reactions, enabling the rapid concentration of dilute prions to improve detection. Furthermore, superparamagnetic nanoparticles clear contaminated solutions of PrPSc. Our findings suggest that coupling magnetic nanoparticle capture with PMCA could accelerate and improve prion detection. Magnetic nanoparticles may also be useful for developing a nontoxic prion decontamination method for biologically derived products. PMID:21228242

  18. Large parametric amplification in an optomechanical system

    NASA Astrophysics Data System (ADS)

    Buters, F. M.; Eerkens, H. J.; Heeck, K.; Weaver, M. J.; Pepper, B.; Sonin, P.; de Man, S.; Bouwmeester, D.

    2015-10-01

    In a Fabry-Perot cavity optomechanical system the mechanical mode can be excited by placing the laser at the blue sideband of the cavity resonance. As soon as the optical driving force overcomes the mechanical losses, self-induced oscillations start to occur. When trying to achieve large parametric amplification in most experiments these self-induced oscillations cross over into chaotic motion due to secondary effects such as heating of the mirror by optical absorption. Here we demonstrate an optomechanical system in which the amplitude of mechanical mode can be greatly amplified due to the use of a low-absorption Bragg mirror on the mechanical oscillator. By scanning the laser across the optical cavity resonance and analyzing the optical output, we demonstrate that we can amplify the mechanical mode to an amplitude 450 times larger than the thermal amplitude at 300 K, without any appearance of unstable or chaotic motion.

  19. Ghost imaging via optical parametric amplification

    NASA Astrophysics Data System (ADS)

    Li, Hong-Guo; Zhang, De-Jian; Xu, De-Qin; Zhao, Qiu-Li; Wang, Sen; Wang, Hai-Bo; Xiong, Jun; Wang, Kaige

    2015-10-01

    We investigate theoretically and experimentally thermal light ghost imaging where the light transmitted through the object as the seed light is amplified by an optical parametric amplifier (OPA). In conventional lens imaging systems with OPA, the spectral bandwidth of OPA dominates the image resolution. Theoretically, we prove that in ghost imaging via optical parametric amplification (GIOPA) the bandwidth of OPA will not affect the image resolution. The experimental results show that for weak seed light the image quality in GIOPA is better than that of conventional ghost imaging. Our work may be valuable in remote sensing with ghost imaging technique, where the light passed through the object is weak after a long-distance propagation.

  20. Cascade DNA nanomachine and exponential amplification biosensing.

    PubMed

    Xu, Jianguo; Wu, Zai-Sheng; Shen, Weiyu; Xu, Huo; Li, Hongling; Jia, Lee

    2015-11-15

    DNA is a versatile scaffold for the assembly of multifunctional nanostructures, and potential applications of various DNA nanodevices have been recently demonstrated for disease diagnosis and treatment. In the current study, a powerful cascade DNA nanomachine was developed that can execute the exponential amplification of p53 tumor suppressor gene. During the operation of the newly-proposed DNA nanomachine, dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) was ingeniously introduced, where the target trigger is repeatedly used as the fuel molecule and the nicked fragments are dramatically accumulated. Moreover, each displaced nicked fragment is able to activate the another type of cyclical strand-displacement amplification, increasing exponentially the value of fluorescence intensity. Essentially, one target binding event can induce considerable number of subsequent reactions, and the nanodevice was called cascade DNA nanomachine. It can implement several functions, including recognition element, signaling probe, polymerization primer and template. Using the developed autonomous operation of DNA nanomachine, the p53 gene can be quantified in the wide concentration range from 0.05 to 150 nM with the detection limit of 50 pM. If taking into account the final volume of mixture, the detection limit is calculated as lower as 6.2 pM, achieving an desirable assay ability. More strikingly, the mutant gene can be easily distinguished from the wild-type one. The proof-of-concept demonstrations reported herein is expected to promote the development and application of DNA nanomachine, showing great potential value in basic biology and medical diagnosis. PMID:26042874

  1. Simple, rapid and accurate molecular diagnosis of acute promyelocytic leukemia by loop mediated amplification technology.

    PubMed

    Spinelli, Orietta; Rambaldi, Alessandro; Rigo, Francesca; Zanghì, Pamela; D'Agostini, Elena; Amicarelli, Giulia; Colotta, Francesco; Divona, Mariadomenica; Ciardi, Claudia; Coco, Francesco Lo; Minnucci, Giulia

    2015-01-01

    The diagnostic work-up of acute promyelocytic leukemia (APL) includes the cytogenetic demonstration of the t(15;17) translocation and/or the PML-RARA chimeric transcript by RQ-PCR or RT-PCR. This latter assays provide suitable results in 3-6 hours. We describe here two new, rapid and specific assays that detect PML-RARA transcripts, based on the RT-QLAMP (Reverse Transcription-Quenching Loop-mediated Isothermal Amplification) technology in which RNA retrotranscription and cDNA amplification are carried out in a single tube with one enzyme at one temperature, in fluorescence and real time format. A single tube triplex assay detects bcr1 and bcr3 PML-RARA transcripts along with GUS housekeeping gene. A single tube duplex assay detects bcr2 and GUSB. In 73 APL cases, these assays detected in 16 minutes bcr1, bcr2 and bcr3 transcripts. All 81 non-APL samples were negative by RT-QLAMP for chimeric transcripts whereas GUSB was detectable. In 11 APL patients in which RT-PCR yielded equivocal breakpoint type results, RT-QLAMP assays unequivocally and accurately defined the breakpoint type (as confirmed by sequencing). Furthermore, RT-QLAMP could amplify two bcr2 transcripts with particularly extended PML exon 6 deletions not amplified by RQ-PCR. RT-QLAMP reproducible sensitivity is 10(-3) for bcr1 and bcr3 and 10(-)2 for bcr2 thus making this assay particularly attractive at diagnosis and leaving RQ-PCR for the molecular monitoring of minimal residual disease during the follow up. In conclusion, PML-RARA RT-QLAMP compared to RT-PCR or RQ-PCR is a valid improvement to perform rapid, simple and accurate molecular diagnosis of APL. PMID:25815362

  2. Simple, rapid and accurate molecular diagnosis of acute promyelocytic leukemia by loop mediated amplification technology

    PubMed Central

    Spinelli, Orietta; Rambaldi, Alessandro; Rigo, Francesca; Zanghì, Pamela; D'Agostini, Elena; Amicarelli, Giulia; Colotta, Francesco; Divona, Mariadomenica; Ciardi, Claudia; Coco, Francesco Lo; Minnucci, Giulia

    2015-01-01

    The diagnostic work-up of acute promyelocytic leukemia (APL) includes the cytogenetic demonstration of the t(15;17) translocation and/or the PML-RARA chimeric transcript by RQ-PCR or RT-PCR. This latter assays provide suitable results in 3-6 hours. We describe here two new, rapid and specific assays that detect PML-RARA transcripts, based on the RT-QLAMP (Reverse Transcription-Quenching Loop-mediated Isothermal Amplification) technology in which RNA retrotranscription and cDNA amplification are carried out in a single tube with one enzyme at one temperature, in fluorescence and real time format. A single tube triplex assay detects bcr1 and bcr3 PML-RARA transcripts along with GUS housekeeping gene. A single tube duplex assay detects bcr2 and GUSB. In 73 APL cases, these assays detected in 16 minutes bcr1, bcr2 and bcr3 transcripts. All 81 non-APL samples were negative by RT-QLAMP for chimeric transcripts whereas GUSB was detectable. In 11 APL patients in which RT-PCR yielded equivocal breakpoint type results, RT-QLAMP assays unequivocally and accurately defined the breakpoint type (as confirmed by sequencing). Furthermore, RT-QLAMP could amplify two bcr2 transcripts with particularly extended PML exon 6 deletions not amplified by RQ-PCR. RT-QLAMP reproducible sensitivity is 10?3 for bcr1 and bcr3 and 10?2 for bcr2 thus making this assay particularly attractive at diagnosis and leaving RQ-PCR for the molecular monitoring of minimal residual disease during the follow up. In conclusion, PML-RARA RT-QLAMP compared to RT-PCR or RQ-PCR is a valid improvement to perform rapid, simple and accurate molecular diagnosis of APL. PMID:25815362

  3. Short communication Demonstration of rapid multiplex PCR amplification involving 16 genetic loci

    E-print Network

    amplification using the polymerase chain reaction (PCR) copies these STR regions to detectable levels and labels DNA extraction, quantification, polymerase chain reaction (PCR) amplification of multiple short tandem

  4. Determination of solubility coefficients and sorption isotherms of gases in polymers by means of isothermal desorption with a chromatographic detection

    NASA Astrophysics Data System (ADS)

    Nizhegorodova, Yu. A.; Belov, N. A.; Berezkin, V. G.; Yampol'skii, Yu. P.

    2015-03-01

    A new method is developed for determining the solubility coefficients of gases in polymers that combines the advantages of the static and dynamic approaches to sorption estimation and allows us to determine the equilibrium characteristics of sorption for small quantities of samples (0.1-0.2 g) and low (<0.5 atm) partial pressures of the investigated gas. Sorption isotherms and solubility coefficients of nitrogen, oxygen, carbon dioxide, methane, ethane, and propane in polyvinyltrimethylsilane are obtained, and in poly[3,4-bis(trimethylsilyl)-tricyclononene-7], polyhexafluoropropylene, and OH-containing polyimide for the first time ever. It is shown that the sorption isotherms of gases for all of the gas-polymer systems in the investigated range of pressures are linear. The obtained solubility coefficients are compared to data for other polymers studied earlier.

  5. Isothermal amplified detection of ATP using Au nanocages capped with a DNA molecular gate and its application in cell lysates.

    PubMed

    Wang, Wei; Zhao, Na; Li, Xiaoxiao; Wan, Jun; Luo, Xiliang

    2015-03-01

    A novel controlled-release biosensor for isothermal amplified detection of ATP using Au nanocages (AuNCs) capped with a DNA molecular gate is reported for the first time, and has been successfully tested in intracellular ATP detection. Two kinds of SH-modified short strand DNAs S1 and S2 were assembled on the surface of the AuNCs by means of Au-thiolate bonding. The hybridization of a long-strand DNA S3 with the two immobilized SH-DNAs leads to the formation of molecular gates. The molecular gates were designed to inhibit the release of the fluorescent molecules such as Rhodamine-B (RhB), which were filled in the hollow interiors of AuNCs. The primer S4 was employed to play the role of a recognition moiety. The specificity recognition reaction between ATP and ATP aptamer gave rise to the primer S4 released from a double-stranded hybrid formed with the ATP aptamer. The released S4 will initiate the autonomous replication-scission-displacement process with the assistance of DNA polymerase and nicking endonuclease. As a result, the DNA synthesis and the DNA cycle achieved the opening of the DNA-based molecular gates and the significant amplification of the release of the guest molecules from AuNCs. In order to realize the cyclic enzymatic amplification of the release of the guest molecules from AuNCs, the long-strand S3 is ingeniously designed in such a way that it contains a Nb.Bpu10I nicking endonuclease recognition sequence and a sequence complementary to the primer S4. The fabricated system was demonstrated to be an efficient biosensor for target molecule detection qualitatively and quantitatively. PMID:25627025

  6. Real-time microfluidic recombinase polymerase amplification for the toxin B gene of Clostridium difficile on a SlipChip platform.

