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Sample records for autocatalytic loop amplification

  1. (PCR) and loop-mediated isothermal amplification

    African Journals Online (AJOL)

    SAM

    2014-03-26

    Mar 26, 2014 ... better alternative for PCR, even in low technology laboratories. In addition, these findings revealed that the possibility of fatal fusariosis due to F. solani is not so rare in HIV positive patients. Key words: Fusarium solani, loop-mediated isothermal amplification (LAMP), HIV, polymerase chain reaction. (PCR).

  2. Evaluation of loop mediated isothermal amplification for diagnosis of ...

    African Journals Online (AJOL)

    Tuberculosis (TB) remains an important global public health problem. The lack of rapid and accurate diagnostic testing is an important impediment to global tuberculosis control. Loop mediated isothermal amplification (LAMP) is a rapid method for nucleic acid amplification. In this study, we assessed the performance of an ...

  3. Combined prokaryotic-eukaryotic delivery and expression of therapeutic factors through a primed autocatalytic positive-feedback loop.

    Science.gov (United States)

    Shi, Lei; Yu, Bin; Cai, Chun-Hui; Huang, Wei; Zheng, Bo-Jian; Smith, David Keith; Huang, Jian-Dong

    2016-01-28

    Progress in bacterial therapy for cancer and infectious diseases is hampered by the absence of safe and efficient vectors. Sustained delivery and high gene expression levels are critical for the therapeutic efficacy. Here we developed a Salmonella typhimrium strain to maintain and safely deliver a plasmid vector to target tissues. This vector is designed to allow dual transcription of therapeutic factors, such as cytotoxic proteins, short hairpin RNAs or combinations, in the nucleus or cytoplasm of eukaryotic cells, with this expression sustained by an autocatalytic positive-feedback loop. Mechanisms to prime the system and maintain the plasmid in the bacterium are also provided. Synergistic effects of attenuated Salmonella and our inter-kingdom system allow the precise expression of Diphtheria toxin A chain (DTA) gene in tumor microenvironment and eradicate large established tumors in immunocompetent animals. In the experiments reported here, 26% of mice (n=5/19) with aggressive tumors were cured and the others all survived until the end of the experiment. We also demonstrated that ST4 packaged with shRNA-encoding plasmids has sustained knockdown effects in nude mice bearing human MDA-MB-231 xenografts. Three weeks after injection of 5×10(6) ST4/pIKT-shPlk, PLK1 transcript levels in tumors were 62.5±18.6% lower than the vector control group (P=0.015). The presence of PLK1 5' RACE-PCR cleavage products confirmed a sustained RNAi-mediated mechanism of action. This innovative technology provides an effective and versatile vehicle for efficient inter-kingdom gene delivery that can be applied to cancer therapy and other purposes. Copyright © 2015. Published by Elsevier B.V.

  4. Application of loop-mediated isothermal amplification (LAMP) of the ...

    African Journals Online (AJOL)

    RIME-LAMP helps increase the probability of detecting the positive cases of camel Trypanosomiasis and it was found to be one useful and alternative molecular diagnostic tool for the detection of T. evansi infections. Key words: Sudan, loop-mediated isothermal amplification (LAMP) - random insertion mobile element (RIME ...

  5. Development of a loop-mediated isothermal amplification assay for ...

    African Journals Online (AJOL)

    Administrator

    2011-09-28

    Sep 28, 2011 ... A loop-mediated isothermal amplification (LAMP) assay targeting uvrC of Mycoplasma bovis was developed and evaluated. The assay specifically amplified only M. bovis; no cross-reactivity was observed for other Mycoplasma species or respiratory bacterial species. The sensitivity of the assay in.

  6. Development of loop-mediated isothermal amplification method for ...

    African Journals Online (AJOL)

    A novel assay method to detect the highly virulent Porcine reproductive and respiratory syndrome virus (PRRSV) termed reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), was reported by using hydroxynaphthol blue (HNB) as the LAMP product colorimetric judgment. By the set of special primers, ...

  7. Progress in multiplex loop-mediated isothermal amplification technology.

    Science.gov (United States)

    Lin, Wen-hui; Zou, Bing-jie; Song, Qin-xin; Zhou, Guo-hua

    2015-09-01

    Loop-mediated isothermal amplification (LAMP) has been widely applied in nucleic acid diagnostics due to its high sensitivity and specificity, high speed and low requirement of equipment. In order to fully leverage these merits, achieve high efficiency and reliability in diagnostics, and expand the applicable fields while keeping low reagent cost, multiplex LAMP technology has been extensively explored in recent years. Common methods for LAMP products detection are mostly based on the double-stranded DNA amplicons or byproducts from the polymerization reaction, so they can only identify the occurrence of amplification reaction but not the origins or specificity of the products. To achieve specific LAMP products detection, researchers developed various multiplex methods by improving the conventional LAMP technology or coupling LAMP with other assays. However, the interference and/or the different amplification efficiencies among different primer sets often lead to biased amplification and thus limited multiplexing level. We here defined these methods as narrow-sensed multiplex LAMP. The research on miniaturized amplification technology which is booming in recent years has given rise to the novel general-sensed multiplex LAMP technology that breaks this limitation by its capability to perform highly parallel and miniaturized simplex reactions in independent compartments. Methods of this type have additional benefits such as lower reagent cost, higher level of automation, lower risk of cross-contamination and better suitability for on-site detection of multiple targets. In this review, we summarize the recent research progress in multiplex LAMP technology from the following aspects: the principle and design of narrow-sensed LAMP and its amplification optimization, the general-sensed LAMP, and the various applications of all multiplex LAMP technologies in diagnostics.

  8. Detection of Botrytis cinerea by loop-mediated isothermal amplification.

    Science.gov (United States)

    Tomlinson, J A; Dickinson, M J; Boonham, N

    2010-12-01

    To develop a sensitive, rapid and simple method for detection of Botrytis cinerea based on loop-mediated isothermal amplification (LAMP) that would be suitable for use outside a conventional laboratory setting. A LAMP assay was designed based on the intergenic spacer of the B. cinerea nuclear ribosomal DNA (rDNA). The resulting assay was characterized in terms of sensitivity and specificity using DNA extracted from cultures. The assay consistently amplified 65 pg B. cinerea DNA. No cross-reactivity was observed with a range of other fungal pathogens, with the exception of the closely related species Botrytis pelargonii. Use of a novel real-time LAMP platform (the OptiGene Genie I) allowed detection of B. cinerea in infected rose petals, with amplification occurring in cut flowers, fruit and vegetables. © 2010 British Crown Copyright. Letters in Applied Microbiology 51, 650-657 © 2010 The Society for Applied Microbiology.

  9. Comparison of nucleic acid sequence-based amplification and loop-mediated isothermal amplification for diagnosis of human African trypanosomiasis

    NARCIS (Netherlands)

    Mugasa, Claire M.; Katiti, Diana; Boobo, Alex; Lubega, George W.; Schallig, Henk D. F. H.; Matovu, Enock

    2014-01-01

    Diagnosis of human African trypanosomiasis (HAT) using molecular tests should ideally achieve high sensitivity without compromising specificity. This study compared 2 simplified tests, nucleic acid sequence-based amplification (NASBA) combined with oligochromatography (OC) and loop-mediated

  10. Detection of fish nocardiosis by loop-mediated isothermal amplification.

    Science.gov (United States)

    Itano, T; Kawakami, H; Kono, T; Sakai, M

    2006-06-01

    Loop-mediated isothermal amplification (LAMP) is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. In this study, using the LAMP method, a protocol for detecting Nocardia seriolae which is a causative agent of fish nocardiosis, was designed. A set of four primers, two inner and two outer, were designed based on the sequence of the 16S-23S ribosomal RNA internal transcribed spacer region of N. seriolae. Time and temperature conditions for detection of N. seriolae were optimized for 60 min at 65 degrees C. Other fish pathogen was not amplified by this LAMP system. The detection of N. seriola using LAMP was found to be more sensitive than that by polymerase chain reaction. LAMP is a highly sensitive and rapid diagnostic procedure for detection of N. seriolae. LAMP is a useful diagnostic method for fish nocardiosis.

  11. Microfluidic continuous flow digital loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Rane, Tushar D; Chen, Liben; Zec, Helena C; Wang, Tza-Huei

    2015-02-07

    Digital nucleic acid detection is rapidly becoming a popular technique for ultra-sensitive and quantitative detection of nucleic acid molecules in a wide range of biomedical studies. Digital polymerase chain reaction (PCR) remains the most popular way of conducting digital nucleic acid detection. However, due to the need for thermocycling, digital PCR is difficult to implement in a streamlined manner on a single microfluidic device, leading to complex fragmented workflows and multiple separate devices and instruments. Loop-mediated isothermal amplification (LAMP) is an excellent isothermal alternative to PCR with potentially better specificity than PCR because of the use of multiple primer sets for a nucleic acid target. Here we report a microfluidic droplet device implementing all the steps required for digital nucleic acid detection including droplet generation, incubation and in-line detection for digital LAMP. As compared to microchamber or droplet array-based digital assays, the continuous flow operation of this device eliminates the constraints on the number of total reactions imposed by the footprint of the device and the analysis throughput caused by the time for lengthy incubation and transfer of materials between instruments.

  12. Loop-mediated isothermal amplification (LAMP) shield for Arduino DNA detection

    NARCIS (Netherlands)

    Velders, Aldrik H.; Schoen, Cor; Saggiomo, Vittorio

    2018-01-01

    Objective: Loop-mediated isothermal amplification (LAMP) of DNA is gaining relevance as a method to detect nucleic acids, as it is easier, faster, and more powerful than conventional Polymerase Chain Reaction. However, LAMP is still mostly used in laboratory settings, because of the lack of a cheap

  13. Rapid Newcastle Disease Virus Detection Based on Loop-Mediated Isothermal Amplification and Optomagnetic Readout

    DEFF Research Database (Denmark)

    Tian, Bo; Ma, Jing; Zardán Gómez de la Torre, Teresa

    2016-01-01

    Rapid and sensitive diagnostic methods based on isothermal amplification are ideal substitutes for PCR in out-of-lab settings. However, there are bottlenecks in terms of establishing low-cost and user-friendly readout methods for isothermal amplification schemes. Combining the high amplification...... efficiency of loop-mediated isothermal amplification (LAMP) with an optomagnetic nanoparticle-based readout system, we demonstrate ultrasensitive and rapid detection of Newcastle disease virus RNA. Biotinylated amplicons of LAMP and reverse transcription LAMP (RT-LAMP) bind to streptavidin-coated magnetic...... nanoparticles (MNPs) resulting in a dramatical increase in the hydrodynamic size of the MNPs. This increase was measured by an optomagnetic readout system and provided quantitative information on the amount of LAMP target sequence. Our assay resulted in a limit of detection of 10 aM of target sequence...

  14. An evaluation of multiple annealing and looping based genome amplification using a synthetic bacterial community

    KAUST Repository

    Wang, Yong

    2016-02-23

    The low biomass in environmental samples is a major challenge for microbial metagenomic studies. The amplification of a genomic DNA was frequently applied to meeting the minimum requirement of the DNA for a high-throughput next-generation-sequencing technology. Using a synthetic bacterial community, the amplification efficiency of the Multiple Annealing and Looping Based Amplification Cycles (MALBAC) kit that is originally developed to amplify the single-cell genomic DNA of mammalian organisms is examined. The DNA template of 10 pg in each reaction of the MALBAC amplification may generate enough DNA for Illumina sequencing. Using 10 pg and 100 pg templates for each reaction set, the MALBAC kit shows a stable and homogeneous amplification as indicated by the highly consistent coverage of the reads from the two amplified samples on the contigs assembled by the original unamplified sample. Although GenomePlex whole genome amplification kit allows one to generate enough DNA using 100 pg of template in each reaction, the minority of the mixed bacterial species is not linearly amplified. For both of the kits, the GC-rich regions of the genomic DNA are not efficiently amplified as suggested by the low coverage of the contigs with the high GC content. The high efficiency of the MALBAC kit is supported for the amplification of environmental microbial DNA samples, and the concerns on its application are also raised to bacterial species with the high GC content.

  15. Loop-mediated isothermal amplification method for a differential identification of human Taenia tapeworms.

    Science.gov (United States)

    Sako, Yasuhito; Nkouawa, Agathe; Yanagida, Tetsuya; Ito, Akira

    2013-01-01

    Loop-mediated isothermal amplification (LAMP), which employs a Bst DNA polymerase with strand-displacement activity and four primers (two inner primers and two outer primers) recognizing six distinct regions on the target DNA, is a highly sensitive, specific, simple, and rapid nucleotide amplification method. Moreover, because the Bst DNA polymerase resists much DNA polymerase inhibitors present in biological specimens, the LAMP method is suitable for the detection of infectious agents from clinical material such as fecal samples. Here, we describe the LAMP method which can differentially detect and identify human Taenia tapeworms, Taenia solium, T. saginata, and T. asiatica, using DNA specimens prepared from parasite tissue and human fecal sample.