    PubMed

    Tsaloglou, M-N; Watson, R J; Rushworth, C M; Zhao, Y; Niu, X; Sutton, J M; Morgan, H

    2015-01-01

    Clostridium difficile is one of the key bacterial pathogens that cause infectious diarrhoea both in the developed and developing world. Isothermal nucleic acid amplification methods are increasingly used for identification of toxinogenic infection by clinical labs. For this purpose, we developed a low-cost microfluidic platform based on the SlipChip concept and implemented real-time isothermal recombinase polymerase amplification (RPA). The on-chip RPA assay targets the Clostridium difficile toxin B gene (tcdB) coding for toxin B, one of the proteins responsible for bacterial toxicity. The device was fabricated in clear acrylic using rapid prototyping methods. It has six replicate 500 nL reaction wells as well as two sets of 500 nL control wells. The reaction can be monitored in real-time using exonuclease fluorescent probes with an initial sample volume of as little as 6.4 ?L. We demonstrated a limit of detection of 1000 DNA copies, corresponding to 1 fg, at a time-to-result of <20 minutes. This miniaturised platform for pathogen detection has potential for use in resource-limited environments or at the point-of-care because of its ease of use and low cost, particularly if combined with preserved reagents. PMID:25371968

  7. HER-2 amplification is highly homogenous in gastric cancer.

    PubMed

    Marx, Andreas H; Tharun, Lars; Muth, Johanna; Dancau, Ana-Maria; Simon, Ronald; Yekebas, Emre; Kaifi, Jussuf T; Mirlacher, Martina; Brümmendorf, Tim H; Bokemeyer, Carsten; Izbicki, Jakob R; Sauter, Guido

    2009-06-01

    Her-2 is the molecular target for antibody-based treatment of breast cancer (trastuzumab). The potential benefit of anti-Her-2 therapy is currently investigated in several other HER-2-amplified cancers including gastric cancer. Although HER-2 amplification occurs in more than 10% of gastric cancers, potential heterogeneity of HER-2 amplification and overexpression could represent a major drawback for anti-Her-2 therapy. To address the potential applicability of trastuzumab in gastric cancer, tissue microarray sections of 166 gastric adenocarcinomas and 69 lymph node metastases were analyzed for Her-2 overexpression and amplification using Food and Drug Administration-approved reagents for immunohistochemistry and fluorescence in situ hybridization. HER-2 amplification was seen in 27 (16%) of 166 gastric adenocarcinomas. Amplification was typically high level with more than 20 HER-2 copies per tumor cell and a HER-2/centromere 17 ratio >3. Amplification was associated with intestinal tumor phenotype but unrelated to survival, grading, pT, pN, or pM. Identical HER-2 status was found in primary tumor and their matched lymph node metastases. Moreover, HER-2 and Topoisomerase IIalpha coamplification analysis of 3 to 16 large sections from 8 Her-2-positive gastric cancers did not reveal any heterogeneity of the amplicon site. The high level of HER-2 amplification in combination with the homogeneity of its expression in primary and metastatic tumors argues for a possible therapeutic utility of trastuzumab in HER-2-amplified gastric adenocarcinomas. PMID:19269014

  8. Analysis of gene amplification in human tumor cell lines

    SciTech Connect

    Fukumoto, M.; Shevrin, D.H.; Roninson, I.B.

    1988-09-01

    Oncogene amplification has been observed in various primary tumors and tumor-derived cell lines. In several types of cancer, amplification of specific oncogenes is correlated with the stage of tumor progression. To estimate the frequency of gene amplification in other tumor types and to determine whether the ability to grow in vivo is associated with gene amplification in tumor cell lines, we have developed a modified version of the in-gel renaturation assay that detects human DNA sequences of unknown nature amplified as little as 7- to 8-fold. This assay was used to screen 16 cell lines derived from various solid tumors and leukemias. Amplified DNA sequences were detected in only one cell line, Calu-3 lung adenocarcinoma. This cell line was found to contain coamplified NGL (formerly termed neu) and ERBA1 oncogenes. However, when one of the amplification-negative cell lines, PC-3 prostatic carcinoma, was selected for in vivo growth in nude mice, amplified DNA sequences became detectable in these cells. The amplified sequences included the MYC oncogene, which showed no amplification in the parental cell line but was amplified 10- to 12-fold in the in vivo-selected cells. MYC amplification may, therefore, provide tumor cells with a selective advantage specific for in vivo growth.

  9. Mechanism of gene amplification via yeast autonomously replicating sequences.

    PubMed

    Sehgal, Shelly; Kaul, Sanjana; Dhar, M K

    2015-01-01

    The present investigation was aimed at understanding the molecular mechanism of gene amplification. Interplay of fragile sites in promoting gene amplification was also elucidated. The amplification promoting sequences were chosen from the Saccharomyces cerevisiae ARS, 5S rRNA regions of Plantago ovata and P. lagopus, proposed sites of replication pausing at Ste20 gene locus of S. cerevisiae, and the bend DNA sequences within fragile site FRA11A in humans. The gene amplification assays showed that plasmid bearing APS from yeast and human beings led to enhanced protein concentration as compared to the wild type. Both the in silico and in vitro analyses were pointed out at the strong bending potential of these APS. In addition, high mitotic stability and presence of TTTT repeats and SAR amongst these sequences encourage gene amplification. Phylogenetic analysis of S. cerevisiae ARS was also conducted. The combinatorial power of different aspects of APS analyzed in the present investigation was harnessed to reach a consensus about the factors which stimulate gene expression, in presence of these sequences. It was concluded that the mechanism of gene amplification was that AT rich tracts present in fragile sites of yeast serve as binding sites for MAR/SAR and DNA unwinding elements. The DNA protein interactions necessary for ORC activation are facilitated by DNA bending. These specific bindings at ORC promote repeated rounds of DNA replication leading to gene amplification. PMID:25685838

  10. Amplification and chromosomal dispersion of human endogenous retroviral sequences

    SciTech Connect

    Steele, P.E.; Martin, M.A.; Rabson, A.B.; Bryan, T.; O'Brien, S.J.

    1986-09-01

    Endogenous retroviral sequences have undergone amplification events involving both viral and flanking cellular sequences. The authors cloned members of an amplified family of full-length endogenous retroviral sequences. Genomic blotting, employing a flanking cellular DNA probe derived from a member of this family, revealed a similar array of reactive bands in both humans and chimpanzees, indicating that an amplification event involving retroviral and associated cellular DNA sequences occurred before the evolutionary separation of these two primates. Southern analyses of restricted somatic cell hybrid DNA preparations suggested that endogenous retroviral segments are widely dispersed in the human genome and that amplification and dispersion events may be linked.

  11. Preparation of DNA-containing extract for PCR amplification

    DOEpatents

    Dunbar, John M.; Kuske, Cheryl R.

    2006-07-11

    Environmental samples typically include impurities that interfere with PCR amplification and DNA quantitation. Samples of soil, river water, and aerosol were taken from the environment and added to an aqueous buffer (with or without detergent). Cells from the sample are lysed, releasing their DNA into the buffer. After removing insoluble cell components, the remaining soluble DNA-containing extract is treated with N-phenacylthiazolium bromide, which causes rapid precipitation of impurities. Centrifugation provides a supernatant that can be used or diluted for PCR amplification of DNA, or further purified. The method may provide a DNA-containing extract sufficiently pure for PCR amplification within 5–10 minutes.

  12. Label-free electrochemical nucleic acid biosensing by tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme amplification.

    PubMed

    Liu, Shufeng; Gong, Hongwei; Wang, Yanqun; Wang, Li

    2016-03-15

    Owing to the intrinsic importance of nucleic acid as bio-targets, the achievement of its simple and sensitive detection with high confidence is very essential for biological studies and diagnostic purposes. Herein, a label-free, isothermal, and ultrasensitive electrochemical detection of target DNA was developed by using a tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme releasing amplification strategy. Upon sensing of the nucleic acid analyte for the assembled hairpin-like probe DNA on the electrode, the DNA polymerase guided the target recycling and simultaneously triggered the lambda exonuclease cleavage, accompanied by the cascade recycling of the released new complementary strand and the amplified liberation of the G-rich sequence of the HRP-mimicking DNAzyme. The electrocatalytic reduction of H2O2 by the generated hemin/G-quadruplex DNAzyme was used for the signal readout and further amplification toward target response. Such tandem functional operation by DNA polymerase, lambda exonuclease and DNAzyme endows the developed biosensor with a high sensitivity and also a high confidence. A low detection limit of 5fM with an excellent selectivity toward target DNA could be achieved. It also exhibits the distinct advantages of simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus may offer a promising avenue for the applications in disease diagnosis and clinical biomedicine. PMID:26513289

  13. Gap formation and stability in non-isothermal protoplanetary discs

    NASA Astrophysics Data System (ADS)

    Les, Robert; Lin, Min-Kai

    2015-06-01

    Several observations of transition discs show lopsided dust distributions. A potential explanation is the formation of a large-scale vortex acting as a dust-trap at the edge of a gap opened by a giant planet. Numerical models of gap-edge vortices have so far employed locally isothermal discs in which the temperature profile is held fixed, but the theory of this vortex-forming or `Rossby wave' instability was originally developed for adiabatic discs. We generalize the study of planetary gap stability to non-isothermal discs using customized numerical simulations of disc-planet systems where the planet opens an unstable gap. We include in the energy equation a simple cooling function with cooling time-scale t_c=? ? _k^{-1}, where ?k is the Keplerian frequency, and examine the effect of ? on the stability of gap edges and vortex lifetimes. We find increasing ? lowers the growth rate of non-axisymmetric perturbations, and the dominant azimuthal wavenumber m decreases. We find a quasi-steady state consisting of one large-scale, overdense vortex circulating the outer gap edge, typically lasting O(103) orbits. We find vortex lifetimes generally increase with the cooling time-scale tc up to an optimal value of tc ˜ 10 orbits, beyond which vortex lifetimes decrease. This non-monotonic dependence is qualitatively consistent with recent studies using strictly isothermal discs that vary the disc aspect ratio. The lifetime and observability of gap-edge vortices in protoplanetary discs is therefore dependent on disc thermodynamics.

  14. Isothermal elastohydrodynamic lubrication of point contacts. 1: Theoretical formulation

    NASA Technical Reports Server (NTRS)

    Hamrock, B. J.; Dowson, D.

    1975-01-01

    The isothermal elastohydrodynamic lubrication (EHL) of a point contact was analyzed numerically by simultaneously solving the elasticity and Reynolds equations. In the elasticity analysis the contact zone was divided into equal rectangular areas, and it was assumed that a uniform pressure was applied over each area. In the numerical analysis of the Reynolds equation, a phi analysis (where phi is equal to the pressure times the film thickness of the 3/2 power) was used to help the relaxation process. The EHL point contact analysis is applicable for the entire range of elliptical parameters and is valid for any combination of rolling and sliding within the contact.