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

    OpenAIRE

    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. Mult...

  17. Comparison of nucleic acid sequence-based amplification and loop-mediated isothermal amplification for diagnosis of human African trypanosomiasis.

    Science.gov (United States)

    Mugasa, Claire M; Katiti, Diana; Boobo, Alex; Lubega, George W; Schallig, Henk D F H; Matovu, Enock

    2014-02-01

    Diagnosis of human African trypanosomiasis (HAT) using molecular tests should ideally achieve high sensitivity without compromising specificity. This study compared 2 simplified tests, nucleic acid sequence-based amplification (NASBA) combined with oligochromatography (OC) and loop-mediated isothermal amplification (LAMP), executed on 181 blood samples from 65 Trypanosoma brucei gambiense HAT patients, 86 controls, and 30 serological suspects from Uganda. Basing on the composite reference standard, the diagnostic sensitivity and specificity of NASBA were 93.9% (95% confidence interval [CI] = 84.9-98.3%) and 100% (95% CI = 94.9-100%), respectively. The same parameters for LAMP were 76.9% (95% CI = 64.8-86.5%) and 100% (95% CI = 91.6-100%), respectively. The level of agreement between LAMP and microscopy was good with a kappa (κ) value of 79.2% (95% CI = 69.4-88.9%), while that of NASBA-OC/microscopy was very good (κ value 94.6%; 95% CI = 89.3-99.8%). The sensitivity of NASBA-OC was significantly higher than that of LAMP (Z = 2.723; P = 0.007). These tests have potential application to HAT surveillance. © 2013.

  18. A novel thermostable polymerase for RNA and DNA Loop-mediated isothermal amplification (LAMP

    Directory of Open Access Journals (Sweden)

    Yogesh eChander

    2014-08-01

    Full Text Available Meeting the goal of providing point of care (POC tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol is a thermostable viral enzyme that enables true POC use in clinics or in field by overcoming important barriers to isothermal amplification. In this paper, we describe the multiple advantages of OmniAmp Pol as an isothermal amplification enzyme and provide examples of its use in loop-mediated isothermal amplification (LAMP for pathogen detection. The inherent reverse transcriptase activity of OmniAmp Pol allows single enzyme detection of RNA targets in RT-LAMP. Common methods of nucleic acid amplification are highly susceptible to sample contaminants, necessitating elaborate nucleic acid purification protocols that are incompatible with POC or field use. OmniAmp Pol was found to be less inhibited by whole blood components typical in certain crude sample preparations . Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions. Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates. Molecular diagnostics in field settings can be challenging due to the lack of refrigeration. The stability of OmniAmp Pol is compatible with a dry format that enables long term storage at ambient temperatures. A final requirement for field operability is compatibility with either commonly available instruments or, in other cases, a simple, inexpensive, portable detection mode requiring minimal training or power. Detection of amplification products is shown using lateral flow strips and analysis on a real-time PCR instrument. Results of this study show that OmniAmp Pol is ideally suited for low resource molecular

  19. Detection of Enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification

    OpenAIRE

    D Wang; X Wang; Y Geng; C An

    2014-01-01

    Purpose : The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD) for an early treatment by using loop-mediated isothermal amplification (LAMP) technique. Materials and Methods : A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for detecting EV71 virus was developed, the specificity and sensitivity of RT-LAMP was tested, and the clinical specimens was assayed by the RT-LAMP comparing with conven...

  20. [Colorimetric detection of HPV6 and HPV16 by loop mediated isothermal amplification].

    Science.gov (United States)

    Lu, Chun-bin; Luo, Le; Yang, Meng-jie; Nie, Kai; Wang, Miao; Ma, Xue-Jun

    2011-01-01

    A simple, rapid and sensitive colorimetric loop mediated isothermal amplification (LAMP) method was established to detect HPV6 and HPV 16 respectively. The method employed a set of four specially designed primers that recognized six distinct sequences of HPV6-E6 or HPV16-E7 for amplification of nucleic acid under isothermal conditions at 63 degrees C for one hour. The amplification process of LAMP was monitored by the addition of HNB (hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by real-time turbidimeter and agarose electrophoresis. Thirteen cervical swab samples having single infection with 13 different HPV genotypes were examined to evaluate the specificity. A serial dilution of a cloned plasmid containing HPV-E6 or HPV-E7 gene was examined to evaluate the sensitivity. The results showed that no cross-reaction with other HPV genotypes was observed. The colorimetric LAMP assay could achieve a sensitivity of 1000 copies, 10-20 times lower than that of real-time PCR. The assay was further evaluated with 62 clinical specimens and consistent results were obtained compared with the detection using Kai Pu HPV Genotyping Kit. We concluded that this colorimetric LAMP assay had potential usefulness for the rapid screening of the HPV6 or HPV16 infection in the laboratories and hospitals of provincial and municipal region in China.

  1. Simple, rapid, inexpensive platform for the diagnosis of malaria by loop mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Surabattula, Rambabu; Vejandla, Manju Pradeep; Mallepaddi, Prudhvi Chand; Faulstich, Konrad; Polavarapu, Rathnagiri

    2013-07-01

    We attempted to improve the loop-mediated isothermal amplification (LAMP) method for malaria diagnosis by using a simple DNA extraction procedure, and a portable device performing both the amplification and detection of LAMP in one platform. Additionally, the device served as a heating block for the DNA preparation. We refer this method as LAMP-Tube scanner, and evaluated using 209 microscopically positive malaria samples and compared them to RDTs and LAMP-Thermocycler. Two most common human infecting Plasmodium species were detected. The LAMP-Tube scanner method is found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was closely less than 60 min. Sensitivity and specificity of LAMP-Tube scanner in detecting Plasmodium falciparum were 95% and 93.3%, compared to microscopy and 98.3% and 100% respectively, compared to standard LAMP-Thermocycler. In addition, it showed a detection limit of 10 and 40 copies of the parasitemia for Plasmodium vivax and P. falciparum. Accordingly, in comparison to the results obtained by microscopy, the LAMP-Tube scanner had a less divergence in sensitivity and specificity, and yielded results similar to those of LAMP-Thermocycler. This method has the great potential as a field usable molecular tool for the diagnosis of malaria and is an alternative to conventional PCR-based diagnostic methods for field use. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Visual detection of murray valley encephalitis virus by reverse transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Gong, Rui; Wang, Han Hua; Qin, Hong; Guo, Xiao Ping; Ma, Xue Jun

    2015-03-01

    A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of Murray valley encephalitis virus (MVEV) infection. The reaction was performed in one step in a single tube at 63 °C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limit of the RT-LAMP assay was 100 copies per reaction based on 10-fold dilutions of in vitro transcribed RNA derived from a synthetic MVEV DNA template. No cross-reaction was observed with other encephalitis-associated viruses. The assay was further evaluated using spiked cerebrospinal fluid sample with pseudotype virus containing the NS5 gene of MVEV. Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  3. Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Detection of Acinetobacter baumannii

    Science.gov (United States)

    Wang, Qinqin; Zhou, Yanbin; Li, Shaoli; Zhuo, Chao; Xu, Siqi; Huang, Lixia; Yang, Ling; Liao, Kang

    2013-01-01

    Background Detection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii. Methodology and Significant Findings Species-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively. Conclusion The RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has

  4. Real-time fluorescence loop mediated isothermal amplification for the detection of Acinetobacter baumannii.

    Directory of Open Access Journals (Sweden)

    Qinqin Wang

    Full Text Available BACKGROUND: Detection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii. METHODOLOGY AND SIGNIFICANT FINDINGS: Species-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively. CONCLUSION: The RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection

  5. Loop-mediated isothermal amplification (LAMP) shield for Arduino DNA detection.

    Science.gov (United States)

    Velders, Aldrik H; Schoen, Cor; Saggiomo, Vittorio

    2018-02-01

    Loop-mediated isothermal amplification (LAMP) of DNA is gaining relevance as a method to detect nucleic acids, as it is easier, faster, and more powerful than conventional Polymerase Chain Reaction. However, LAMP is still mostly used in laboratory settings, because of the lack of a cheap and easy, one-button device that can perform LAMP experiments. Here we show how to build and program an Arduino shield for a LAMP and detection of DNA. The here described Arduino Shield is cheap, easy to assemble, to program and use, it is battery operated and the detection of DNA is done by naked-eye so that it can be used in field.

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

    Directory of Open Access Journals (Sweden)

    Siwon Lee

    2015-12-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2013-11-01

    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.

  8. LAVA: An Open-Source Approach To Designing LAMP (Loop-Mediated Isothermal Amplification DNA Signatures

    Directory of Open Access Journals (Sweden)

    Gardner Shea N

    2011-06-01

    Full Text Available Abstract Background We developed an extendable open-source Loop-mediated isothermal AMPlification (LAMP signature design program called LAVA (LAMP Assay Versatile Analysis. LAVA was created in response to limitations of existing LAMP signature programs. Results LAVA identifies combinations of six primer regions for basic LAMP signatures, or combinations of eight primer regions for LAMP signatures with loop primers, which can be used as LAMP signatures. The identified primers are conserved among target organism sequences. Primer combinations are optimized based on lengths, melting temperatures, and spacing among primer sites. We compare LAMP signature candidates for Staphylococcus aureus created both by LAVA and by PrimerExplorer. We also include signatures from a sample run targeting all strains of Mycobacterium tuberculosis. Conclusions We have designed and demonstrated new software for identifying signature candidates appropriate for LAMP assays. The software is available for download at http://lava-dna.googlecode.com/.

  9. Simultaneous elimination of carryover contamination and detection of DNA with uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP).

    Science.gov (United States)

    Hsieh, Kuangwen; Mage, Peter L; Csordas, Andrew T; Eisenstein, Michael; Soh, H Tom

    2014-04-11

    We report a one-pot, closed-vessel enzymatic assay that eliminates carryover contamination while preserving robust DNA amplification in loop-mediated isothermal amplification (LAMP), providing reliable and rapid detection of target DNA in contaminated samples.

  10. Rapid and sensitive diagnosis of Acanthamoeba keratitis by loop-mediated isothermal amplification.

    Science.gov (United States)

    Ge, Z; Qing, Y; Zicheng, S; Shiying, S

    2013-11-01

    A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of Acanthamoeba. The sensitivity of the LAMP assay was tested using different copies of positive DNA. The specificity of the assay was tested using DNA extracted from Acanthamoeba, Pseudomonas aeruginosa, Candida albicans, herpes simplex virus-1 and human corneal epithelial cells. Its effectiveness was evaluated and compared with culture, corneal smear examination and real-time PCR in corneal samples from mice with Acanthamoeba keratitis. We also tested three corneal samples from patients with suspected Acanthamoeba or fungal infection using LAMP. Loop-mediated isothermal amplification was confirmed to be very sensitive, with the lowest detection limit being ten copies/tube of Acanthamoeba DNA. The LAMP primers only amplified Acanthamoeba DNA. During the development of Acanthamoeba keratitis in mice, almost all of the positive rates of LAMP at each time post-infection were higher than those of culture or corneal smear examination. The total positive rate of LAMP was significantly higher than those of culture and corneal smear examination (p Acanthamoeba keratitis tested positive for Acanthamoeba using LAMP along with culture or corneal smear examination, whereas the other suspected fungal keratitis tested negative. The LAMP assay is a simple, rapid, highly specific and sensitive method for the diagnosis of keratitis caused by Acanthamoeba. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  13. A Digital Microfluidics Platform for Loop-Mediated Isothermal Amplification Detection

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    Beatriz Jorge Coelho

    2017-11-01

    Full Text Available Digital microfluidics (DMF arises as the next step in the fast-evolving field of operation platforms for molecular diagnostics. Moreover, isothermal schemes, such as loop-mediated isothermal amplification (LAMP, allow for further simplification of amplification protocols. Integrating DMF with LAMP will be at the core of a new generation of detection devices for effective molecular diagnostics at point-of-care (POC, providing simple, fast, and automated nucleic acid amplification with exceptional integration capabilities. Here, we demonstrate for the first time the role of coupling DMF and LAMP, in a dedicated device that allows straightforward mixing of LAMP reagents and target DNA, as well as optimum temperature control (reaction droplets undergo a temperature variation of just 0.3 °C, for 65 °C at the bottom plate. This device is produced using low-temperature and low-cost production processes, adaptable to disposable and flexible substrates. DMF-LAMP is performed with enhanced sensitivity without compromising reaction efficacy or losing reliability and efficiency, by LAMP-amplifying 0.5 ng/µL of target DNA in just 45 min. Moreover, on-chip LAMP was performed in 1.5 µL, a considerably lower volume than standard bench-top reactions.

  14. Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification: A Novel Analytically Rapid, Sensitive, Multiplex Loop-Mediated Isothermal Amplification Detection Technique.

    Science.gov (United States)

    Wang, Yi; Wang, Yan; Lan, Ruiting; Xu, Huaqing; Ma, Aijing; Li, Dongxun; Dai, Hang; Yuan, Xuejiao; Xu, Jianguo; Ye, Changyun

    2015-07-01

    Loop-mediated isothermal amplification (LAMP) is restricted to detecting a single target, limiting the usefulness of this method. To achieve multiplex LAMP-based detection, we developed a novel approach we called the multiple endonuclease restriction real-time-LAMP assay. In this system, the LAMP forward or backward inner primers contain 5' end short sequences that are recognized by the restriction endonuclease Nb.BsrDI, and the new forward or backward inner primers were modified at the 5' end with a fluorophore and in the middle with a dark quencher. Nb.BsrDI digests the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which releases the quenching, resulting in a gain of signal. The assay permitted real-time detection of single or multiple target sequences in a single tube, and the positive results can be obtained in as short as 12 minutes. The novel methodology is highly efficient and specific, detecting down to 250 fg of DNA per reaction of Listeria DNA tested, and was successful in evaluating raw meat samples. The multiple endonuclease restriction real-time-LAMP technology, which is an extension of LAMP to accommodate robust, target-specific, and multiplex detection, provides a molecular diagnostic tool with less detection time and high sensitivity and specificity compared with those of LAMP and quantitative real-time PCR. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  15. Self-organized critical noise amplification in human closed loop control

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    Felix Patzelt

    2007-11-01

    Full Text Available When humans perform closed loop control tasks like in upright standing or while balancing a stick, their behavior exhibits non-Gaussian fluctuations with long-tailed distributions. The origin of these fluctuations is not known. Here, we investigate if they are caused by selforganized critical noise amplification which emerges in control systems when an unstable dynamics becomes stabilized by an adaptive controller that has finite memory. Starting from this theory, we formulate a realistic model of adaptive closed loop control by including constraints on memory and delays. To test this model, we performed psychophysical experiments where humans balanced an unstable target on a screen. It turned out that the model reproduces the long tails of the distributions together with other characteristic features of the human control dynamics. Fine-tuning the model to match the experimental dynamics identifies parameters characterizing a subject’s control system which can be independently tested. Our results suggest that the nervous system involved in closed loop motor control nearly optimally estimates system parameters on-line from very short epochs of past observations.

  16. Detection of swine transmissible gastroenteritis coronavirus using loop-mediated isothermal amplification

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    Chen Qin

    2010-08-01

    Full Text Available Abstract A conserved nucleic acid fragment of the nucleocapsid gene of Swine Transmissible Gastroenteritis Coronavirus (TGEV was chosen as the target, six special primers were designed successfully. Loop-mediated isothermal amplification (LAMP was developed to detect the TGEV by incubation at 60°C for 1 h and the product specificity was confirmed by HphI digestion. Standard curves with high accuracy for TGEV quantization was constructed by adding 1 × SYBR greenI in the LAMP reaction. The assay established in this study was found to detect only the TGEV and no cross-reaction with other viruses, demonstrating its high specificity. By using serial sample dilutions as templates, the detection limit of LAMP was about 10 pg RNA, 10 times more sensitive than that of PCR and could be comparable to the nest-PCR.

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

    Science.gov (United States)

    Kinoshita, Yuta; Niwa, Hidekazu; Katayama, Yoshinari; Hariu, Kazuhisa

    2015-01-01

    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.

  18. An investigation of the open-loop amplification of Reynolds number dependent processes by wave distortion

    Science.gov (United States)

    Purdy, K. R.; Ventrice, M. B.; Fang, J.

    1972-01-01

    Analytical and experimental studies were initiated to determine if the response of a constant temperature hot wire anemometer to acoustic oscillations could serve as an analog to the response of the drop vaporization burning rate process to acoustic oscillations, and, perhaps, also as an analog to any Reynolds number dependent process. The motivation behind this study was a recent analytical study which showed that distorted acoustic oscillations could amplify the open-loop response of vaporization limited combustion. This type of amplification may be the cause of unstable combustion in liquid propellant rocket engines. The analytical results obtained for the constant temperature anemometer are similar in nature to those previously obtained for vaporization limited combustion and indicate that the response is dependent on the amount and type of distortion as well as other factors, such as sound pressure level, Mach number and hot wire temperature. Preliminary results indicate qualitative agreement between theory and experiment.

  19. Detection of Mycobacterium ulcerans by the loop mediated isothermal amplification method.

    Science.gov (United States)

    Ablordey, Anthony; Amissah, Diana Ackon; Aboagye, Isaac Frimpong; Hatano, Ben; Yamazaki, Toshio; Sata, Tetsutaro; Ishikawa, Koichi; Katano, Harutaka

    2012-01-01

    Buruli ulcer (BU) caused by Mycobacterium ulcerans (M. ulcerans) has emerged as an important public health problem in several rural communities in sub-Saharan Africa. Early diagnosis and prompt treatment are important in preventing disfiguring complications associated with late stages of the disease progression. Presently there is no simple and rapid test that is appropriate for early diagnosis and use in the low-resource settings where M. ulcerans is most prevalent. We compared conventional and pocket warmer loop mediated isothermal amplification (LAMP) methods (using a heat block and a pocket warmer respectively as heat source for amplification reaction) for the detection of M. ulcerans in clinical specimens. The effect of purified and crude DNA preparations on the detection rate of the LAMP assays were also investigated and compared with that of IS2404 PCR, a reference assay for the detection of M. ulcerans. Thirty clinical specimens from suspected BU cases were examined by LAMP and IS2404 PCR. The lower detection limit of both LAMP methods at 60°C was 300 copies of IS2404 and 30 copies of IS2404 for the conventional LAMP at 65°C. When purified DNA extracts were used, both the conventional LAMP and IS2404 PCR concordantly detected 21 positive cases, while the pocket warmer LAMP detected 19 cases. Nine of 30 samples were positive by both the LAMP assays as well as IS2404 PCR when crude extracts of clinical specimens were used. The LAMP method can be used as a simple and rapid test for the detection of M. ulcerans in clinical specimens. However, obtaining purified DNA, as well as generating isothermal conditions, remains a major challenge for the use of the LAMP method under field conditions. With further improvement in DNA extraction and amplification conditions, the pwLAMP could be used as a point of care diagnostic test for BU.

  20. Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay.

    Science.gov (United States)

    Zhou, Dinggang; Wang, Chunfeng; Li, Zhu; Chen, Yun; Gao, Shiwu; Guo, Jinlong; Lu, Wenying; Su, Yachun; Xu, Liping; Que, Youxiong

    2016-01-01

    Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg(2+), 6:1 ratio of inner vs. outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was 10-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100%) by LAMP and 97/100 cases (97%) by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable, and cost-effective for detection of the bar specific transgenic sugarcane.

  1. Detection of bar transgenic sugarcane with a rapid and visual loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Dinggang eZhou

    2016-03-01

    Full Text Available Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg2+, 6:1 ratio of inner vs outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was ten-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100% by LAMP and 97/100 cases (97% by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable and cost-effective for detection of the bar specific transgenic sugarcane.

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

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    Ryo Kubota

    2015-03-01

    Full Text Available 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 λ-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.

  3. Loop-mediated isothermal amplification for rapid and convenient detection of Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Li, Jiahe; Minion, F Chris; Petersen, Andrew C; Jiang, Fei; Yang, Sheng; Guo, Panpan; Li, Jinxiang; Wu, Wenxue

    2013-04-01

    Loop-mediated isothermal amplification (LAMP), a novel method of gene amplification, was employed in this study for detecting Mycoplasma hyopneumoniae in the respiratory tract or lungs of swine. The pathogen can be detected in LAMP reactions containing as few as 10 fg purified target DNA (10 copies of M. hyopneumoniae genome) within 30 min, which was comparable to real-time PCR. After 30-min reaction at 63 °C, the addition of a certain amount of dye (SYBR Green I and hydroxyl naphthol blue at a proper ratio) into the LAMP reaction system makes the results easily determined as positive or negative by visual inspection. In addition, the LAMP was able to distinguish between M. hyopneumoniae and other closely-related mycoplasma strains, indicating a high degree of specificity. The LAMP assay was more simple and cheap, since the reaction could be completed under isothermal conditions and less laboratorial infrastructure are required. And, it was proven reliable for M. hyopneumoniae diagnosis of nasal swab and lung samples from the field.

  4. Rapid, Loop-Mediated Isothermal Amplification Detection of Coeliac Disease Risk Alleles.

    Science.gov (United States)

    Erlichster, Michael; Tye-Din, Jason A; Varney, Michael D; Skafidas, Efstratios; Kwan, Patrick

    2018-02-16

    Human leukocyte antigen (HLA) genotyping has become a useful investigation in the diagnostic work-up of coeliac disease (CD), with utility in risk stratification and screening. However, broad application of this technology has been hindered by the cost and time burden of conventional laboratory-based assays. We have developed and validated CD-loop-mediated isothermal amplification (CD-LAMP), a LAMP assay, which enables rapid identification of the signature CD risk genotypes, HLA-DQ2.5, HLA-DQ8, HLA-DQ2.2, and HLA-DQA1*05. Sample-to-answer is achieved in approximately 65 minutes without DNA purification, thermal cycling, or specialized analytical equipment. CD-LAMP genotyping of samples was 100% concordant with accredited pathology genotyping on a panel of 40 blood and 20 saliva samples. In a panel of 100 purified DNA samples, genotyping of the high risk DQ2.5 genotype was 100% concordant with accredited pathology genotyping, with slightly reduced sensitivity for the DQ8 genotype (97.1%), and reduced specificity for the DQ8 (93.9%) and DQ2.2 genotypes (95.1%). CD-LAMP results are easily visualized, instrument free, through the addition of a DNA intercalating dye following amplification. Combined with point-of-care antibody testing, CD-LAMP may enable immediate, confident CD diagnosis at a low cost in the clinical setting. Copyright © 2018. Published by Elsevier Inc.

  5. Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of BK Virus▿

    Science.gov (United States)

    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

  6. Development of a loop-mediated isothermal amplification assay for rapid detection of BK virus.

    Science.gov (United States)

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

    2007-05-01

    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.

  7. Detection of Soybean mosaic virus by Reverse Transcription Loop-mediated Isothermal Amplification

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    Yeong-Hoon Lee

    2015-12-01

    Full Text Available Soybean mosaic virus (SMV is a prevalent pathogen that causes significant yield reduction in soybean production worldwide. SMV belongs to potyvirus and causes typical symptoms such as mild mosaic, mosaic and necrosis. SMV is seed-borne and also transmitted by aphid. Eleven SMV strains, G1 to G7, G5H, G6H, G7H, and G7a were reported in soybean varieties in Korea. A reverse transcription loop-mediated isothermal amplification (RT-LAMP method allowed one-step detection of gene amplification by simple procedure and needed only a simple incubator for isothermal template. This RT-LAMP method allowed direct detection of RNA from virus-infected plants without thermal cycling and gel electrophoresis. In this study, we designed RT-LAMP primers named SML-F3/B3/FIP/BIP from coat protein gene sequence of SMV. After the reaction of RT-LAMP, products were identified by electrophoresis and with the detective fluorescent dye, SYBR Green I under daylight and UV light. Optimal reaction condition was at 58°C for 60 min and the primers of RT-LAMP showed the specificity for nine SMV strains tested in this study.

  8. Visual Detection of Potato leafroll virus by One-step Reverse Transcription Loop-Mediated Isothermal Amplification of DNA with Hydroxynaphthol Blue Dye

    NARCIS (Netherlands)

    Ahmadi, S.; Almasi, A.M.; Fatehi, F.; Struik, P.C.; Moradi, A.

    2013-01-01

    Loop-mediated isothermal amplification (LAMP) assay is a novel technique for amplifying DNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency. We applied reverse transcription loop-mediated isothermal amplification (RT-LAMP) to visually detect Potato leafroll

  9. A loop-mediated isothermal amplification assay and sample preparation procedure for sensitive detection of Xanthomonas fragariae in strawberry

    Science.gov (United States)

    Xanthomonas fragariae is a bacterium that causes angular leaf spot of strawberry. Asymptomatic infections are common and contribute to the difficulties in disease management. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay with a bacterial enrichment proced...