  15. Giant planet formation. A first classification of isothermal protoplanetary equilibria

    NASA Astrophysics Data System (ADS)

    Pe?nik, B.; Wuchterl, G.

    2005-09-01

    We present a model for the equilibrium of solid planetary cores embedded in a gaseous nebula. From this model we are able to extract an idealized roadmap of all hydrostatic states of the isothermal protoplanets. The complete classification of the isothermal protoplanetary equilibria should improve the understanding of the general problem of giant planet formation, within the framework of the nucleated instability hypothesis. We approximate the protoplanet as a spherically symmetric, isothermal, self-gravitating classical ideal gas envelope in equilibrium, around a rigid body of given mass and density, with the gaseous envelope required to fill the Hill-sphere. Starting only with a core of given mass and an envelope gas density at the core surface, the equilibria are calculated without prescribing the total protoplanetary mass or nebula density. In this way, a variety of hydrostatic core-envelope equilibria has been obtained. Two types of envelope equilibria can be distinguished: uniform equilibrium, were the density of the envelope gas drops approximately an order of magnitude as the radial distance increases to the outer boundary, and compact equilibrium, having a small but very dense gas layer wrapped around the core and very low, exponentially decreasing gas density further out. The effect of the envelope mass on the planetary gravitational potential further discriminates the models into the self-gravitating and the non-self gravitating ones. The static critical core masses of the protoplanets for the typical orbits of 1, 5.2, and 30 AU, around a parent star of 1 solar mass ({M_?}) are found to be 0.1524, 0.0948, and 0.0335 Earth masses ({M_oplus}), respectively, for standard nebula conditions (Kusaka et al. 1970). These values are much lower than currently admitted ones primarily because our model is isothermal and the envelope is in thermal equilibrium with the nebula. Our solutions show a wide range of possible envelopes. For a given core, multiple solutions (at least two) are found to fit into the same nebula. Some of those solutions posses equal envelope mass. This variety is a consequence of the envelope's self-gravity. We extend the concept of the static critical core mass to the local and global critical core mass. Above the global critical mass, only compact solutions exist. We conclude that the “global static critical core mass” marks the meeting point of all four qualitatively different envelope regions.

  16. Wave Properties of Isothermal Magneto-Rotational Fluids

    E-print Network

    M. Sharif; Umber Sheikh

    2009-08-28

    In this paper, the isothermal plasma wave properties in the neighborhood of the pair production region for the Kerr black hole magnetosphere are discussed. We have considered the Fourier analyzed form of the perturbed general relativistic magnetohydrodynamical equations whose determinant leads to a dispersion relation. For the special scenario, the $x$-component of the complex wave vectors are numerically calculated. Respective components of the propagation vector, attenuation vector, phase and group velocities are shown in graphs. We have particularly investigated the existence of a Veselago medium and wave behavior (modes of waves dispersion

  17. Macroscopic Velocity Amplification in Stacked Disks

    NASA Astrophysics Data System (ADS)

    Murthy, Srividya; White, Gary

    2015-04-01

    When a small sphere rests atop a larger sphere (for example, a basketball with a tennis ball balanced on top), and both are released from a height, the resulting ``velocity amplification'' of the small sphere when the pair rebound from a hard floor, is a staple of the physics demonstration toolkit--usually impressive, sometimes dangerous. While this phenomenon has been studied in the literature in some detail, we set out to explore this effect by constructing a device involving stacked disks falling in a plane, fashioned after an online design by Wayne Peterson of Brigham Young University. When two disks, stacked edge to edge atop one another and confined to a vertical plane, are dropped, the top disk rebounds to a much greater height than it started from, as expected. In this talk, we report on experiments conducted by dropping the disks and recording the heights to which they rise on rebound, and the comparison of these results with our theoretical predictions and computer simulations. Frances E. Walker Fellowship.

  18. Small Sample Whole-Genome Amplification

    SciTech Connect

    Hara, C A; Nguyen, C P; Wheeler, E K; Sorensen, K J; Arroyo, E S; Vrankovich, G P; Christian, A T

    2005-09-20

    Many challenges arise when trying to amplify and analyze human samples collected in the field due to limitations in sample quantity, and contamination of the starting material. Tests such as DNA fingerprinting and mitochondrial typing require a certain sample size and are carried out in large volume reactions; in cases where insufficient sample is present whole genome amplification (WGA) can be used. WGA allows very small quantities of DNA to be amplified in a way that enables subsequent DNA-based tests to be performed. A limiting step to WGA is sample preparation. To minimize the necessary sample size, we have developed two modifications of WGA: the first allows for an increase in amplified product from small, nanoscale, purified samples with the use of carrier DNA while the second is a single-step method for cleaning and amplifying samples all in one column. Conventional DNA cleanup involves binding the DNA to silica, washing away impurities, and then releasing the DNA for subsequent testing. We have eliminated losses associated with incomplete sample release, thereby decreasing the required amount of starting template for DNA testing. Both techniques address the limitations of sample size by providing ample copies of genomic samples. Carrier DNA, included in our WGA reactions, can be used when amplifying samples with the standard purification method, or can be used in conjunction with our single-step DNA purification technique to potentially further decrease the amount of starting sample necessary for future forensic DNA-based assays.

  19. Small sample whole-genome amplification

    NASA Astrophysics Data System (ADS)

    Hara, Christine; Nguyen, Christine; Wheeler, Elizabeth; Sorensen, Karen; Arroyo, Erin; Vrankovich, Greg; Christian, Allen

    2005-11-01

    Many challenges arise when trying to amplify and analyze human samples collected in the field due to limitations in sample quantity, and contamination of the starting material. Tests such as DNA fingerprinting and mitochondrial typing require a certain sample size and are carried out in large volume reactions; in cases where insufficient sample is present whole genome amplification (WGA) can be used. WGA allows very small quantities of DNA to be amplified in a way that enables subsequent DNA-based tests to be performed. A limiting step to WGA is sample preparation. To minimize the necessary sample size, we have developed two modifications of WGA: the first allows for an increase in amplified product from small, nanoscale, purified samples with the use of carrier DNA while the second is a single-step method for cleaning and amplifying samples all in one column. Conventional DNA cleanup involves binding the DNA to silica, washing away impurities, and then releasing the DNA for subsequent testing. We have eliminated losses associated with incomplete sample release, thereby decreasing the required amount of starting template for DNA testing. Both techniques address the limitations of sample size by providing ample copies of genomic samples. Carrier DNA, included in our WGA reactions, can be used when amplifying samples with the standard purification method, or can be used in conjunction with our single-step DNA purification technique to potentially further decrease the amount of starting sample necessary for future forensic DNA-based assays.

  20. Research highlights: enhancing whole genome amplification using compartmentalization.

    PubMed

    Tay, Andy; Kulkarni, Rajan P; Karimi, Armin; Di Carlo, Dino

    2015-12-01

    The ability to break up a larger liquid volume into an array of smaller confined volumes that do not chemically communicate is a key enabling technology driving microfluidic innovations. We highlight recent work using drop-based confinement to improve on whole genome amplification, reducing amplification bias and contaminant amplification by bringing reactions to saturation within each confined drop. We also highlight a complementary technique to target whole genome amplification to a subset of nucleic acids within a sample by combining drop-based PCR with sorting and downstream sequencing. These new approaches have the potential to enhance our ability to categorize the diversity of microorganisms (especially difficult to culture species) that contribute to complex microbial communities, and in particular assemble the individual genomes of the species involved in biologically and environmentally important microbiomes. PMID:26486454

  1. Amplification of surface temperature trends and variability in thetropical atmosphere

    SciTech Connect

    Santer, B.D.; Wigley, T.M.L.; Mears, C.; Wentz, F.J.; Klein,S.A.; Seidel, D.J.; Taylor, K.E.; Thorne, P.W.; Wehner, M.F.; Gleckler,P.J.; Boyle, J.S.; Collins, W.D.; Dixon, K.W.; Doutriaux, C.; Free, M.; Fu, Q.; Hansen, J.E.; Jones, G.S.; Ruedy, R.; Karl, T.R.; Lanzante, J.R.; Meehl, G.A.; Ramaswamy, V.; Russell, G.; Schmidt, G.A.

    2005-08-11

    The month-to-month variability of tropical temperatures is larger in the troposphere than at the Earth's surface. This amplification behavior is similar in a range of observations and climate model simulations, and is consistent with basic theory. On multi-decadal timescales, tropospheric amplification of surface warming is a robust feature of model simulations, but occurs in only one observational dataset. Other observations show weak or even negative amplification. These results suggest that either different physical mechanisms control amplification processes on monthly and decadal timescales, and models fail to capture such behavior, or (more plausibly) that residual errors in several observational datasets used here affect their representation of long-term trends.

  2. Drag amplification and fatigue damage in vortex-induced vibrations

    E-print Network

    Jhingran, Vikas Gopal

    2008-01-01

    Fatigue damage and drag force amplification due to Vortex-Induced-Vibrations (VIV) continue to cause significant problems in the design of structures which operate in ocean current environments. These problems are magnified ...

  3. Controlled power compression in materials for X-ray amplification

    SciTech Connect

    Borisov, A. B.; McPherson, A.; Boyer, K.; Rhodes, C. K.

    1995-05-01

    New nonlinear phenomena involving (1) multiphoton excited X-ray emission from clusters and (2) stable channeled electromagnetic propagation in plasmas have combined scaling properties highly conducive for X-ray amplification in the kilovolt range.

  4. DNA amplification is rare in normal human cells

    SciTech Connect

    Wright, J.A.; Watt, F.M.; Hudson, D.L.; Stark, G.R. ); Smith, H.S.; Hancock, M.C. )

    1990-03-01

    Three types of normal human cells were selected in tissue culture with three drugs without observing a single amplification event from a total of 5 x 10{sup 8} cells. No drug-resistant colonies were observed when normal foreskin keratinocytes were selected with N-(phosphonacetyl)-L-aspartate or with hydroxyurea or when normal mammary epithelial cells were selected with methotrexate. Some slightly resistant colonies with limited potential for growth were obtained when normal diploid fibroblast cells derived from fetal lung were selected with methotrexate or hydroxyurea but careful copy-number analysis of the dihydrofolate reductase and ribonucleotide reductase genes revealed no evidence of amplification. The rarity of DNA amplification in normal human cells contrasts strongly with the situation in tumors and in established cell lines, where amplification of onogenes and of genes mediating drug resistance is frequent. The results suggest that tumors and cell lines have acquired the abnormal ability to amplify DNA with high frequency.

  5. Nonlinearity management in fiber transmission systems with hybrid amplification

    NASA Astrophysics Data System (ADS)

    Ania-Castañón, J. D.; Nasieva, I. O.; Kurukitkoson, N.; Turitsyn, S. K.; Borsier, C.; Pincemin, E.

    2004-04-01

    Nonlinearity management in transmission lines with periodic dispersion compensation and hybrid Raman-Erbium doped fiber amplification is studied both analytically and numerically. Different transmission/compensating fiber pairs are considered, with particular focus on the SMF/DCF case.