  10. One-step reverse transcription loop mediated isothermal amplification assay for detection of Apple chlorotic leaf spot virus

    Science.gov (United States)

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of Apple chlorotic leaf spot virus (ACLSV) was developed. In this method, a set of four primers was designed based on the conserved regions in the coat protein gene of ACLSV, and was synthesized for the ...

  11. Evaluation of a loop-mediated isothermal amplification (LAMP) method for rapid on-site detection of horse meat

    NARCIS (Netherlands)

    Aartse, Aafke; Scholtens-Toma, Ingrid; A, van der Hans J.G.; Boersma-Greve, Monique M.; Prins, Theo W.; Ginkel, van Leen A.; Kok, Esther J.; Bovee, Toine F.H.

    2017-01-01

    Detection of horse DNA by loop-mediated isothermal amplification (LAMP) seems one of the most promising methods to meet the criteria of fast, robust, cost efficient, specific, and sensitive on-site detection. In the present study an assessment of the specificity and sensitivity of the LAMP horse

  12. Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites.

    Science.gov (United States)

    Lucchi, Naomi W; Ljolje, Dragan; Silva-Flannery, Luciana; Udhayakumar, Venkatachalam

    2016-01-01

    Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1-8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed.

  13. Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites.

    Directory of Open Access Journals (Sweden)

    Naomi W Lucchi

    Full Text Available Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP, are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1-8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed.

  14. Rapid and sensitive detection of Didymella bryoniae by visual loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Xiefeng Yao

    2016-08-01

    Full Text Available Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB in Cucurbitaceae crops (e.g. cantaloupe, muskmelon, cucumber, and watermelon. GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462 common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII of D. bryoniae; ideal conditions for detection were optimized for completion in 45 min at 63°C. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with those of a conventional polymerase chain reaction (PCR. The sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 0.1 fg μL−1 of targeted DNA. The LAMP assay could be accomplished in about 45 min, with the results visible to the naked eye. The assay showed high specificity in discriminating all D. bryoniae isolates from seven other fungal pathogens that occur in Cucurbitaceae crops. The LAMP assay also detected D. bryoniae infection in young muskmelon leaves with suspected early symptoms of GSB disease. Hence, the technique has great potential for developing rapid and sensitive visual detection methods for the D. bryoniae pathogen in crops and seeds. This method has potential application in early prediction of disease and reducing the risk of epidemics.

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

    Science.gov (United States)

    Hasiów-Jaroszewska, Beata; Budzyńska, 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.

  16. Development of a Loop Mediated Isothermal Amplification for Diagnosis ofAscaris lumbricoidesin Fecal Samples.

    Science.gov (United States)

    Shiraho, Esther A; Eric, Agola L; Mwangi, Ibrahim N; Maina, Geoffrey M; Kinuthia, Joseph M; Mutuku, Martin W; Mugambi, Robert M; Mwandi, Jackson M; Mkoji, Gerald M

    2016-01-01

    Ascaris lumbricoides is a nematode parasite that causes the common tropical infection ascariasis in humans. It is also considered among the neglected tropical diseases. Diagnosis relies mainly on microscopy-based methods which are laborious, are limited by low sensitivity, and require high expertise. We have developed a loop mediated isothermal amplification (LAMP) for diagnosis of ascariasis in fecal samples, based on the first internal transcribed (ITS-1) spacer region of the ribosomal DNA. We used Primer Explorer V4 software to design primers. Ascaris adult and ova were obtained from naturally infected school children, whose parents/guardians gave consent for their participation in the study. Genomic DNA was extracted using alkaline lysis method and amplified by LAMP at 63°C for 45 minutes. LAMP products were visualized by naked eyes after adding SYBR Green dye and also on agarose gel. LAMP successfully and reliably detected Ascaris DNA from a single egg and in fecal samples. The assay specifically detected Ascaris DNA without amplifying DNA from ova of other parasites which commonly coexist with A. lumbricoides in feces. The developed LAMP assay has great potential for use in ascariasis diagnosis at the point of care and in low infection intensity situation that characterize control and elimination campaigns.

  17. Development of a Loop Mediated Isothermal Amplification for Diagnosis of Ascaris lumbricoides in Fecal Samples

    Science.gov (United States)

    Eric, Agola L.; Mwangi, Ibrahim N.; Maina, Geoffrey M.; Kinuthia, Joseph M.; Mutuku, Martin W.; Mugambi, Robert M.; Mwandi, Jackson M.; Mkoji, Gerald M.

    2016-01-01

    Ascaris lumbricoides is a nematode parasite that causes the common tropical infection ascariasis in humans. It is also considered among the neglected tropical diseases. Diagnosis relies mainly on microscopy-based methods which are laborious, are limited by low sensitivity, and require high expertise. We have developed a loop mediated isothermal amplification (LAMP) for diagnosis of ascariasis in fecal samples, based on the first internal transcribed (ITS-1) spacer region of the ribosomal DNA. We used Primer Explorer V4 software to design primers. Ascaris adult and ova were obtained from naturally infected school children, whose parents/guardians gave consent for their participation in the study. Genomic DNA was extracted using alkaline lysis method and amplified by LAMP at 63°C for 45 minutes. LAMP products were visualized by naked eyes after adding SYBR Green dye and also on agarose gel. LAMP successfully and reliably detected Ascaris DNA from a single egg and in fecal samples. The assay specifically detected Ascaris DNA without amplifying DNA from ova of other parasites which commonly coexist with A. lumbricoides in feces. The developed LAMP assay has great potential for use in ascariasis diagnosis at the point of care and in low infection intensity situation that characterize control and elimination campaigns. PMID:27882242

  18. Loop-mediated isothermal amplification: an emerging technology for detection of fish and shellfish pathogens.

    Science.gov (United States)

    Savan, R; Kono, T; Itami, T; Sakai, M

    2005-10-01

    Fish and shellfish diseases are a constant threat to the sustainability and economic viability of aquaculture. Early diagnosis plays a vital role in management of fish and shellfish diseases. Traditionally, various biochemical and serological tests have been used for fish disease diagnosis. However, the time and expertise required for such diagnoses makes it difficult for aquaculturists to easily adopt them under production conditions. Polymerase chain reaction and probe-based nucleic acid detection have become increasingly popular in fish and shellfish diagnostics. Recently, a novel technique called loop-mediated isothermal amplification (LAMP) has been developed, which is highly sensitive and rapid. LAMP has been used for the detection of bacterial, viral, fungal and parasitic diseases in both animal and plants. In aquaculture, LAMP-based detection of pathogens like Edwardsiella tarda, E. ictaluri, Nocardia seriolae, Tetracapsuloides bryosalmonae, white spot syndrome virus and infectious haematopoietic necrosis virus have been reported. In this review, the application of LAMP for the detection of aquaculture-associated pathogens is discussed.

  19. Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Staphylococcus aureus

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    King Ting Lim

    2013-01-01

    Full Text Available Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA, is an important human pathogen that produces a variety of toxins and causes a wide range of infections, including soft-tissue infections, bacteremia, and staphylococcal food poisoning. A loop-mediated isothermal amplification (LAMP assay targeting the arcC gene of S. aureus was developed and evaluated with 119 S. aureus and 25 non-S. aureus strains. The usefulness of the assay was compared with the PCR method that targets spa and arcC genes. The optimal temperature for the LAMP assay was 58.5°C with a detection limit of 2.5 ng/μL and 102 CFU/mL when compared to 12.5 ng/μL and 103 CFU/mL for PCR (spa and arcC. Both LAMP and PCR assays were 100% specific, 100% sensitive, 100% positive predictive value (PPV, and 100% negative predictive value (NPV. When tested on 30 spiked blood specimens (21 MRSA, eight non-S. aureus and one negative control, the performance of LAMP and PCR was comparable: 100% specific, 100% sensitive, 100% PPV, and 100% NPV. In conclusion, the LAMP assay was equally specific with a shorter detection time when compared to PCR in the identification of S. aureus. The LAMP assay is a promising alternative method for the rapid identification of S. aureus and could be used in resource-limited laboratories and fields.

  20. Yersinia pestis detection by loop-mediated isothermal amplification combined with magnetic bead capture of DNA

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    Na Feng

    Full Text Available ABSTRACT We developed a loop-mediated isothermal amplification (LAMP assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23 CFU for pure culture, whereas 2.3 × 104 or 2.3 × 106 CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3 × 106 CFU, but PCR was negative at the level of 2.3 × 107 CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3 × 103 or 2.3 × 106 CFU, whereas 2.3 × 105 or 2.3 × 107 CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.

  1. Development and evaluation of loop-mediated isothermal amplification assay for rapid detection of Capripoxvirus

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    Kanisht Batra

    2015-11-01

    Full Text Available Aim: The present study was undertaken to develop a nucleic acid-based diagnostic assay loop-mediated isothermal amplification assay (LAMP targeting highly conserved genomic regions of Capripoxvirus (CaPVs and its comparative evaluation with real-time polymerase chain reaction (PCR. Material and Methods: Lyophilized vaccine strain of sheeppox virus (SPPV was used for optimization of LAMP assay. The LAMP assay was designed using envelope immunogenic protein (P32 coding gene targeting highly conserved genomic regions of CaPV responsible for causing sheep pox, goat pox, and lumpy skin disease in sheep, goat and cattle respectively. Serial tenfold dilution of SPPV recombinant plasmid DNA was used for a calculating limit of detection. Analytical sensitivity and specificity were performed. Results: The test described is quick (30 min, sensitive and specific for detection of CaPVs. The described assay did not show any cross-reactivity to other related viruses that cause apparently similar clinical signs. It was found to be ten times more sensitive than conventional PCR however, 100 times less sensitive than quantitative PCR (qPCR. LAMP assay results were monitored by color change method using picogreen dye and agarose gel electrophoresis. Conclusion: LAMP assay can be a very good alternative for CaPV detection to other molecular techniques requiring sophisticated equipments.

  2. Loop-Mediated Isothermal Amplification Method for Differentiation and Rapid Detection of Taenia Species▿

    Science.gov (United States)

    Nkouawa, Agathe; Sako, Yasuhito; Nakao, Minoru; Nakaya, Kazuhiro; Ito, Akira

    2009-01-01

    Rapid detection and differentiation of Taenia species are required for the control and prevention of taeniasis and cysticercosis in areas where these diseases are endemic. Because of the lower sensitivity and specificity of the conventional diagnosis based on microscopical examination, molecular tools are more reliable for differential diagnosis of these diseases. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) assay for differential diagnosis of infections with Taenia species with cathepsin L-like cysteine peptidase (clp) and cytochrome c oxidase subunit 1 (cox1) genes. LAMP with primer sets to the cox1 gene could differentiate between three species, and LAMP with primer sets to the clp gene could differentiate Taenia solium from Taenia saginata/Taenia asiatica. Restriction enzyme digestion of the LAMP products from primer set Tsag-clp allowed the differentiation of Taenia saginata from Taenia asiatica. We demonstrated the high specificity of LAMP by testing known parasite DNA samples extracted from proglottids (n = 100) and cysticerci (n = 68). LAMP could detect one copy of the target gene or five eggs of T. asiatica and T. saginata per gram of feces, showing sensitivity similar to that of PCR methods. Furthermore, LAMP could detect parasite DNA in all taeniid egg-positive fecal samples (n = 6). Due to the rapid, simple, specific, and sensitive detection of Taenia species, the LAMP assays are valuable tools which might be easily applicable for the control and prevention of taeniasis and cysticercosis in countries where these diseases are endemic. PMID:19005142

  3. Loop-mediated isothermal amplification method for differentiation and rapid detection of Taenia species.

    Science.gov (United States)

    Nkouawa, Agathe; Sako, Yasuhito; Nakao, Minoru; Nakaya, Kazuhiro; Ito, Akira

    2009-01-01

    Rapid detection and differentiation of Taenia species are required for the control and prevention of taeniasis and cysticercosis in areas where these diseases are endemic. Because of the lower sensitivity and specificity of the conventional diagnosis based on microscopical examination, molecular tools are more reliable for differential diagnosis of these diseases. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) assay for differential diagnosis of infections with Taenia species with cathepsin L-like cysteine peptidase (clp) and cytochrome c oxidase subunit 1 (cox1) genes. LAMP with primer sets to the cox1 gene could differentiate between three species, and LAMP with primer sets to the clp gene could differentiate Taenia solium from Taenia saginata/Taenia asiatica. Restriction enzyme digestion of the LAMP products from primer set Tsag-clp allowed the differentiation of Taenia saginata from Taenia asiatica. We demonstrated the high specificity of LAMP by testing known parasite DNA samples extracted from proglottids (n = 100) and cysticerci (n = 68). LAMP could detect one copy of the target gene or five eggs of T. asiatica and T. saginata per gram of feces, showing sensitivity similar to that of PCR methods. Furthermore, LAMP could detect parasite DNA in all taeniid egg-positive fecal samples (n = 6). Due to the rapid, simple, specific, and sensitive detection of Taenia species, the LAMP assays are valuable tools which might be easily applicable for the control and prevention of taeniasis and cysticercosis in countries where these diseases are endemic.