  6. Three-dimensional Simulation of Backward Raman Amplification

    SciTech Connect

    A.A. Balakin; G.M. Fraiman; N.J. Fisch

    2005-11-12

    Three-dimensional (3-D) simulations for the Backward Raman Amplification (BRA) are presented. The images illustrate the effects of pump depletion, pulse diffraction, non-homogeneous plasma density, and plasma ionization.

  7. A facile and pragmatic electrochemical biosensing strategy for ultrasensitive detection of DNA in real sample based on defective T junction induced transcription amplification.

    PubMed

    Yuan, Rui; Ding, Shijia; Yan, Yurong; Zhang, Ye; Zhang, Yuhong; Cheng, Wei

    2016-03-15

    A novel and pragmatic electrochemical sensing strategy was developed for ultrasensitive and specific detection of nucleic acids by combining with defective T junction induced transcription amplification (DTITA). The homogeneous recognition and specific binding of target DNA with a pair of designed probes formed a defective T junction, further triggered primer extension reaction and in vitro transcription amplification to produce numerous single-stranded RNA. These RNA products of DTITA could hybridized with the biotinylated detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the surface of the biosensor. The proposed isothermal DTITA strategy displayed remarkable signal amplification performance and reproducibility. The electrochemical DNA biosensor showed very high sensitivity for target DNA with a low detection limit of 0.4fM (240 molecules of the synthetic DNA), and can directly detect target pathogenic gene of Group B Streptococci (GBS) from as low as 400 copies of genomic DNA. Moreover, the established biosensor was successfully verified for directly identifying GBS in clinical samples. This proposed strategy presented a simple and pragmatic platform toward ultrasensitive and handy nucleic acids detection, and would become a potential tool for general application in point-of-care setting. PMID:26385733

  8. Detection of biological molecules using chemical amplification and optical sensors

    DOEpatents

    Van Antwerp, William Peter (Valencia, CA); Mastrototaro, John Joseph (Los Angeles, CA)

    2000-01-01

    Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal.

  9. Detection of biological molecules using chemical amplification and optical sensors

    DOEpatents

    Van Antwerp, William Peter; Mastrototaro, John Joseph

    2004-10-12

    Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal.

  10. The amplification of weak measurements under quantum noise

    E-print Network

    Xuanmin Zhu; Yu-Xiang Zhang

    2015-05-08

    The influence of outside quantum noises on the amplification of weak measurements is investigated. Three typical quantum noises are discussed. The maximum values of the pointer's shifts decrease sharply with the strength of the depolarizing channel and phase damping. In order to obtain significant amplified signals, the preselection quantum systems must be kept away from the two quantum noises. Interestingly, the amplification effect is immune to the amplitude damping noise.

  11. Simulation study of electron response amplification in coherent electron cooling

    SciTech Connect

    Hao Y.; Litvinenko, V.N.

    2012-05-20

    In Coherent Electron Cooling (CEC), it is essential to study the amplification of electron response to a single ion in the FEL process, in order to proper align the electron beam and the ion beam in the kicker to maximize the cooling effect. In this paper, we use Genesis to simulate the amplified electron beam response of single ion in FEL amplification process, which acts as Green's function of the FEL amplifier.

  12. MYCN gene amplification in patients with neuroblastic tumors.

    PubMed

    Estiar, M A; Fazilaty, H; Aslanabadi, S; Seifi, M; Varghaei, P; Rezamand, A

    2014-01-01

    Although neuroblastic tumors are the most prevalent solid tumors, little is known about the genetic basis underlying their progression. The prognostic role for the MYCN gene in neuroblastic tumors is irrefutable. The aim of this study is to identify the frequency of MYCN gene amplification and its relationship with clinicopathological and prognostic factors in 40 patients with neuroblastic tumors by using real-time quantitative PCR. There was significant association between the age of older than 18 months and the high number of metastasis. 83.3% of metastatic neuroblastic tumors in patients aged more than 18 months were in stage 4, while it was about 12.5% in patients aged less than 18 months. We found an amplification of MYCN in 19 out of 40 patients. Also, we found MYCN gene amplification in 64% of neuroblastoma (NB) and 8% of gangelioneuroblastoma (GNB) cases. There was a significant association between the histological type of samples with MYCN gene amplification. Neuroblastic tumors have a varied range of MYCN gene amplification depend on histopathology types. No significant associations have been found between MYCN gene amplification and tumor evaluation, CNS involvement, metastasis, stage of disease and patients outcome. PMID:25231001

  13. Empirical evidence for acceleration-dependent amplification factors

    USGS Publications Warehouse

    Borcherdt, R.D.

    2002-01-01

    Site-specific amplification factors, Fa and Fv, used in current U.S. building codes decrease with increasing base acceleration level as implied by the Loma Prieta earthquake at 0.1g and extrapolated using numerical models and laboratory results. The Northridge earthquake recordings of 17 January 1994 and subsequent geotechnical data permit empirical estimates of amplification at base acceleration levels up to 0.5g. Distance measures and normalization procedures used to infer amplification ratios from soil-rock pairs in predetermined azimuth-distance bins significantly influence the dependence of amplification estimates on base acceleration. Factors inferred using a hypocentral distance norm do not show a statistically significant dependence on base acceleration. Factors inferred using norms implied by the attenuation functions of Abrahamson and Silva show a statistically significant decrease with increasing base acceleration. The decrease is statistically more significant for stiff clay and sandy soil (site class D) sites than for stiffer sites underlain by gravely soils and soft rock (site class C). The decrease in amplification with increasing base acceleration is more pronounced for the short-period amplification factor, Fa, than for the midperiod factor, Fv.

  14. Oncogene-like induction of cellular invasion from centrosome amplification

    PubMed Central

    Godinho, Susana A.; Picone, Remigio; Burute, Mithila; Dagher, Regina; Su, Ying; Leung, Cheuk T.; Polyak, Kornelia; Brugge, Joan S.; Thery, Manuel; Pellman, David

    2014-01-01

    Centrosome amplification has long been recognized as a feature of human tumors, however its role in tumorigenesis remains unclear1. Centrosome amplification is poorly tolerated by non-transformed cells, and, in the absence of selection, extra centrosomes are spontaneously lost2. Thus, the high frequency of centrosome amplification, particularly in more aggressive tumors3, raises the possibility that extra centrosomes could, in some contexts, confer advantageous characteristics that promote tumor progression. Using a three-dimensional model system and other approaches to culture human mammary epithelial cells, we find that centrosome amplification triggers cell invasion. This invasive behavior is similar to that induced by overexpression of the breast cancer oncogene ErbB24 and indeed enhances invasiveness triggered by ErbB2. We show that, through increased centrosomal microtubule nucleation, centrosome amplification increases Rac1 activity, which disrupts normal cell-cell adhesion and promotes invasion. These findings demonstrate that centrosome amplification, a structural alteration of the cytoskeleton, can promote features of malignant transformation. PMID:24739973

  15. Multiple pathways of selected gene amplification during adaptive mutation.

    PubMed

    Kugelberg, Elisabeth; Kofoid, Eric; Reams, Andrew B; Andersson, Dan I; Roth, John R

    2006-11-14

    In a phenomenon referred to as "adaptive mutation," a population of bacterial cells with a mutation in the lac operon (lac-) accumulates Lac+ revertants during prolonged exposure to selective growth conditions (lactose). Evidence was provided that selective conditions do not increase the mutation rate but instead favor the growth of rare cells with a duplication of the leaky lac allele. A further increase in copy number (amplification) improves growth and increases the likelihood of a sequence change by adding more mutational targets to the clone (cells and lac copies per cell). These duplications and amplifications are described here. Before selection, cells with large (134-kb) lac duplications and long junction sequences (>1 kb) were common (0.2%). The same large repeats were found after selection in cells with a low-copy-number lac amplification. Surprisingly, smaller repeats (average, 34 kb) were found in high-copy-number amplifications. The small-repeat duplications form when deletions modify a preexisting large-repeat duplication. The shorter repeat size allowed higher lac amplification and better growth on lactose. Thus, selection favors a succession of gene-amplification types that make sequence changes more probable by adding targets. These findings are relevant to genetic adaptation in any biological systems in which fitness can be increased by adding gene copies (e.g., cancer and bacterial drug resistance). PMID:17082307

  16. Laser velocimetry measurements in non-isothermal CVD systems

    NASA Technical Reports Server (NTRS)

    Johnson, E. J.; Hyer, P. V.; Culotta, P. W.; Clark, I. O.

    1991-01-01

    Researchers at the NASA Langley Research Center are applying laser velocimetry (LV) techniques to characterize the fluid dynamics of non-isothermal flows inside fused silica chambers designed for chemical vapor deposition (CVD). Experimental issues involved in the application of LV techniques to this task include thermophoretic effects on the LV seed particles, seeding the hazardous gases, index of refraction gradients in the flow field and surrounding media, optical access, relatively low flow velocities, and analysis and presentation of sparse data. An overview of the practical difficulties these issues represent to the use of laser velocimetry instrumentation for CVD applications is given. A fundamental limitation on the application of LV techniques in non-isothermal systems is addressed which involves a measurement bias due to the presence of thermal gradients. This bias results from thermophoretic effects which cause seed particle trajectories to deviate from gas streamlines. Data from a research CVD reactor are presented which indicate that current models for the interaction of forces such as Stokes drag, inertia, gravity, and thermophoresis are not adequate to predict thermophoretic effects on particle-based velocimetry measurements in arbitrary flow configurations.

  17. Bondi-like Accretion in Magnetized Supersonic Isothermal Turbulence

    NASA Astrophysics Data System (ADS)

    Burleigh, Kaylan J.; McKee, Christopher F.; Klein, Richard I.

    2016-01-01

    The Bondi and Bondi-Hoyle-Lytlleton formulas give the order of magnitude steady-accretion rate onto a point mass at rest or moving, respectively, in a uniform density gas in the limit of negligible gas self-gravity. This applies in star-forming clouds where self-gravity is negligible near protostars and new-born stars, but instead of being uniform the gas is supersonically turbulent and threaded by dynamically important (Alven Mach number ? 1) large-scale magnetic fields. To determine the Bondi-like accretion rate in these environments, we used the ORION2 code to carry out grid-based 3D adaptive mesh refinement (AMR) magnetohydrodynamic (MHD) simulations of accretion onto sink particles embedded in an environment of fully developed, magnetized supersonic isothermal turbulence. We evolved the models until the median and mean accretion rates, over particles, became steady. We present a simple semi-analytic model that predicts the median and mean accretion rate from the turbulent properties of the background medium, such as the 3D Mach number and RMS plasma-?, and show that it is highly consistent with our simulations. Numerical codes can use our semi-analytic model as an accurate sub-grid model for accretion in magnetized supersonic isothermal turbulence.