  4. Evaluation of loop-mediated isothermal amplification assay for rapid diagnosis ofAcanthamoebakeratitis.

    Science.gov (United States)

    Mewara, Abhishek; Khurana, Sumeeta; Yoonus, Shakila; Megha, Kirti; Tanwar, Parveen; Gupta, Amit; Sehgal, Rakesh

    2017-01-01

    The clinical features of Acanthamoeba keratitis (AK) are non-specific and closely resemble bacterial, viral and fungal keratitis. We compared loop-mediated isothermal amplification (LAMP) with microscopy, non-nutrient agar (NNA) culture and polymerase chain reaction (PCR) in clinical suspects of AK. Of 52 clinical samples (42 AK suspects and 10 proven bacterial, viral or fungal keratitis), 3 were positive by direct microscopy (sensitivity 60%, confidence interval [CI]: 17%-92.7%), and 5 by NNA culture, 18S rDNA PCR and LAMP (sensitivity 100%, CI: 46.3%-100%). The limit of detection of Acanthamoeba DNA was 1 pg/μl by both LAMP and PCR. PCR and LAMP assays targeting 18S rDNA gene were found particularly suitable for a rapid and accurate diagnosis of AK. LAMP assay takes 2-3 h lesser than PCR, and thus offers a rapid, highly sensitive and specific, simple and affordable diagnostic modality for patients suspected of AK, especially in resource limited settings.

  5. Loop-Mediated Isothermal Amplification as a Fast Noninvasive Method of Helicobacter pylori Diagnosis.

    Science.gov (United States)

    Yari, Farideh; Abiri, Ramin; Aryan, Ehsan; Ahmadi Jouybari, Touraj; Navabi, Jafar; Alvandi, Amirhooshang

    2016-09-01

    Helicobacter pylori infection is etiologically associated with some important health problems such as gastric cancer. Because of the high clinical importance of H. pylori infection, development of a noninvasive test for the detection of H. pylori is desirable. In this study, a loop-mediated isothermal amplification (LAMP) targeted ureC of H. pylori was evaluated on 100 stool specimens and compared with a stool antigen test. Culture and rapid urease test were considered as gold standards. The overall detection rate of the fecal antigen test and LAMP was 58% and 82%, respectively. The analytical sensitivity of the fecal antigen test and LAMP was 500 and 10 H. pylori cells/g and 10 fg DNA/reaction, which is equal to six H. pylori genome. LAMP technique has been characterized by high sensitivity and low detection limit for the detection of H. pylori in stool specimen. Clinical diagnostic performance of LAMP was better than the stool antigen test. © 2015 Wiley Periodicals, Inc.

  6. Colorimetric Nucleic Acid Detection on Paper Microchip Using Loop Mediated Isothermal Amplification and Crystal Violet Dye.

    Science.gov (United States)

    Roy, Sharmili; Mohd-Naim, Noor Faizah; Safavieh, Mohammadali; Ahmed, Minhaz Uddin

    2017-11-22

    Nucleic acid detection is of paramount importance in monitoring of microbial pathogens in food safety and infectious disease diagnostic applications. To address these challenges, a rapid, cost-effective label-free technique for nucleic acid detection with minimal instrumentations is highly desired. Here, we present paper microchip to detect and quantify nucleic acid using colorimetric sensing modality. The extracted DNA from food samples of meat as well as microbial pathogens was amplified utilizing loop-mediated isothermal amplification (LAMP). LAMP amplicon was then detected and quantified on a paper microchip fabricated in a cellulose paper and a small wax chamber utilizing crystal violet dye. The affinity of crystal violet dye toward dsDNA and positive signal were identified by changing the color from colorless to purple. Using this method, detection of Sus scrofa (porcine) and Bacillus subtilis (bacteria) DNA was possible at concentrations as low as 1 pg/μL (3.43 × 10 -1 copies/μL) and 10 pg/μL (2.2 × 10 3 copies/μL), respectively. This strategy can be adapted for detection of other DNA samples, with potential for development of a new breed of simple and inexpensive paper microchip at the point-of-need.

  7. Loop-Mediated Isothermal Amplification (LAMP): Emergence As an Alternative Technology for Herbal Medicine Identification.

    Science.gov (United States)

    Li, Jing-Jian; Xiong, Chao; Liu, Yue; Liang, Jun-Song; Zhou, Xing-Wen

    2016-01-01

    Correct identification of medicinal plant ingredients is essential for their safe use and for the regulation of herbal drug supply chain. Loop-mediated isothermal amplification (LAMP) is a recently developed approach to identify herbal medicine species. This novel molecular biology technique enables timely and accurate testing, especially in settings where infrastructures to support polymerase chain reaction facilities are lacking. Studies that used this method have altered our view on the extent and complexity of herbal medicine identification. In this review, we give an introduction into LAMP analysis, covers the basic principles and important aspects in the development of LAMP analysis method. Then we presented a critical review of the application of LAMP-based methods in detecting and identifying raw medicinal plant materials and their processed products. We also provide a practical standard operating procedure (SOP) for the utilization of the LAMP protocol in herbal authentication, and consider the prospects of LAMP technology in the future developments of herbal medicine identification and the challenges associated with its application.

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

    Science.gov (United States)

    Chen, Rui; Tong, QunBo; Zhang, Yi; Lou, Di; Kong, QingMing; Lv, Shan; Zhuo, MingMing; Wen, LiYong; Lu, ShaoHong

    2011-10-25

    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. 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. 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.

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

    Directory of Open Access Journals (Sweden)

    Zhuo MingMing

    2011-10-01

    Full Text Available Abstract 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.

  10. Rapid, sensitive, and specific detection of Clostridium tetani by loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Jiang, Dongneng; Pu, Xiaoyun; Wu, Jiehong; Li, Meng; Liu, Ping

    2013-01-01

    Tetanus is a specific infectious disease, which is often associated with catastrophic events such as earthquakes, traumas, and war wounds. The obligate anaerobe Clostridium tetani is the pathogen that causes tetanus. Once the infection of tetanus progresses to an advanced stage within the wounds of limbs, the rates of amputation and mortality increase manifold. Therefore, it is necessary to devise a rapid and sensitive point-of-care detection method for C. tetani so as to ensure an early diagnosis and clinical treatment of tetanus. In this study, we developed a detection method for C. tetani using loop-mediated isothermal amplification (LAMP) assay, wherein the C. tetani tetanus toxin gene was used as the target gene. The method was highly specific and sensitive, with a detection limit of 10 colony forming units (CFU)/ml, and allowed quantitative analysis. While detecting C. tetani in clinical samples, it was found that the LAMP results completely agreed with those of the traditional API 20A anaerobic bacteria identification test. As compared with the traditional API test and PCR assay, LAMP detection of C. tetani is simple and rapid, and the results can be identified through naked-eye observation. Therefore, it is an ideal and rapid point-of-care testing method for tetanus.

  11. Development of a Loop Mediated Isothermal Amplification for Diagnosis of Ascaris lumbricoides in Fecal Samples

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    Esther A. Shiraho

    2016-01-01

    Full Text Available Ascaris lumbricoides is a nematode parasite that causes the common tropical infection ascariasis in humans. It is also considered among the neglected tropical diseases. Diagnosis relies mainly on microscopy-based methods which are laborious, are limited by low sensitivity, and require high expertise. We have developed a loop mediated isothermal amplification (LAMP for diagnosis of ascariasis in fecal samples, based on the first internal transcribed (ITS-1 spacer region of the ribosomal DNA. We used Primer Explorer V4 software to design primers. Ascaris adult and ova were obtained from naturally infected school children, whose parents/guardians gave consent for their participation in the study. Genomic DNA was extracted using alkaline lysis method and amplified by LAMP at 63°C for 45 minutes. LAMP products were visualized by naked eyes after adding SYBR Green dye and also on agarose gel. LAMP successfully and reliably detected Ascaris DNA from a single egg and in fecal samples. The assay specifically detected Ascaris DNA without amplifying DNA from ova of other parasites which commonly coexist with A. lumbricoides in feces. The developed LAMP assay has great potential for use in ascariasis diagnosis at the point of care and in low infection intensity situation that characterize control and elimination campaigns.

  12. Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Acanthamoeba

    Science.gov (United States)

    Yang, Hye-Won; Lee, Yu-Ran; Inoue, Noboru; Jha, Bijay Kumar; Danne, Dinzouna-Boutamba Sylvatrie; Kim, Hong-Kyun; Lee, Junhun; Goo, Youn-Kyoung; Kong, Hyun-Hee; Chung, Dong-Il

    2013-01-01

    Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner. PMID:23864737

  13. Evaluation of loop-mediated isothermal amplification assay for rapid diagnosis of Acanthamoeba keratitis

    Directory of Open Access Journals (Sweden)

    Abhishek Mewara

    2017-01-01

    Full Text Available Background: The clinical features of Acanthamoeba keratitis (AK are non-specific and closely resemble bacterial, viral and fungal keratitis. Materials and Methods: We compared loop-mediated isothermal amplification (LAMP with microscopy, non-nutrient agar (NNA culture and polymerase chain reaction (PCR in clinical suspects of AK. Results: Of 52 clinical samples (42 AK suspects and 10 proven bacterial, viral or fungal keratitis, 3 were positive by direct microscopy (sensitivity 60%, confidence interval [CI]: 17%–92.7%, and 5 by NNA culture, 18S rDNA PCR and LAMP (sensitivity 100%, CI: 46.3%–100%. The limit of detection of Acanthamoeba DNA was 1 pg/μl by both LAMP and PCR. Conclusion: PCR and LAMP assays targeting 18S rDNA gene were found particularly suitable for a rapid and accurate diagnosis of AK. LAMP assay takes 2–3 h lesser than PCR, and thus offers a rapid, highly sensitive and specific, simple and affordable diagnostic modality for patients suspected of AK, especially in resource limited settings

  14. Rapid and sensitive detection of Bordetella bronchiseptica by loop-mediated isothermal amplification (LAMP

    Directory of Open Access Journals (Sweden)

    Hui Zhang

    2013-10-01

    Full Text Available Bordetella bronchiseptica causes acute and chronic respiratory infections in diverse animal species and occasionally in humans. In this study, we described the establishment of a simple, sensitive and cost-efficient loop-mediated isothermal amplification (LAMP assay for the detection of B. bronchiseptica. A set of primers towards a 235 bp region within the flagellum gene of B. bronchiseptica was designed with online software.. The specificity of the LAMP assay was examined by using 6 porcine pathogens and 100 nasal swabs collected from healthy pigs and suspect infected pigs. The results indicated that positive reactions were confirmed for all B. bronchiseptica and no cross-reactivity was observed from other non-B. bronchiseptica. In sensitivity evaluations, the technique successfully detected a serial dilutions of extracted B. bronchiseptica DNA with a detection limit of 9 copies, which was 10 times more sensitive than that of PCR. Compared with conventional PCR, the higher sensitivity of LAMP method and no need for the complex instrumentation make this LAMP assay a promising alternative for the diagnosis of B. bronchiseptica in rural areas and developing countries where there lacks of complex laboratory services.

  15. Detection of Brucellosis in Sika Deer ( Cervus nippon ) through Loop-mediated Isothermal Amplification (LAMP).

    Science.gov (United States)

    Liu, Qianhong; Wei, Jie; Sun, Qingsong; Wang, Ben; Wang, Yuting; Hu, Ying; Wu, Wenrong

    2017-07-01

    Brucellosis (Brucella bovis) in sika deer ( Cervus nippon ) can cause enormous losses to stag breeding, especially in areas in which stag breeding has become an important industry. It also poses a threat to humans because it is a zoonotic disease. Use of the loop-mediated isothermal amplification (LAMP) assay has been poorly described in the diagnosis of brucellosis in deer. We developed a LAMP assay targeting the omp25 gene sequence to detect brucellosis in sika deer. The reaction can be completed in 60 min at 63 C and, with a detection limit of 17 pg, it was more sensitive than conventional PCR, with its detection limit of 1.7 ng. No cross-reactivity was observed with four bacteria: Escherichia coli , Salmonella enterica subsp. enterica, Clostridium pasteurianum , and Pseudomonas aeruginosa . We used 263 samples of blood to evaluate the reaction. The percentage of agreement between LAMP and PCR reached 91%; relative specificity reached 87%, and relative sensitivity reached 100%. The results indicate LAMP can be a simple and rapid diagnostic tool for detecting brucellosis in sika deer, particularly in the field, where it is essential to control brucellosis in deer with a rapid and accurate diagnosis for removal of positive animals.

  16. Detection of the quarantine species Thrips palmi by loop-mediated isothermal amplification.

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    Arnika Przybylska

    Full Text Available 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.

  17. Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus

    DEFF Research Database (Denmark)

    Dukes, J.P.; King, D.P.; Alexandersen, Søren

    2006-01-01

    Speed is paramount in the diagnosis of foot-and-mouth disease (FMD) and simplicity is required if a test is to be deployed in the field. The development of a one-step, reverse transcription loop-mediated amplification (RT-LAMP) assay enables FMD virus (FMDV) to be detected in under an hour...... in a single tube without thermal cycling. A fragment of the 3D RNA polymerase gene of the virus is amplified at 65 degrees C in the presence of a primer mixture and both reverse transcriptase and Bst DNA polymerase. Compared with real-time RT-PCR, RT-LAMP was consistently faster, and ten copies of FMDV...... transcript were detected in twenty-two minutes. Amplification products were detected by visual inspection, agarose gel electrophoresis, or in real-time by the addition of a fluorescent dye. The specificity of the reaction was demonstrated by the absence of amplification of RNA from other viruses that cause...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. Detection of phytoplasma by loop-mediated isothermal amplification of DNA (LAMP).

    Science.gov (United States)

    Obura, E; Masiga, D; Wachira, F; Gurja, B; Khan, Z R

    2011-02-01

    Napier stunt phytoplasma (16SrXI and 16SrIII) in eastern Africa is a serious threat to the expansion of Napier grass (Pennisetum purpureum) farming in the region, where it is widely cultivated as fodder in zero grazing livestock systems. The grass has high potential for bio-fuel production, and has been adopted by farmers as a countermeasure to cereal stem borer Lepidoptera, since it attracts and traps the insect. Diagnosis of stunt phytoplasma have been largely by nested polymerase chain reaction (nPCR) targeting the 16S rRNA gene. However, the method is laborious, costly and technically demanding. This investigation has developed a simpler but effective phytoplasma diagnostic tool, called; loop-mediated isothermal amplification of DNA (LAMP). The assay was tested on 8 symptomatic and 8 asymptomatic plants, while its detection limit was compared to nested PCR using samples serially diluted from 3 ng/μl to 0.38 pg/μl. Molecular typing of LAMP products was determined by BsrI restriction digestion and Southern blot analysis. The assay sensitivity, positive and negative predictive values were estimated, while the specificity was tested on 11 phytoplasma groups. LAMP was specific to 5 phytoplasma groups: 16SrVI, X, XI and XVI. BsrI restriction digestion produced two predicted fragments, and there was specific binding of probe DNA to the LAMP amplicons in Southern blot analysis. The assay sensitivity was 100%, while the positive and negative predictive values were 63 and 100% respectively. LAMP was 20-fold more sensitive than nested PCR. This study validates LAMP for routine diagnosis of Napier stunt and other closely related phytoplasmas. Copyright © 2010 Elsevier B.V. All rights reserved.

  20. Loop-mediated isothermal amplification (LAMP: Early detection of Toxoplasma gondii infection in mice

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    Kong Qing-Ming

    2012-01-01

    Full Text Available Abstract Background Toxoplasmosis is a widespread zoonotic parasitic disease that occurs in both animals and humans. Traditional molecular assays are often difficult to perform, especially for the early diagnosis of Toxoplasma gondii infections. Here, we established a novel loop-mediated isothermal amplification targeting the 529 bp repeat element (529 bp-LAMP to detect T. gondii DNA in blood samples of experimental mice infected with tachyzoites of the RH strain. Findings The assay was performed with Bst DNA polymerase at 65°C for 1 h. The detection limit of the 529 bp-LAMP assay was as low as 0.6 fg of T. gondii DNA. The sensitivity of this assay was 100 and 1000 fold higher than that of the LAMP targeting B1 gene (B1-LAMP and nested PCR targeting 529 bp repeat element (529 bp-nested PCR, respectively. The specificity of the 529 bp-LAMP assay was determined using the DNA samples of Trypanosoma evansi, Plasmodium falciparum, Paragonimus westermani, Schistosoma japonicum, Fasciola hepatica and Angiostrongylus cantonensis. No cross-reactivity with the DNA of any parasites was found. The assay was able to detect T. gondii DNA in all mouse blood samples at one day post infection (dpi. Conclusions We report the following findings: (i The detection limit of the 529 bp-LAMP assay is 0.6 fg of T. gondii DNA; (ii The assay does not involve any cross-reactivity with the DNA of other parasites; (iii This is the first report on the application of the LAMP assay for early diagnosis of toxoplasmosis in blood samples from experimentally infected mice. Due to its simplicity, sensitivity and cost-effectiveness for common use, we suggest that this assay should be used as an early diagnostic tool for health control of toxoplasmosis.

  1. Loop-mediated isothermal amplification as a reliable assay for Toxocara canis infection in pet dogs.

    Science.gov (United States)

    Khoshakhlagh, Paria; Spotin, Adel; Mahami-Oskouei, Mahmoud; Shahbazi, Abbas; Ozlati, Maryam

    2017-09-01

    Keeping of infected dogs as pet results in the potential transmission risk factors for shedding helminthic infections such as toxocariasis. Lack of accurate identification of Toxocara canis eggs in non-dewormed infected pet dogs remains a diagnostic concern among researchers. In this study, dog owners were asked to fill up a questionnaire regarding their pets and their attitude towards the deworming regimen. One hundred faecal samples were collected from pet dogs (Northwest Iran) and were subsequently identified by the ZnSo4 flotation technique, PCR and loop-mediated isothermal amplification (LAMP) assays. The DNA of the recovered T. canis eggs was then extracted and amplified by LAMP and PCR. Furthermore, ITS2 amplicons were sequenced for appraisal of the phylogenetic analysis. Nine, 5 and 11% of T. canis infections were identified by microscopy, PCR and LAMP, respectively. It was detected that LAMP was 10 times (10 -10 to 10 -13  g/μl) more sensitive than PCR (10 -10 to 10 -12  g/μl). The kappa value between LAMP and PCR indicated a faint concurrence (0.463). The kappa coefficient between LAMP and flotation technique indicated a strong agreement (0.667). The highest infection rate (n = 11) was detected in non-dewormed pet dogs, particularly those less than 3 months old (P canis eggs in infected pet dogs. It was proposed that the dog holder's awareness is insufficient to implement regular deworming schedules. Additionally, regional policymakers should broadly revise anthelmintic treatment guidelines.

  2. Specific detection of Pectobacterium carotovorum by loop-mediated isothermal amplification.

    Science.gov (United States)

    Yasuhara-Bell, Jarred; Marrero, Glorimar; De Silva, Asoka; Alvarez, Anne M

    2016-12-01

    Potatoes are an important agroeconomic crop worldwide and maceration diseases caused by pectolytic bacterial pathogens result in significant pre- and post-harvest losses. Pectobacterium carotovorum shares a common host range with other Pectobacterium spp. and other members of the Enterobacteriaceae, such as Dickeya spp. As these pathogens cannot be clearly differentiated on the basis of the symptoms they cause, improved methods of identification are critical for the determination of sources of contamination. Current standardized methods for the differentiation of pectolytic species are time consuming and require trained personnel, as they rely on traditional bacteriological practices that do not always produce conclusive results. In this growing world market, there is a need for rapid diagnostic tests that can differentiate between pectolytic pathogens, as well as separate them from non-pectolytic enteric bacteria associated with soft rots of potato. An assay has been designed previously to detect the temperate pathogen Pectobacterium atrosepticum, but there is currently no recognized rapid assay for the detection of the tropical/subtropical counterpart, Pectobacterium carotovorum. This report describes the development of a loop-mediated isothermal amplification (LAMP) assay that detects P. carotovorum with high specificity. The assay was evaluated using all known species of Pectobacterium and only showed positive reactions for P. carotovorum. This assay was also tested against 15 non-target genera of plant-associated bacteria and did not produce any false positives. The LAMP assay described here can be used as a rapid test for the differentiation of P. carotovorum from other pectolytic pathogens, and its gene target can be the basis for the development of other molecular-based detection assays. © 2016 BSPP and John Wiley & Sons Ltd.

  3. Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Papaya ringspot virus.

    Science.gov (United States)

    Shen, Wentao; Tuo, Decai; Yan, Pu; Yang, Yong; Li, Xiaoying; Zhou, Peng

    2014-08-01

    Papaya ringspot virus (PRSV) and Papaya leaf distortion mosaic virus (PLDMV), which causes disease symptoms similar to PRSV, threaten commercial production of both non-transgenic-papaya and PRSV-resistant transgenic papaya in China. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect PLDMV was developed previously. In this study, the development of another RT-LAMP assay to distinguish among transgenic, PRSV-infected and PLDMV-infected papaya by detection of PRSV is reported. A set of four RT-LAMP primers was designed based on the highly conserved region of the P3 gene of PRSV. The RT-LAMP method was specific and sensitive in detecting PRSV, with a detection limit of 1.15×10(-6)μg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR. Field application of the RT-LAMP assay demonstrated that samples positive for PRSV were detected only in non-transgenic papaya, whereas samples positive for PLDMV were detected only in commercialized PRSV-resistant transgenic papaya. This suggests that PRSV remains the major limiting factor for non-transgenic-papaya production, and the emergence of PLDMV threatens the commercial transgenic cultivar in China. However, this study, combined with the earlier development of an RT-LAMP assay for PLDMV, will provide a rapid, sensitive and cost-effective diagnostic power to distinguish virus infections in papaya. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Detection of HbsAg and hATIII genetically modified goats (Caprahircus) by loop-mediated isothermal amplification.

    Science.gov (United States)

    Tao, Chenyu; Zhang, Qingde; Zhai, Shanli; Liu, Bang

    2013-11-01

    In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.

  5. Using the ubiquitous pH meter combined with a loop mediated isothermal amplification method for facile and sensitive detection of Nosema bombycis genomic DNA PTP1.

    Science.gov (United States)

    Xie, Shunbi; Yuan, Yali; Song, Yue; Zhuo, Ying; Li, Tian; Chai, Yaqin; Yuan, Ruo

    2014-12-28

    Here we show an amplification-coupled detection method for directly measuring released hydrogen ions during the loop mediated isothermal amplification (LAMP) procedure by using a pH meter. The genomic DNA of Nosema bombycis (N. bombycis) was amplified and detected by employing this LAMP-pH meter platform for the first time.

  6. Detection of enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Wang, D; Wang, X; Geng, Y; An, C

    2014-01-01

    The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD) for an early treatment by using loop-mediated isothermal amplification (LAMP) technique. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for detecting EV71 virus was developed, the specificity and sensitivity of RT-LAMP was tested, and the clinical specimens was assayed by the RT-LAMP comparing with conventional reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR. A total of 116 clinical specimens from the suspected HFMD individual were detected with the RT-LAMP. The detection rate for EV71 was 56.89% by RT-LAMP, 41.38% by real-time PCR and 34.48% by RT-PCR. The minimum detection limit of RT-LAMP was 0.01 PFU, both of RT-PCR and real-time PCR was 0.1PFU. Non-cross-reactive amplification with other enteroviruses was detected in the survey reports. The effectiveness of RT-LAMP is higher than RT-PCR and real-time PCR. The protocol is easy to operate and time saving. It was not an expensive instrument, which was needed; it is an applicable method for rapid diagnosis of the disease, especially in resource-poor countries or in developing countries.

  7. Detection of Enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification

    Directory of Open Access Journals (Sweden)

    D Wang

    2014-01-01

    Full Text Available Purpose : The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD for an early treatment by using loop-mediated isothermal amplification (LAMP technique. Materials and Methods : A reverse-transcription loop-mediated isothermal amplification (RT-LAMP for detecting EV71 virus was developed, the specificity and sensitivity of RT-LAMP was tested, and the clinical specimens was assayed by the RT-LAMP comparing with conventional reverse-transcription polymerase chain reaction (RT-PCR and real-time PCR. Results : A total of 116 clinical specimens from the suspected HFMD individual were detected with the RT-LAMP. The detection rate for EV71 was 56.89% by RT-LAMP, 41.38% by real-time PCR and 34.48% by RT-PCR. The minimum detection limit of RT-LAMP was 0.01 PFU, both of RT-PCR and real-time PCR was 0.1PFU. Non-cross-reactive amplification with other enteroviruses was detected in the survey reports. Conclusions : The effectiveness of RT-LAMP is higher than RT-PCR and real-time PCR. The protocol is easy to operate and time saving. It was not an expensive instrument, which was needed; it is an applicable method for rapid diagnosis of the disease, especially in resource-poor countries or in developing countries.