  18. A microfabrication-based approach to quantitative isothermal titration calorimetry.

    PubMed

    Wang, Bin; Jia, Yuan; Lin, Qiao

    2016-04-15

    Isothermal titration calorimetry (ITC) directly measures heat evolved in a chemical reaction to determine equilibrium binding properties of biomolecular systems. Conventional ITC instruments are expensive, use complicated design and construction, and require long analysis times. Microfabricated calorimetric devices are promising, although they have yet to allow accurate, quantitative ITC measurements of biochemical reactions. This paper presents a microfabrication-based approach to integrated, quantitative ITC characterization of biomolecular interactions. The approach integrates microfabricated differential calorimetric sensors with microfluidic titration. Biomolecules and reagents are introduced at each of a series of molar ratios, mixed, and allowed to react. The reaction thermal power is differentially measured, and used to determine the thermodynamic profile of the biomolecular interactions. Implemented in a microdevice featuring thermally isolated, well-defined reaction volumes with minimized fluid evaporation as well as highly sensitive thermoelectric sensing, the approach enables accurate and quantitative ITC measurements of protein-ligand interactions under different isothermal conditions. Using the approach, we demonstrate ITC characterization of the binding of 18-Crown-6 with barium chloride, and the binding of ribonuclease A with cytidine 2'-monophosphate within reaction volumes of approximately 0.7µL and at concentrations down to 2mM. For each binding system, the ITC measurements were completed with considerably reduced analysis times and material consumption, and yielded a complete thermodynamic profile of the molecular interaction in agreement with published data. This demonstrates the potential usefulness of our approach for biomolecular characterization in biomedical applications. PMID:26655185

  19. Sorption isotherms and isosteric heats of sorption of Malaysian paddy.

    PubMed

    Mousa, Wael; Ghazali, Farinazleen Mohamad; Jinap, S; Ghazali, Hasanah Mohd; Radu, Son

    2014-10-01

    Understanding the water sorption characteristics of cereal is extremely essential for optimizing the drying process and ensuring storage stability. Water relation of rough rice was studied at 20, 30, 40 and 50 °C over relative humidity (RH.) between 0.113 and 0.976 using the gravimetric technique. The isotherms displayed the general sigmoid, Type II pattern and exhibited the phenomenon of hysteresis where it was more pronounced at lower temperatures. The sorption characteristics were temperature dependence where the sorption capacity of the paddy increased as the temperature was decreased at fixed (RH). Among the models assessed for their ability to fit the sorption data, Oswin equation was the best followed by the third order polynomial, GAB, Smith, Chung-Pfost, and Henderson models. The monolayer moisture content was higher for desorption than adsorption and tend to decrease with the increase in temperature. Given the temperature dependence of the sorption isotherms the isosteric heats of sorption were calculated using Claussius-Clapeyron equation. The net isosteric heats decreased as the moisture content was increased and heats of desorption were greater than that of adsorption. PMID:25328208

  20. A New Potential-Density Pair for Isothermal Star Clusters

    E-print Network

    Stone, Nicholas C

    2015-01-01

    We present a new potential-density pair designed to model nearly isothermal star clusters (and similar self-gravitating systems) with a central core and an outer turnover radius, beyond which density falls off as $r^{-4}$. In the intermediate zone, the profile is similar to that of an isothermal sphere (density $\\rho \\propto r^{-2}$), somewhat less steep than the King 1962 profile, and with the advantage that many dynamical quantities can be written in a simple closed form. We derive analytic expressions for the cluster binding energy, central velocity dispersion, and escape velocity, and apply these to create toy models for cluster core collapse and evaporation. We rederive classical results for evaporating, collapsing, and quasi-equilibrium (heated) clusters, and fit our projected surface brightness profiles to observed globular and open clusters. We find that the quality of the fit is generally at least as good as that for the surface brightness profiles of King 1962. This model can be used for convenient ...

  1. Isothermal nitridation kinetics of TiSi{sub 2} powders

    SciTech Connect

    Roger, J. Maillé, L.; Dourges, M.A.

    2014-04-01

    The aim of the present work is to determine the kinetics of reaction between TiSi{sub 2} powder and gaseous nitrogen. Isothermal nitridation of TiSi{sub 2} powders with fine (1.4 µm) and medium (4.5 µm) particle size has been studied in pure nitrogen atmosphere from 1000 to 1200 °C for duration up to 50 h. The isothermal nitridation kinetics of TiSi{sub 2} powders were investigated by thermogravimetry. The nitridation rate strongly depends on the particle size and temperature. Smaller size particle exhibits higher nitridation rate due to its larger surface area. The conversion process is complex with nucleation and growth of TiN at the surface of the grain and Si{sub 3}N{sub 4} inside the grain promoted by the Kirkendall effect with an influence of the volume increase. - Graphical abstract: Backscattered electrons image of a transverse TiSi{sub 2} grain nitrurated at 1100 °C for 50 h. - Highlights: • Influence of grain size on TiSi{sub 2} powder nitridation. • Influence of temperature on TiSi{sub 2} powder nitridation. • Experimental measurements of the nitridation kinetics. • An explanation of the nitridation mechanism.

  2. Solubilization isotherms of aromatic solutes in surfactant aggregates

    SciTech Connect

    Gadelle, F.; Koros, W.J.; Schechter, R.S. . Dept. of Chemical Engineering)

    1995-03-01

    Several factors affecting solubilization of aromatic solutes in surfactant micelles have been investigated. Solubilization isotherms of benzene, toluene, and chlorobenzene in various aqueous micellar solutions were determined using head space gas chromatography. Cationic surfactants such as cetylpyridinium chloride or cetyltrimethylammonium bromide present high solubilization capacities. Comparable anionic surfactants exhibit lower solubilization and a greater tendency to precipitate. It was observed that nonionic surfactants show high solubilization on a molar basis. Solubilization in mixed cationic-anionic micelles was also investigated. It also appears that the molecular size of the solute determines the extent of the solubilization. Finally, the shape of the different isotherms indicates that knowing the amount solubilized at saturation of the micellar solution is not sufficient to estimate solubilization at solute concentrations lower than the solute aqueous solubility. Solubilization of organics in surfactant micelles is of major importance in many applications. One new application is micellar-enhanced ultrafiltration. Another application of interest is the surfactant-enhanced aquifer remediation, a process in which a micellar aqueous solution is flushed into contaminated groundwaters to enhance recovery of pollutants by micellar solubilization.

  3. ISOTHERMAL AND MULTITHERMAL ANALYSIS OF CORONAL LOOPS OBSERVED WITH AIA

    SciTech Connect

    Schmelz, J. T.; Jenkins, B. S.; Worley, B. T.; Anderson, D. J.; Pathak, S.; Kimble, J. A.

    2011-04-10

    The coronal filters in the Atmospheric Imaging Assembly (AIA) aboard the Solar Dynamics Observatory peak at different temperatures; the series covers the entire active region temperature range, making AIA ideal for multithermal analysis. Here, we analyze coronal loops from several active regions that have been observed by AIA. We have specifically targeted cool loops (or at least loops with a cool component) that were chosen in the 171 A channel of AIA, which has a peak response temperature of log T = 5.8. We wanted to determine if the loops could be described as isothermal or multithermal. We find that several of our 12 loops have narrow temperature distributions, which may be consistent with isothermal plasma; these can be modeled with a single flux tube. Other loops have intermediate-width temperature distributions, appear well-constrained, and should be multi-stranded. The remaining loops, however, have unrealistically broad differential emission measures. We find that this problem is the result of missing low-temperature lines in the AIA 131 A channel. If we repeat the analysis without the 131 A data, these loops also appear to be well-constrained and multi-stranded.

  4. Strong Lensing by Binary Galaxies Modelled as Isothermal Spheres

    E-print Network

    E. M. Shin; N. W. Evans

    2008-07-30

    We study the problem of gravitational lensing by binary galaxies, idealized as two isothermal spheres. In a wide binary, each galaxy possesses individual tangential, nearly astroidal, caustics and roundish radial caustics. As the separation of the binary is made smaller, the caustics undergo a sequence of metamorphoses. The first metamorphosis occurs when the tangential caustics merge to form a single six-cusped caustic, lying interior to the radial caustics. At still smaller separations, the six-cusped caustic undergoes the second metamorphosis and splits into a four-cusped caustic and two three-cusped caustics, which shrink to zero size (an elliptic umbilic catastrophe) before they enlarge again and move away from the origin perpendicular to the binary axis. Finally, a third metamorphosis occurs as the three-cusp caustics join the radial caustics, leaving an inner distorted astroid caustic enclosed by two outer caustics. The maximum number of images possible is 7. Classifying the multiple imaging according to critical isochrones, there are only 8 possibilities: 2 three-image cases, 3 five-image cases, and 3 seven-image cases. When the isothermal spheres are singular, the core images vanish into the central singularity. The number of images may then be 1, 2, 3, 4 or 5, depending on the source location, and the separation and masses of the pair of lensing galaxies. The locations of metamorphoses, and the onset of threefold and fivefold multiple imaging, can be worked out analytically in this case.

  5. Initiation and amplification of the Ningaloo Niño

    NASA Astrophysics Data System (ADS)

    Marshall, Andrew G.; Hendon, Harry H.; Feng, Ming; Schiller, Andreas

    2015-11-01

    Marine heat waves along the Western Australian coast are potentially damaging to the marine environment especially coastal fisheries and the Ningaloo Reef. Initiation and amplification mechanisms for marine heat waves (referred to as `Ningaloo Niño' events) are explored using ocean and atmosphere reanalyses for the period 1960-2011. We find that the onset stage from October to November is promoted by wind-evaporation-SST feedback that operates to the northwest of the coast on the north-eastern flank of the Mascarene subtropical high: cyclonic anomalies act to reduce the surface wind speed and warm the ocean surface, thereby driving increased rainfall and stronger cyclonic anomalies. The growth and southward expansion of positive SST anomalies along the Australian west coast is further supplemented by anomalous poleward advection of heat by the Leeuwin Current, which is coupled with the cyclonic anomalies off the coast. The strongest Ningaloo Niño events, such as the record strong 2011 event, occur in conjunction with La Niña conditions in the Pacific, which drives westerly wind anomalies to the northwest of Australia that can promote the WES feedback and accelerate the Leeuwin Current via transmission of thermocline anomalies from the western Pacific onto the west Australian coast. However, many Ningaloo Niño events occur independent of La Niña and some Ningaloo Niño events even occur during certain El Niños. We explain this general independence from ENSO because the triggering of Ningaloo Niño events from the Pacific is most sensitive to antecedent SST anomalies in the far western Pacific, rather than in the central Pacific where ENSO typically has greatest magnitude.

  6. Tomography-based monitoring of isothermal snow metamorphism under advective conditions

    NASA Astrophysics Data System (ADS)

    Ebner, P. P.; Schneebeli, M.; Steinfeld, A.

    2015-02-01

    Time-lapse X-ray micro-tomography was used to investigate the structural dynamics of isothermal snow metamorphism exposed to an advective airflow. Diffusion and advection across the snow pores were analysed in controlled laboratory experiments. The 3-D digital geometry obtained by tomographic scans was used in direct pore-level numerical simulations to determine the effective transport properties. The results showed that isothermal advection with saturated air have no influence on the coarsening rate that is typical for isothermal snow metamorphism. Diffusion originating in the Kelvin effect between snow structures dominates and is the main transport process in isothermal snow packs.