  8. Validation of a Salmonella loop-mediated isothermal amplification assay in animal food.

    Science.gov (United States)

    Domesle, Kelly J; Yang, Qianru; Hammack, Thomas S; Ge, Beilei

    2018-01-02

    Loop-mediated isothermal amplification (LAMP) has emerged as a promising alternative to PCR for pathogen detection in food testing and clinical diagnostics. This study aimed to validate a Salmonella LAMP method run on both turbidimetry (LAMP I) and fluorescence (LAMP II) platforms in representative animal food commodities. The U.S. Food and Drug Administration (FDA)'s culture-based Bacteriological Analytical Manual (BAM) method was used as the reference method and a real-time quantitative PCR (qPCR) assay was also performed. The method comparison study followed the FDA's microbiological methods validation guidelines, which align well with those from the AOAC International and ISO. Both LAMP assays were 100% specific among 300 strains (247 Salmonella of 185 serovars and 53 non-Salmonella) tested. The detection limits ranged from 1.3 to 28 cells for six Salmonella strains of various serovars. Six commodities consisting of four animal feed items (cattle feed, chicken feed, horse feed, and swine feed) and two pet food items (dry cat food and dry dog food) all yielded satisfactory results. Compared to the BAM method, the relative levels of detection (RLODs) for LAMP I ranged from 0.317 to 1 with a combined value of 0.610, while those for LAMP II ranged from 0.394 to 1.152 with a combined value of 0.783, which all fell within the acceptability limit (2.5) for an unpaired study. This also suggests that LAMP was more sensitive than the BAM method at detecting low-level Salmonella contamination in animal food and results were available 3days sooner. The performance of LAMP on both platforms was comparable to that of qPCR but notably faster, particularly LAMP II. Given the importance of Salmonella in animal food safety, the LAMP assays validated in this study holds great promise as a rapid, reliable, and robust method for routine screening of Salmonella in these commodities. Published by Elsevier B.V.

  9. A Tripartite Amplification Loop Involving the Transcription Factor WRKY75, Salicylic Acid, and Reactive Oxygen Species Accelerates Leaf Senescence.

    Science.gov (United States)

    Guo, Pengru; Li, Zhonghai; Huang, Peixin; Li, Bosheng; Fang, Shuang; Chu, Jinfang; Guo, Hongwei

    2017-11-01

    Leaf senescence is a highly coordinated, complicated process involving the integration of numerous internal and environmental signals. Salicylic acid (SA) and reactive oxygen species (ROS) are two well-defined inducers of leaf senescence whose contents progressively and interdependently increase during leaf senescence via an unknown mechanism. Here, we characterized the transcription factor WRKY75 as a positive regulator of leaf senescence in Arabidopsis thaliana. Knockdown or knockout of WRKY75 delayed age-dependent leaf senescence, while overexpression of WRKY75 accelerated this process. WRKY75 transcription is induced by age, SA, H 2 O 2 , and multiple plant hormones. Meanwhile, WRKY75 promotes SA production by inducing the transcription of SA INDUCTION-DEFICIENT2 ( SID2 ) and suppresses H 2 O 2 scavenging, partly by repressing the transcription of CATALASE2 ( CAT2 ). Genetic analysis revealed that the mutation of SID2 or an increase in catalase activity rescued the precocious leaf senescence phenotype evoked by WRKY75 overexpression. Based on these results, we propose a tripartite amplification loop model in which WRKY75, SA, and ROS undergo a gradual but self-sustained rise driven by three interlinking positive feedback loops. This tripartite amplification loop provides a molecular framework connecting upstream signals, such as age and plant hormones, to the downstream regulatory network executed by SA- and H 2 O 2 -responsive transcription factors during leaf senescence. © 2017 American Society of Plant Biologists. All rights reserved.

  10. A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods

    Directory of Open Access Journals (Sweden)

    Gavin J. Nixon

    2014-12-01

    Full Text Available Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR. There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These ‘isothermal’ methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT, akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

  11. LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP FOR THE DETECTION OF SALMONELLA SPP. ISOLATED FROM DIFFERENT FOOD TYPES

    Directory of Open Access Journals (Sweden)

    Kostas Papanotas

    2012-08-01

    Full Text Available The objective of this study was the application and evaluation of a loop-mediated isothermal amplification (LAMP method for the detection of Salmonella spp. strains isolated from food samples. Salmonella specific invA gene sequences (50 strains, 15 serotypes were amplified at 65oC in 60 min. All of the strains of Salmonella subsp. Enterica were shown to be positive using the LAMP reaction assay, whereas, all other bacteria, virus and yeasts tested in this study were negative. LAMP products could be visually detected under day light or ultraviolet light, while the specific amplification of the DNA of Salmonella strains generated ladder-like pattern bands on agarose gel. LAMP is suitable for the sensitive, rapid, and inexpensive detection of Salmonella spp. in food analytical laboratories.

  12. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid diagnosis of chilli veinal mottle virus.

    Science.gov (United States)

    Banerjee, Amrita; Roy, Somnath; Sharma, Susheel Kumar; Dutta, Sudip Kumar; Chandra, Satish; Ngachan, S V

    2016-07-01

    Chilli veinal mottle virus (ChiVMV) causes significant economic loss to chilli cultivation in northeastern India, as well as in eastern Asia. In this study, we have developed a single-tube one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid, sensitive and specific diagnosis of ChiVMV. Amplification could be visualized after adding SYBR Green I (1000×) dye within 60 min under isothermal conditions at 63 °C, with a set of four primers designed based on the large nuclear inclusion protein (NIb) domain of ChiVMV (isolate KC-ML1). The RT-LAMP method was 100 times more sensitive than one-step reverse transcription polymerase chain reaction (RT-PCR), with a detection limit of 0.0001 ng of total RNA per reaction.

  13. Concurrent reactivation of herpes simplex and varicella zoster viruses confirmed by the loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Kobayashi, Tsukane; Yagami, Akiko; Suzuki, Kayoko; Yoshikawa, Tetsushi; Matsunaga, Kayoko

    2014-01-01

    Concurrent reactivation of herpes simplex and varicella zoster viruses is rare. Here, we describe the case of an elderly patient with herpes labialis and herpes zoster manifesting as a right-side facial eruption with vesicles and crusting. The loop-mediated isothermal amplification (LAMP) assay demonstrated the presence of both herpes simplex virus type 1 and varicella zoster virus in swab samples taken from the face, which was confirmed by real-time PCR, suggesting concurrent reactivation of both viruses. The use of the LAMP assay in the present case indicates its usefulness in the diagnosis of atypical herpes infections.

  14. Amplification activation loop between caspase-8 and -9 dominates artemisinin-induced apoptosis of ASTC-a-1 cells.

    Science.gov (United States)

    Xiao, Fenglian; Gao, Weijie; Wang, Xiaoping; Chen, Tongsheng

    2012-06-01

    Although caspases have been demonstrated to be involved in artemisinin (ARTE)-induced apoptosis, their exact functions are not well understood. The aim of this report is to explore the roles of caspase-8, -9 and -3 during ARTE-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cells. ARTE treatment induces a rapid generation of reactive oxygen species (ROS), and ROS-dependent apoptosis as well as the activation of caspase-8, -9 and -3 via time- and dose-dependent fashion. Of upmost importance, inhibition of caspase-8 or -9, but not caspase-3, almost completely blocks the ARTE-induced not only activation of the caspase-8, -9 and -3 but also apoptosis. In addition, the apoptotic process triggered by ARTE does not involve the Bid cleavage, tBid translocation, significant loss of mitochondrial membrane potential and cytochrome c release from mitochondria. Moreover, silencing Bax/Bak does not prevent the ATRE-induced cell death as well as the activation of caspase-8, -9 and -3. Collectively, our data firstly demonstrate that ARTE triggers a ROS-mediated positive feedback amplification activation loop between caspase-8 and -9 independent of mitochondria, which dominantly mediated the ARTE-induced apoptosis via a caspase-3-independent apoptotic pathway in ASTC-a-1 cells. Our findings imply a potential to develop new derivatives from artemisinin to effectively initiate the amplification activation loop of caspases.

  15. Loop-Mediated Isothermal Amplification Procedure (LAMP) for Detection of the Potato Zebra Chip Pathogen "Candidatus Liberibacter solanacearum".

    Science.gov (United States)

    Ravindran, Aravind; Lévy, Julien; Pierson, Elizabeth; Gross, Dennis C

    2015-01-01

    An efficient loop-mediated isothermal amplification procedure (LAMP) for the detection of "Candidatus Liberibacter solanacearum" (Lso), the bacterial causal agent of potato zebra chip (ZC) disease, is described in this chapter. Similar to the polymerase chain reaction (PCR), the LAMP employs a bacterial polymerase to amplify specific DNA sequences. However, the method differs from conventional PCR in that it uses six primers specific to the target region to generate a loop structure and autocycling strand displacement rather than thermocycling for sequence amplification. Moreover, unlike PCR that requires agarose gel electrophoresis for resolution, the positive LAMP results can be visualized directly as a precipitate within the reaction tubes. The 16S rDNA gene of "Ca. Liberibacter solanacearum" was used as the target for the design of the six LAMP primers. The LAMP technique is a reliable, rapid, and cost-effective method of detecting the "Ca. Liberibacter solanacearum" pathogen in the potato/tomato psyllid, Bactericera cockerelli, and in field-grown potato plants and tubers.

  16. Loop-mediated isothermal amplification for the rapid detection of Chrysanthemum chlorotic mottle viroid (CChMVd).

    Science.gov (United States)

    Park, Jungan; Jung, Yuchul; Kil, Eui-Joon; Kim, Jaedeok; Thi Tran, Dung; Choi, Seung-Kook; Yoon, Ju-Yeon; Cho, Won Kyong; Lee, Sukchan

    2013-10-01

    Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering rapid, sensitive, and convenient diagnosis of infectious diseases. Chrysanthemum chlorotic mottle viroid (CChMVd) causes one of the most serious viral diseases in chrysanthemum in Korea. A sensitive LAMP assay was developed for rapidly detecting CChMVd infection. The assay was based on a set of four primers matching the specific region of the CChMVd genome. The CChMVd LAMP primer sets were designed using the sequences from nonsymptomatic and symptomatic CChMVd isolates in Korea. The efficiency and specificity of this method were optimized using Bst DNA polymerase, which allowed for increased viroid detection sensitivity. The reaction was carried out at 65 °C for 90 min, and was improved by adding SYBR Green I dye to the inside of the reaction tube lid prior to amplification. The results indicate that this LAMP method will be useful for chrysanthemum viroid disease monitoring and detecting CChMVd infectious disease. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  17. Sensitive and rapid detection of Paragonimus westermani infection in humans and animals by loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Chen, M X; Ai, L; Zhang, R L; Xia, J J; Wang, K; Chen, S H; Zhang, Y N; Xu, M J; Li, X; Zhu, X Q; Chen, J X

    2011-05-01

    In the present study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for the detection of Paragonimus westermani adults, metacercariae, and eggs in human and animal samples. The LAMP amplification can be finished in 45 min under isothermal condition at 60°C by employing a set of four species-specific primer mixtures and the results can be checked by naked-eye visualization. No amplification products were detected with deoxyribunucleic acid (DNA) of related trematode species including Fasciola hepatica, Fasciola gigantica, Clonorchis sinensis, Opisthorchis viverrini, Schistosoma mansoni, and Schistosoma japonicum. The method was further validated by examining P. westermani DNA in intermediate hosts including freshwater crabs and crayfish, as well as in sputum and pleural fluid samples from patients of paragonimiasis. These results indicated that the LAMP assay was highly specific, sensitive, and rapid, and it was approximately 100 times more sensitive than conventional specific PCR. The LAMP assay established in this study provides a rapid and sensitive tool for the detection of P. westermani DNA in freshwater crabs, crayfish, sputum, and pleural fluid samples, which has important implications for effective control of human paragonimiasis.