  7. Somatic amplifications and deletions in genome of papillary thyroid carcinomas.

    PubMed

    Passon, Nadia; Bregant, Elisa; Sponziello, Marialuisa; Dima, Maria; Rosignolo, Francesca; Durante, Cosimo; Celano, Marilena; Russo, Diego; Filetti, Sebastiano; Damante, Giuseppe

    2015-11-01

    Somatic gene copy number variation contributes to tumor progression. Using comparative genomic hybridization (CGH) array, the presence of genomic imbalances was evaluated in a series of 27 papillary thyroid carcinomas (PTCs). To detect only somatic imbalances, for each sample, the reference DNA was from normal thyroid tissue of the same patient. The presence of the BRAF V600E mutation was also evaluated. Both amplifications and deletions showed an uneven distribution along the entire PTC cohort; amplifications were more frequent than deletions (mean values of 17.5 and 7.2, respectively). Number of aberration events was not even among samples, the majority of them occurring only in a small fraction of PTCs. Most frequent amplifications were detected at regions 2q35, 4q26, and 4q34.1, containing FN1, PDE5A, and GALNTL6 genes, respectively. Most frequent deletions occurred at regions 6q25.2, containing OPMR1 and IPCEF1 genes and 7q14.2, containing AOAH and ELMO1 genes. Amplification of FN1 and PDE5A genomic regions was confirmed by quantitative PCR. Frequency of amplifications and deletions was in relationship with clinical features and BRAF mutation status of tumor. In fact, according to the American Joint Committee on Cancer stage and American Thyroid Association (ATA) risk classification, amplifications are more frequent in higher risk samples, while deletions tend to prevail in the lower risk tumors. Analysis of single aberrations according to the ATA risk grouping shows that amplifications containing PDE5A, GALNTL6, DHRS3, and DOCK9 genes are significantly more frequent in the intermediate/high risk group than in the low risk group. Thus, our data would indicate that analysis of somatic genome aberrations by CGH array can be useful to identify additional prognostic variables. PMID:25863487

  8. Regulation of ribosomal DNA amplification by the TOR pathway.

    PubMed

    Jack, Carmen V; Cruz, Cristina; Hull, Ryan M; Keller, Markus A; Ralser, Markus; Houseley, Jonathan

    2015-08-01

    Repeated regions are widespread in eukaryotic genomes, and key functional elements such as the ribosomal DNA tend to be formed of high copy repeated sequences organized in tandem arrays. In general, high copy repeats are remarkably stable, but a number of organisms display rapid ribosomal DNA amplification at specific times or under specific conditions. Here we demonstrate that target of rapamycin (TOR) signaling stimulates ribosomal DNA amplification in budding yeast, linking external nutrient availability to ribosomal DNA copy number. We show that ribosomal DNA amplification is regulated by three histone deacetylases: Sir2, Hst3, and Hst4. These enzymes control homologous recombination-dependent and nonhomologous recombination-dependent amplification pathways that act in concert to mediate rapid, directional ribosomal DNA copy number change. Amplification is completely repressed by rapamycin, an inhibitor of the nutrient-responsive TOR pathway; this effect is separable from growth rate and is mediated directly through Sir2, Hst3, and Hst4. Caloric restriction is known to up-regulate expression of nicotinamidase Pnc1, an enzyme that enhances Sir2, Hst3, and Hst4 activity. In contrast, normal glucose concentrations stretch the ribosome synthesis capacity of cells with low ribosomal DNA copy number, and we find that these cells show a previously unrecognized transcriptional response to caloric excess by reducing PNC1 expression. PNC1 down-regulation forms a key element in the control of ribosomal DNA amplification as overexpression of PNC1 substantially reduces ribosomal DNA amplification rate. Our results reveal how a signaling pathway can orchestrate specific genome changes and demonstrate that the copy number of repetitive DNA can be altered to suit environmental conditions. PMID:26195783

  9. Regulation of ribosomal DNA amplification by the TOR pathway

    PubMed Central

    Jack, Carmen V.; Cruz, Cristina; Hull, Ryan M.; Keller, Markus A.; Ralser, Markus; Houseley, Jonathan

    2015-01-01

    Repeated regions are widespread in eukaryotic genomes, and key functional elements such as the ribosomal DNA tend to be formed of high copy repeated sequences organized in tandem arrays. In general, high copy repeats are remarkably stable, but a number of organisms display rapid ribosomal DNA amplification at specific times or under specific conditions. Here we demonstrate that target of rapamycin (TOR) signaling stimulates ribosomal DNA amplification in budding yeast, linking external nutrient availability to ribosomal DNA copy number. We show that ribosomal DNA amplification is regulated by three histone deacetylases: Sir2, Hst3, and Hst4. These enzymes control homologous recombination-dependent and nonhomologous recombination-dependent amplification pathways that act in concert to mediate rapid, directional ribosomal DNA copy number change. Amplification is completely repressed by rapamycin, an inhibitor of the nutrient-responsive TOR pathway; this effect is separable from growth rate and is mediated directly through Sir2, Hst3, and Hst4. Caloric restriction is known to up-regulate expression of nicotinamidase Pnc1, an enzyme that enhances Sir2, Hst3, and Hst4 activity. In contrast, normal glucose concentrations stretch the ribosome synthesis capacity of cells with low ribosomal DNA copy number, and we find that these cells show a previously unrecognized transcriptional response to caloric excess by reducing PNC1 expression. PNC1 down-regulation forms a key element in the control of ribosomal DNA amplification as overexpression of PNC1 substantially reduces ribosomal DNA amplification rate. Our results reveal how a signaling pathway can orchestrate specific genome changes and demonstrate that the copy number of repetitive DNA can be altered to suit environmental conditions. PMID:26195783

  10. Alternative Chemical Amplification Methods for Peroxy Radical Detection

    NASA Astrophysics Data System (ADS)

    Wood, E. C. D.

    2014-12-01

    Peroxy radicals (HO2, CH3O2, etc.) are commonly detected by the chemical amplification technique, in which ambient air is mixed with high concentrations of CO and NO, initiating a chain reaction that produces 30 - 200 NO2 molecules per sampled peroxy radical. The NO2 is then measured by one of several techniques. With the exception of CIMS-based techniques, the chemical amplification method has undergone only incremental improvements since it was first introduced in 1982. The disadvantages of the technique include the need to use high concentrations of CO and the greatly reduced sensitivity of the amplification chain length in the presence of water vapor. We present a new chemical amplification scheme in which either ethane or acetaldehyde is used in place of CO, with the NO2 product detected using Cavity Attenuated Phase Shift spectroscopy (CAPS). Under dry conditions, the amplification factor of the alternative amplifiers are approximately six times lower than the CO-based amplifier. The relative humidity "penalty" is not as severe, however, such that at typical ambient relative humidity (RH) values the amplification factor is within a factor of three of the CO-based amplifier. Combined with the NO2 sensitivity of CAPS and a dual-channel design, the detection limit of the ethane amplifier is less than 2 ppt (1 minute average, signal-to-noise ratio 2). The advantages of these alternative chemical amplification schemes are improved safety, a reduced RH correction, and increased sensitivity to organic peroxy radicals relative to HO2.

  11. Advanced Yellow Fever Virus Genome Detection in Point-of-Care Facilities and Reference Laboratories

    PubMed Central

    Patel, Pranav; Yillah, Jasmin; Weidmann, Manfred; Méndez, Jairo A.; Nakouné, Emmanuel Rivalyn; Niedrig, Matthias

    2012-01-01

    Reported methods for the detection of the yellow fever viral genome are beset by limitations in sensitivity, specificity, strain detection spectra, and suitability to laboratories with simple infrastructure in areas of endemicity. We describe the development of two different approaches affording sensitive and specific detection of the yellow fever genome: a real-time reverse transcription-quantitative PCR (RT-qPCR) and an isothermal protocol employing the same primer-probe set but based on helicase-dependent amplification technology (RT-tHDA). Both assays were evaluated using yellow fever cell culture supernatants as well as spiked and clinical samples. We demonstrate reliable detection by both assays of different strains of yellow fever virus with improved sensitivity and specificity. The RT-qPCR assay is a powerful tool for reference or diagnostic laboratories with real-time PCR capability, while the isothermal RT-tHDA assay represents a useful alternative to earlier amplification techniques for the molecular diagnosis of yellow fever by field or point-of-care laboratories. PMID:23052311

  12. Isothermal decomposition of gamma-irradiated dysprosium acetate

    NASA Astrophysics Data System (ADS)

    Mahfouz, R. M.; Al-Shehri, S. M.; Monshi, M. A. S.; Abd El-Salam, N. M.

    Isothermal decomposition of un-irradiated and pre-gamma-irradiated dysprosium acetate [Dy(CH3COO)(3)] has been investigated at different temperatures between 603-623 K. Irradiation was observed to enhance the rate of decomposition without modifying the mechanism of the thermal decomposition. Thermal decomposition of dysposium acetate is shown to proceed by a nucleation and growth mechanism (Avarmi-Erofe'ev equation) both for un-irradiated and pre-gamma-irradiated samples. The enhancement of the decomposition was found to increase with an increase in the gamma-ray dose applied to the sample and may be attributed to an increase in point defects and formation of additional nucleation centers generated in the host lattice. Thermodynamic values of the main decomposition process were calculated and evaluated.

  13. Isothermal elastohydrodynamic lubrication of point contacts. 2: Ellipticity parameter results

    NASA Technical Reports Server (NTRS)

    Hamrock, B. J.; Dowson, D.

    1976-01-01

    A numerical solution of the isothermal elastohydrodynamic problem for point contacts is presented which reproduces all the essential features of experimental observations based upon optical interferometry. In particular, the two side lobes, in which minimum film thickness regions occur, emerge in the theoretical solutions. The influence of the ellipticity parameter on solutions to the point contact problem is explored. The ellipticity parameter k was varied from 1 (a ball on a plate) to 8 (a configuration approaching line contact). It is shown that the minimum film thickness can be related to the well known line contact solutions by a remarkably simple expression involving either k or the effective radius of curvature ratio R sub y/R sub x.

  14. Isothermal Titration Calorimetry: Assisted Crystallization of RNA-Ligand Complexes.

    PubMed

    Da Veiga, Cyrielle; Mezher, Joelle; Dumas, Philippe; Ennifar, Eric

    2016-01-01

    The success rate of nucleic acids/ligands co-crystallization can be significantly improved by performing preliminary biophysical analyses. Among suitable biophysical approaches, isothermal titration calorimetry (ITC) is certainly a method of choice. ITC can be used in a wide range of experimental conditions to monitor in real time the formation of the RNA- or DNA-ligand complex, with the advantage of providing in addition the complete binding profile of the interaction. Following the ITC experiment, the complex is ready to be concentrated for crystallization trials. This chapter describes a detailed experimental protocol for using ITC as a tool for monitoring RNA/small molecule binding, followed by co-crystallization. PMID:26227041

  15. Advanced magnetic anisotropy determination through isothermal remanent magnetization of nanoparticles

    NASA Astrophysics Data System (ADS)

    Hillion, A.; Tamion, A.; Tournus, F.; Gaier, O.; Bonet, E.; Albin, C.; Dupuis, V.

    2013-09-01

    We propose a theoretical framework enabling the simulation of isothermal remanence magnetization (IRM) curves, based on the Stoner-Wohlfarth model combined with the Néel macrospin relaxation time description. We show how low temperature IRM curves, which have many advantages compared to hysteresis loops, can be efficiently computed for realistic assemblies of magnetic particles with both a size and anisotropy constant distribution, and a biaxial anisotropy. The IRM curves, which probe the irreversible switching provoked by an applied field, are shown to be complementary to other usual measurements (in particular low-field susceptibility curves where a thermal switching is involved). As an application, the experimental IRM curve of Co clusters embedded in a carbon matrix is analyzed. We demonstrate how powerful such an analysis can be, which in the present case allows us to put into evidence an anisotropy constant dispersion among the Co nanoparticles.

  16. Capillarity in isothermal infiltration of alumina fiber preforms with aluminum

    SciTech Connect

    Michaud, V.J.; Mortensen, A. . Dept. of Materials Science); Compton, L.M. )

    1994-10-01

    Models derived in petroleum engineering and soil science for flow of two immiscible fluids in a porous medium are extended to the infiltration of ceramic preforms by a liquid metal. SAFFIL alumina fiber preforms are infiltrated with an aluminum matrix in a series of interrupted unidirectional and isothermal experiments at various low applied pressures, to measure profiles of the volume fraction of metal along the length of the preforms. Comparison of experimental data with theory reveals the existence of a pressure-dependent incubation time for wetting of the alumina preforms by molten aluminum at 973 K. If this incubation time is taken into account, experimental curves of metal distribution are well predicted by theory, confirming the validity of the models after initiation of flow.

  17. SSB Binding to ssDNA Using Isothermal Titration Calorimetry

    PubMed Central

    Kozlov, Alexander G.; Lohman, Timothy M.

    2015-01-01

    Isothermal titration calorimetry (ITC) is a powerful method for studying protein–DNA interactions in solution. As long as binding is accompanied by an appreciable enthalpy change, ITC studies can yield quantitative information on stoichiometries, binding energetics (affinity, binding enthalpy and entropy) and potential site–site interactions (cooperativity). This can provide a full thermodynamic description of an interacting system which is necessary to understand the stability and specificity of protein–DNA interactions and to correlate the activities or functions of different species. Here we describe procedures to perform and analyze ITC studies using as examples, the E. coli SSB (homotetramer with 4 OB-folds) and D. radiodurans SSB (homodimer with 4 OB-folds). For oligomeric protein systems such as these, we emphasize the need to be aware of the likelihood that solution conditions will influence not only the affinity and enthalpy of binding but also the mode by which the SSB oligomer binds ssDNA. PMID:22976176

  18. Optimal smoothing of site-energy distributions from adsorption isotherms

    SciTech Connect

    Brown, L.F.; Travis, B.J.

    1983-01-01

    The equation for the adsorption isotherm on a heterogeneous surface is a Fredholm integral equation. In solving it for the site-energy distribution (SED), some sort of smoothing must be carried out. The optimal amount of smoothing will give the most information that is possible without introducing nonexistent structure into the SED. Recently, Butler, Reeds, and Dawson proposed a criterion (the BRD criterion) for choosing the optimal smoothing parameter when using regularization to solve Fredholm equations. The BRD criterion is tested for its suitability in obtaining optimal SED's. This criterion is found to be too conservative. While using it never introduces nonexistent structure into the SED, significant information is often lost. At present, no simple criterion for choosing the optimal smoothing parameter exists, and a modeling approach is recommended.

  19. Experimental strain of isothermal remanent magnetization in ductile sandstone

    NASA Astrophysics Data System (ADS)

    Borradaile, Graham J.; Mothersill, John S.

    A macroscopically ductile sandstone, to which a homogeneous isothermal remanent magnetization (IRM) was applied, has been deformed at 150 MPa confining pressure and constant strain rate of 10 -5 s -1. Hydrostatic compaction does not produce a deflection of the IRM vector although it is reduced in intensity. Pure shear producing shortening in the range 2 to 35% steadily reduces the intensity of magnetization but also homogeneously rotates the remanence vector toward the plane of flattening. The amount of rotation is slightly less than that expected for a non-material line undergoing homogeneous strain. Deformation selectively removes weakly coercive components of remanence, as revealed by alternating field (AF) demagnetization. During deformation a weak deformational viscous remanent magnetization (DVRM) is acquired from the pressure vessel. This is different from a conventional viscous remanent magnetization (VRM) in that it is not acquired when the specimen is subject to hydrostatic confining pressure alone, even for periods three times longer than the longest deformation test.

  20. Liouvillian integrability of gravitating static isothermal fluid spheres

    NASA Astrophysics Data System (ADS)

    Iacono, Roberto; Llibre, Jaume

    2014-10-01

    We examine the integrability properties of the Einstein field equations for static, spherically symmetric fluid spheres, complemented with an isothermal equation of state, ? = np. In this case, Einstein's equations can be reduced to a nonlinear, autonomous second order ordinary differential equation (ODE) for m/R (m is the mass inside the radius R) that has been solved analytically only for n = -1 and n = -3, yielding the cosmological solutions by De Sitter and Einstein, respectively, and for n = -5, case for which the solution can be derived from the De Sitter's one using a symmetry of Einstein's equations. The solutions for these three cases are of Liouvillian type, since they can be expressed in terms of elementary functions. Here, we address the question of whether Liouvillian solutions can be obtained for other values of n. To do so, we transform the second order equation into an equivalent autonomous Lotka-Volterra quadratic polynomial differential system in {R}^2, and characterize the Liouvillian integrability of this system using Darboux theory. We find that the Lotka-Volterra system possesses Liouvillian first integrals for n = -1, -3, -5, which descend from the existence of invariant algebraic curves of degree one, and for n = -6, a new solvable case, associated to an invariant algebraic curve of higher degree (second). For any other value of n, eventual first integrals of the Lotka-Volterra system, and consequently of the second order ODE for the mass function must be non-Liouvillian. This makes the existence of other solutions of the isothermal fluid sphere problem with a Liouvillian metric quite unlikely.

  1. Coronal Alfvén speeds in an isothermal atmosphere. I. Global properties

    NASA Astrophysics Data System (ADS)

    Régnier, S.; Priest, E. R.; Hood, A. W.

    2008-11-01

    Aims: Estimating Alfvén speeds is of interest in modelling the solar corona, studying the coronal heating problem and understanding the initiation and propagation of coronal mass ejections (CMEs). Methods: We assume here that the corona is in a magnetohydrostatic equilibrium and that, because of the low plasma ?, one may decouple the magnetic forces from pressure and gravity. The magnetic field is then described by a force-free field for which we perform a statistical study of the magnetic field strength with height for four different active regions. The plasma along each field line is assumed to be in a hydrostatic equilibrium. As a first approximation, the coronal plasma is assumed to be isothermal with a constant or varying gravity with height. We study a bipolar magnetic field with a ring distribution of currents, and apply this method to four active regions associated with different eruptive events. Results: By studying the global properties of the magnetic field strength above active regions, we conclude that (i) most of the magnetic flux is localized within 50 Mm of the photosphere; (ii) most of the energy is stored in the corona below 150 Mm; (iii) most of the magnetic field strength decays with height for a nonlinear force-free field slower than for a potential field. The Alfvén speed values in an isothermal atmosphere can vary by two orders of magnitude (up to 100 000 km s-1). The global properties of the Alfvén speed are sensitive to the nature of the magnetic configuration. For an active region with highly twisted flux tubes, the Alfvén speed is significantly increased at the typical height of the twisted flux bundles; in flaring regions, the average Alfvén speeds are above 5000 km s-1 and depart highly from potential field values. Conclusions: We discuss the implications of this model for the reconnection rate and inflow speed, the coronal plasma ? and the Alfvén transit time.

  2. Liouvillian integrability of gravitating static isothermal fluid spheres

    SciTech Connect

    Iacono, Roberto; Llibre, Jaume

    2014-10-01

    We examine the integrability properties of the Einstein field equations for static, spherically symmetric fluid spheres, complemented with an isothermal equation of state, ? = np. In this case, Einstein's equations can be reduced to a nonlinear, autonomous second order ordinary differential equation (ODE) for m/R (m is the mass inside the radius R) that has been solved analytically only for n = -1 and n = -3, yielding the cosmological solutions by De Sitter and Einstein, respectively, and for n = -5, case for which the solution can be derived from the De Sitter's one using a symmetry of Einstein's equations. The solutions for these three cases are of Liouvillian type, since they can be expressed in terms of elementary functions. Here, we address the question of whether Liouvillian solutions can be obtained for other values of n. To do so, we transform the second order equation into an equivalent autonomous Lotka–Volterra quadratic polynomial differential system in R² and characterize the Liouvillian integrability of this system using Darboux theory. We find that the Lotka–Volterra system possesses Liouvillian first integrals for n = -1, -3, -5, which descend from the existence of invariant algebraic curves of degree one, and for n = -6, a new solvable case, associated to an invariant algebraic curve of higher degree (second). For any other value of n, eventual first integrals of the Lotka–Volterra system, and consequently of the second order ODE for the mass function must be non-Liouvillian. This makes the existence of other solutions of the isothermal fluid sphere problem with a Liouvillian metric quite unlikely.

  3. Precision retrieval of non-isothermal exo-atmospheres

    NASA Astrophysics Data System (ADS)

    Waldmann, Ingo Peter; Rocchetto, Marco

    2015-12-01

    Spectroscopy of extrasolar planets is as fast moving as it is new. When trying to characterise the atmospheres of these foreign worlds, we are faced with three challenges: 1) The correct treatment of atmospheric opacities at high temperatures, 2) Low signal-to-noise of the observed data, and 3) Large, degenerate parameter spaces. To advance in the interpretation of exoplanetary atmospheres, one must address these challenges in one coherent framework. This is particularly true for emission spectroscopy, where the need for non-isothermal temperature-pressure profiles significantly increases degeneracies in low signal-to-noise data. In the light of these challenges, we developed a novel, bayesian atmospheric retrieval suite, Tau-REx (Waldmann et al. 2015a,b). Tau-REx is a full line-by-line emission/transmission spectroscopy retrieval code based on the most complete hot line-lists from the ExoMol project. For emission spectroscopy, the correct retrieval of the atmosphere’s thermal gradient is extremely challenging with sparse and/or low SNR data. Tau-REx implements a novel two-stage retrieval algorithm which allows the code to iteratively adapt its retrieval complexity to the likelihood surface of the observed data. This way we achieve a very high retrieval accuracy and robustness to low SNR data. Using nested-sampling in conjunction with large scale cluster computing, Tau-REx integrates the full Bayesian Evidence, which allows for precise model selection of the exoplanet’s chemistry and thermal dynamics. Precision and statistical rigour is paramount in the measurement of quantities such as the carbon-oxygen ratio of planets which allow insights into the formation history of these exotic worlds. In this conference I will discuss the intricacies of retrieving the thermal emission of non-isothermal atmospheres and what can be learned from data of current and future facilities.

  4. Recent warming amplification over high elevation regions across the globe

    NASA Astrophysics Data System (ADS)

    Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

    2014-07-01

    Despite numerous studies in recent decades, our understanding of whether warming amplification is prevalent in high-elevation regions remains uncertain. In this work, on the basis of annual mean temperature series (1961-2010) of 2,367 stations around the globe, we examine both altitudinal amplification and regional amplification in the high elevation regions across the globe using new methodology. We develop the function equations of warming components of altitude, latitude and longitude and station warming rates for individual stations within a high-elevation region based on basic mathematic and physical principles, and find a significant altitudinal amplification trend for the Tibetan Plateau, Loess Plateau, Yunnan-Guizhou Plateau, Alps, United States Rockies, Appalachian Mountains, South American Andes and Mongolian Plateau. At the same time, we detect a greater warming for four high-elevation regions than their low elevation counterparts for the paired regions available. These suggest that warming amplification in high-elevation regions is an intrinsic feature of recent global warming.

  5. Study of chirped pulse amplification based on Raman backscattering

    NASA Astrophysics Data System (ADS)

    Yang, X.; Vieux, G.; Lyachev, A.; Farmer, J.; Raj, G.; Ersfeld, B.; Brunetti, E.; Wiggins, M.; Issac, R.; Jaroszynski, D. A.

    2009-05-01

    Raman backscattering (RBS) in plasma is an attractive source of intense, ultrashort laser pulses, which has the potential asa basic for a new generation of laser amplifiers.1 Taking advantage of plasma, which can withstand extremely high power densities and can offer high efficiencies over short distances, Raman amplification in plasma could lead to significant reductions in both size and cost of high power laser systems. Chirped laser pulse amplification through RBS could be an effective way to transfer energy from a long pump pulse to a resonant counter propagating short probe pulse. The probe pulse is spectrally broadened in a controlled manner through self-phase modulation. Mechanism of chirped pulse Raman amplification has been studied, and features of supperradiant growth associated with the nonlinear stage are observed in the linear regime. Gain measurements are briefly summarized. The experimental measurements are in qualitative agreement with simulations and theoretical predictions.

  6. Parametric amplification of attosecond pulse trains at 11?nm

    PubMed Central

    Seres, J.; Seres, E.; Landgraf, B.; Ecker, B.; Aurand, B.; Hoffmann, A.; Winkler, G.; Namba, S.; Kuehl, T.; Spielmann, C.

    2014-01-01

    We report the first experimental demonstration of the parametric amplification of attosecond pulse trains at around 11?nm. The helium amplifier is driven by intense laser pulses and seeded by high-order harmonics pulses generated in a neon gas jet. Our measurements suggest that amplification takes place only if the seed pulse-trains are perfectly synchronized in time with the driving laser field in the amplifier. Varying the delay, we estimate the durations of the individual extreme ultraviolet pulses within the train to be on the order of 0.2?fs. Our results demonstrate that strong-field parametric amplification can be a suitable tool to amplify weak attosecond pulses from non-destructive pump-probe experiments and it is an important step towards designing amplifiers for realization of energetic XUV pulses with sub-femtosecond duration using compact lasers fitting in university laboratories. PMID:24594502

  7. Weak-value amplification as an optimal metrological protocol

    NASA Astrophysics Data System (ADS)

    Alves, G. Bié; Escher, B. M.; de Matos Filho, R. L.; Zagury, N.; Davidovich, L.

    2015-06-01

    The implementation of weak-value amplification requires the pre- and postselection of states of a quantum system, followed by the observation of the response of the meter, which interacts weakly with the system. Data acquisition from the meter is conditioned to successful postselection events. Here we derive an optimal postselection procedure for estimating the coupling constant between system and meter and show that it leads both to weak-value amplification and to the saturation of the quantum Fisher information, under conditions fulfilled by all previously reported experiments on the amplification of weak signals. For most of the preselected states, full information on the coupling constant can be extracted from the meter data set alone, while for a small fraction of the space of preselected states, it must be obtained from the postselection statistics.

  8. Divided-pulse amplification for terawatt-class fiber lasers

    NASA Astrophysics Data System (ADS)

    Eidam, T.; Kienel, M.; Klenke, A.; Limpert, J.; Tünnermann, A.

    2015-10-01

    The coherent combination of ultra short laser pulses is a promising approach for scaling the average and peak power of ultrafast lasers. Fiber lasers and amplifiers are especially suited for this technique due to their simple singe-pass setups that can be easily parallelized. Here we propose the combination of the well-known approach of spatially separated amplification with the technique of divided-pulse amplification, i.e. an additionally performed temporally separated amplification. With the help of this multidimensional pulse stacking, laser systems come into reach capable of emitting 10's of joules of energy at multi-kW average powers that simultaneously employ a manageable number of fibers.

  9. Linear Amplification of Optical Signal in Coupled Photonic Crystal Waveguides

    E-print Network

    Jandieri, Vakhtang

    2015-01-01

    We introduce a weakly coupled photonic crystal waveguide as a promising and realistic model for all-optical amplification. A symmetric pillar type coupled photonic crystal waveguide consisting of dielectric rods periodically distributed in a free space is proposed as all-optical amplifier. Using the unique features of the photonic crystals to control and guide the light, we have properly chosen the frequency at which only one mode (odd mode) becomes the propagating mode in the coupled photonic crystal waveguide, whereas another mode (even mode) is completely reflected from the guiding structure. Under this condition, the all-optical amplification is fully realized. The amplification coefficient for the continuous signal and the Gaussian pulse is calculated.

  10. Numerical Simulation of a Single-Wafer Isothermal Plasma Etching Reactor

    E-print Network

    Economou, Demetre J.

    Numerical Simulation of a Single-Wafer Isothermal Plasma Etching Reactor Sang-Kyu Parkand Demetre J-plate single- wafer isothermal reactor was conducted. The oxygen plasma etching of polymer under high pressure as the flow rate increased. Etching rate increased but etching uniformity degraded as the wafer reactivity

  11. A Sixth-Form Teaching Unit on the Langmuir Adsorption Isotherm

    ERIC Educational Resources Information Center

    Walkley, G. H.

    1973-01-01

    Presents a teaching unit on the Langmuir absorption isotherm suitable for advanced secondary school chemistry classes. Describes the experimental investigation of the isothermal adsorption of sulfur dioxide on charcoal, and discusses the derivation of the Langmuir equation and some applications. (JR)

  12. Heat and mass transport in non-isothermal partially saturated oil-wax Antonio Fasano1

    E-print Network

    Fasano, Antonio

    Heat and mass transport in non-isothermal partially saturated oil-wax solutions Antonio Fasano1 of the main mechanisms at the origin of wax deposition, i.e. diffusion in non-isothermal solutions. We that wax migration to the wall is mainly driven by two mechanisms: (1) displacement of crystals suspended

  13. of the isothermal EulerPoisson system to the DriftDiffusion Equations

    E-print Network

    Relaxation of the isothermal Euler­Poisson system to the Drift­Diffusion Equations S. Junca and M06108 Nice C'edex 2 Abstract : We consider the one dimensional Euler­Poisson system, in the isothermal describes the collisions between the electrons and the atoms of the crystal. Obviously, equation (1

  14. Determination of thermal inactivation kinetics of Listeria monocytogenes in chicken meat by isothermal and dynamic methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this research is to determine the thermal inactivation kinetics of Listeria monocytogenes in chicken breast meat using both isothermal and dynamic conditions. A four-strain cocktail of L. monocytogenes was inoculated to chicken breast meat. Isothermal studies were performed by sub...

  15. Revisiting Isotherm Analyses Using R: Comparison of Linear, Non-linear, and Bayesian Techniques

    EPA Science Inventory

    Extensive adsorption isotherm data exist for an array of chemicals of concern on a variety of engineered and natural sorbents. Several isotherm models exist that can accurately describe these data from which the resultant fitting parameters may subsequently be used in numerical ...

  16. Surface Adsorption Isotherms and Surface Excess Densities of n-Butane in Silicalite-1

    E-print Network

    Kjelstrup, Signe

    Surface Adsorption Isotherms and Surface Excess Densities of n-Butane in Silicalite-1 Isabella 27, 2008. ReVised Manuscript ReceiVed NoVember 13, 2008 We present isotherms for the adsorption of n were considered: a flat surface having only one adsorption site and a surface with a zigzag texture

  17. EFFECTS OF TEMPERATURE ON TRICHLOROETHYLENE DESORPTION FROM SILICA GEL AND NATURAL SEDIMENTS. 1. ISOTHERMS. (R822626)

    EPA Science Inventory

    Aqueous phase isotherms were calculated from vapor phase desorption isotherms
    measured at 15, 30, and 60 C for
    trichloroethylene on a silica gel, an aquifer sediment, a soil, a sand fraction,
    and a clay and silt fraction, all at...

  18. FREUNDLICH ISOTHERM: SOME LIMITATIONS IN ITS USE FOR PESTICIDE ENVIRONMENTAL FATE MODELING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Freundlich isotherm robustly fits most experimental adsorption-desorption isotherm curves, and is especially good for fitting data from highly heterogeneous sorbent systems, including soils. In a "batch" aqueous pesticide soil adsorption experiment, the data may be fitted to the Freundlich equa...

  19. The Langmuir isotherm: a commonly applied but misleading approach for the analysis of protein adsorption behavior.

    PubMed

    Latour, Robert A

    2015-03-01

    The Langmuir adsorption isotherm provides one of the simplest and most direct methods to quantify an adsorption process. Because isotherm data from protein adsorption studies often appear to be fit well by the Langmuir isotherm model, estimates of protein binding affinity have often been made from its use despite that fact that none of the conditions required for a Langmuir adsorption process may be satisfied for this type of application. The physical events that cause protein adsorption isotherms to often provide a Langmuir-shaped isotherm can be explained as being due to changes in adsorption-induced spreading, reorientation, clustering, and aggregation of the protein on a surface as a function of solution concentration in contrast to being due to a dynamic equilibrium adsorption process, which is required for Langmuir adsorption. Unless the requirements of the Langmuir adsorption process can be confirmed, fitting of the Langmuir model to protein adsorption isotherm data to obtain thermodynamic properties, such as the equilibrium constant for adsorption and adsorption free energy, may provide erroneous values that have little to do with the actual protein adsorption process, and should be avoided. In this article, a detailed analysis of the Langmuir isotherm model is presented along with a quantitative analysis of the level of error that can arise in derived parameters when the Langmuir isotherm is inappropriately applied to characterize a protein adsorption process. PMID:24853075

  20. Using Compression Isotherms of Phospholipid Monolayers to Explore Critical Phenomena: A Biophysical Chemistry Experiment

    ERIC Educational Resources Information Center

    Gragson, Derek E.; Beaman, Dan; Porter, Rhiannon

    2008-01-01

    Two experiments are described in which students explore phase transitions and critical phenomena by obtaining compression isotherms of phospholipid monolayers using a Langmuir trough. Through relatively simple analysis of their data students gain a better understanding of compression isotherms, the application of the Clapeyron equation, the…