  18. groEL is a suitable genetic marker for detecting Vibrio parahaemolyticus by loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Siddique, M P; Jang, W J; Lee, J M; Ahn, S H; Suraiya, S; Kim, C H; Kong, I S

    2017-08-01

    A groEL gene-based loop-mediated isothermal amplification (LAMP) assay was developed to detect Vibrio parahaemolyticus in contaminated seafood and water. The assay was optimized and conducted at 63°C for 40 min using Bacillus stearothermophilus (Bst) DNA polymerase, large fragment. Amplification was analysed via multiple detection methods, including opacity, formation of white precipitate, DNA intercalating dyes (ethidium bromide and SYBR Green I), metal ion-binding indicator dye, calcein, and 2% agarose gel electrophoresis. A characteristic ladder-like band pattern on agarose gel and the desired colour changes when using different dyes were observed in positive cases, and these were species-specific for V. parahaemolyticus when compared with other closely related Vibrio spp. The limit of detection (LoD) of this assay was 100 fg per reaction, 100-fold higher than that for conventional polymerase chain reaction (PCR). When tested on artificially contaminated seafood and seawater, the LoDs of the LAMP assay were 120 and 150 fg per reaction respectively, and those of conventional PCR were 120 and 150 pg per reaction respectively. Based on our results, the groEL gene-based LAMP assay is rapid, specific, sensitive, and reliable for detecting V. parahaemolyticus, and it could be used in field diagnosis. The loop-mediated isothermal amplification (LAMP) assay using groEL gene (an abundant, highly conserved gene and member of the groESL chaperone gene family) provided rapid, species-specific and highly sensitive method for detecting Vibrio parahaemolyticus, the leading causal agent of seafood-borne diseases worldwide. Moreover, groEL LAMP revealed high efficiency than conventional PCR assay for V. parahaemolyticus using template both from pure culture and artificially contaminated seafood and water, which indicated the applicability in the field and environmental screening purpose for the organism. © 2017 The Society for Applied Microbiology.

  19. Algorithms for detecting and analysing autocatalytic sets.

    Science.gov (United States)

    Hordijk, Wim; Smith, Joshua I; Steel, Mike

    2015-01-01

    Autocatalytic sets are considered to be fundamental to the origin of life. Prior theoretical and computational work on the existence and properties of these sets has relied on a fast algorithm for detectingself-sustaining autocatalytic sets in chemical reaction systems. Here, we introduce and apply a modified version and several extensions of the basic algorithm: (i) a modification aimed at reducing the number of calls to the computationally most expensive part of the algorithm, (ii) the application of a previously introduced extension of the basic algorithm to sample the smallest possible autocatalytic sets within a reaction network, and the application of a statistical test which provides a probable lower bound on the number of such smallest sets, (iii) the introduction and application of another extension of the basic algorithm to detect autocatalytic sets in a reaction system where molecules can also inhibit (as well as catalyse) reactions, (iv) a further, more abstract, extension of the theory behind searching for autocatalytic sets. (i) The modified algorithm outperforms the original one in the number of calls to the computationally most expensive procedure, which, in some cases also leads to a significant improvement in overall running time, (ii) our statistical test provides strong support for the existence of very large numbers (even millions) of minimal autocatalytic sets in a well-studied polymer model, where these minimal sets share about half of their reactions on average, (iii) "uninhibited" autocatalytic sets can be found in reaction systems that allow inhibition, but their number and sizes depend on the level of inhibition relative to the level of catalysis. (i) Improvements in the overall running time when searching for autocatalytic sets can potentially be obtained by using a modified version of the algorithm, (ii) the existence of large numbers of minimal autocatalytic sets can have important consequences for the possible evolvability of

  20. An empirical approach for quantifying loop-mediated isothermal amplification (LAMP) using Escherichia coli as a model system.

    Science.gov (United States)

    Subramanian, Sowmya; Gomez, Romel D

    2014-01-01

    Loop mediated isothermal amplification (LAMP) is a highly efficient, selective and rapid DNA amplification technique for genetic screening of pathogens. However, despite its popularity, there is yet no mathematical model to quantify the outcome and no well-defined metric for comparing results that are available. LAMP is intrinsically complex and involves multiple pathways for gene replication, making fundamental modelling nearly intractable. To circumvent this difficulty, an alternate, empirical model is introduced that will allow one to extract a set of parameters from the concentration versus time curves. A simple recipe to deduce the time to positive, Tp--a parameter analogous to the threshold cycling time in polymerase chain reaction (PCR), is also provided. These parameters can be regarded as objective and unambiguous indicators of LAMP amplification. The model is exemplified on Escherichia coli strains by using the two gene fragments responsible for vero-toxin (VT) production and tested against VT-producing (O157 and O45) and non-VT producing (DH5 alpha) strains. Selective amplification of appropriate target sequences was made using well established LAMP primers and protocols, and the concentrations of the amplicons were measured using a Qubit 2.0 fluorometer at specific intervals of time. The data is fitted to a generalized logistic function. Apart from providing precise screening indicators, representing the data with a small set of numbers offers significant advantages. It facilitates comparisons of LAMP reactions independently of the sampling technique. It also eliminates subjectivity in interpretation, simplifies data analysis, and allows easy data archival, retrieval and statistical analysis for large sample populations. To our knowledge this work represents a first attempt to quantitatively model LAMP and offer a standard method that could pave the way towards high throughput automated screening.

  1. An empirical approach for quantifying loop-mediated isothermal amplification (LAMP using Escherichia coli as a model system.

    Directory of Open Access Journals (Sweden)

    Sowmya Subramanian

    Full Text Available Loop mediated isothermal amplification (LAMP is a highly efficient, selective and rapid DNA amplification technique for genetic screening of pathogens. However, despite its popularity, there is yet no mathematical model to quantify the outcome and no well-defined metric for comparing results that are available. LAMP is intrinsically complex and involves multiple pathways for gene replication, making fundamental modelling nearly intractable. To circumvent this difficulty, an alternate, empirical model is introduced that will allow one to extract a set of parameters from the concentration versus time curves. A simple recipe to deduce the time to positive, Tp--a parameter analogous to the threshold cycling time in polymerase chain reaction (PCR, is also provided. These parameters can be regarded as objective and unambiguous indicators of LAMP amplification. The model is exemplified on Escherichia coli strains by using the two gene fragments responsible for vero-toxin (VT production and tested against VT-producing (O157 and O45 and non-VT producing (DH5 alpha strains. Selective amplification of appropriate target sequences was made using well established LAMP primers and protocols, and the concentrations of the amplicons were measured using a Qubit 2.0 fluorometer at specific intervals of time. The data is fitted to a generalized logistic function. Apart from providing precise screening indicators, representing the data with a small set of numbers offers significant advantages. It facilitates comparisons of LAMP reactions independently of the sampling technique. It also eliminates subjectivity in interpretation, simplifies data analysis, and allows easy data archival, retrieval and statistical analysis for large sample populations. To our knowledge this work represents a first attempt to quantitatively model LAMP and offer a standard method that could pave the way towards high throughput automated screening.

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

    OpenAIRE

    Villari, C.; Tomlinson, J. A.; Battisti, A.; Boonham, N.; Capretti, P.; Faccoli, M.

    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 ...

  3. When the collective acts on its components: economic crisis autocatalytic percolation

    Science.gov (United States)

    Cantono, S.; Solomon, S.

    2010-07-01

    Agent-based models have improved the standards for empirical support and validation criteria in social, biological, cognitive and human sciences. Yet, the inclusion, in these models, of vertical interactions between various aggregation levels remains a challenge. We study analytically, numerically and by simulation the generic consequences of interactions between the collective and its individual components: the appearance of an autocatalytic loop between the dynamics of the collective and its components; the system, which is dominated by a limited number of factors amplified by this collectiveindividuals autocatalytic loop; the microscopic features, which are not involved in the autocatalytic loop and are irrelevant at the systemic level; and how the above clarify the interplay between macroscopic predictable features and the ones dependent on random unpredictable individual events. Using the social and market percolation framework, we study the dramatic effects of the collectiveindividuals autocatalytic loop on economic crisis propagation: the percolation transition becomes discontinuous; there are a few relevant regions and regimes corresponding to a quite diverse range of response policy options; there are stability ranges where appropriate policies can help to avoid macroscopic crisis percolation; and beyond those regions the systemic crisis might become unstoppable.

  4. Loop-mediated isothermal amplification in disposable polyester-toner microdevices.

    Science.gov (United States)

    de Oliveira, Kezia Gomes; Borba, Juliane Cristina; Bailão, Alexandre Melo; de Almeida Soares, Célia Maria; Carrilho, Emanuel; Duarte, Gabriela Rodrigues Mendes

    2017-10-01

    In recent years, isothermal DNA amplification methods have emerged as an alternative in molecular diagnostics due to its ease of operation. The purpose of using isothermal amplification is to simplify the diagnostics work by i) eliminating heating cycles, ii) reducing equipment costs, and iii) providing rapid and accurate results in laboratories with limited resources. Here we show a simple and fast method for E. coli detection in disposable polyester-toner (PeT) microdevice. The amplification by LAMP of the malB gene from E. coli was carried out in a microchamber with 5-μL capacity and the reaction was thermally controlled with a thermoblock at 66 °C for 60 min. The passivation of the surface of PeT channels with BSA improved the efficiency of the LAMP reaction. The detection of amplified LAMP fragments was performed directly on-chip by visual detection and validated with off-chip detection to compare results. Visualization of amplicons directly in the microchip yielded positive reactions as low as 10 double-stranded DNA copies. Separation by gel electrophoresis was able to detect amplicons in reactions that initiated only with one copy of double-stranded DNA. We demonstrate that LAMP in PeT microchip is an important tool for molecular diagnostics at the point-of-care. Copyright © 2017. Published by Elsevier Inc.

  5. Loop-Mediated Isothermal Amplification Assay Targeting the MOMP Gene for Rapid Detection of Chlamydia psittaci Abortus Strain

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    Guo-Zhen Lin, Fu-Ying Zheng, Ji-Zhang Zhou, Guang-Hua Wang, Xiao-An Cao, Xiao-Wei Gong and Chang-Qing Qiu*

    2012-05-01

    Full Text Available For rapid detection of the Chlamydia psittaci abortus strain, a loop-mediated isothermal amplification (LAMP assay was developed and evaluated in this study. The primers for the LAMP assay were designed on the basis of the main outer membrane protein (MOMP gene sequence of C. psittaci. Analysis showed that the assay could detect the abortus strain of C. psittaci with adequate specificity. The sensitivity of the test was the same as that of the nested-conventional PCR and higher than that of chick embryo isolation. Testing of 153 samples indicated that the LAMP assay could detect the genome of the C. psittaci abortus strain effectively in clinical samples. This assay is a useful tool for rapid diagnosis of C. psittaci infection in sheep, swine and cattle.

  6. Development and application of a quantitative loop-mediated isothermal amplification method for detecting genetically modified maize MON863.

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    Huang, Sicong; Xu, Yuancong; Yan, Xinghua; Shang, Ying; Zhu, Pengyu; Tian, Wenying; Xu, Wentao

    2015-01-01

    A SYBR Green I-based quantitative loop-mediated isothermal amplification (LAMP) assay was developed for the rapid detection of genetically modified maize MON863. A set of primers was designed based on the integration region of the Cry3Bb1 and tahsp17 genes. The qualitative and quantitative reaction conditions (dNTPs, betaine, primers, Mg(2+), Bst polymerase, temperature, reaction time) were optimized. The concentrations of Mg(2+) and betaine were found to be important to the LAMP assay. The detection limits of both qualitative and quantitative LAMP for MON863 were as low as 4 haploid genomic DNA, and the LAMP reactions can be completed within 1 h at an isothermal temperature of 65 °C. The results of this study demonstrate that this new SYBR Green I-based quantitative LAMP assay system is reliable, sensitive and accurate. © 2014 Society of Chemical Industry.

  7. Loop-Mediated Isothermal Amplification (LAMP) for Detection of Campylobacter jejuni and C. coli in Thai Children with Diarrhea.

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    Pham, Ngan Thi Kim; Trinh, Quang Duy; Khamrin, Pattara; Ukarapol, Nuthapong; Kongsricharoern, Tipachan; Yamazaki, Wataru; Komine-Aizawa, Shihoko; Okitsu, Shoko; Maneekarn, Niwat; Hayakawa, Satoshi; Ushijima, Hiroshi

    2015-01-01

    Campylobacter species are common causes of bacterial diarrhea, and Campylobacter jejuni and C. coli are known as the predominant causative agents in humans. Recent studies suggested that loop-mediated isothermal amplification (LAMP) is an efficient and practical tool for rapid detection of C. jejuni and C. coli in clinical samples. We used LAMP to screen 151 stool samples for Campylobacter; these samples were collected in 2012 from Thai children with diarrhea. The PCR method discriminated C. jejuni and C. coli among the detected Campylobacter strains; these species were subjected to sequencing of the hipO gene (in C. jejuni) or the ask gene (in C. coli). The results suggest that the prevalence of Campylobacter infection among Thai children with diarrhea is 8.6%, and C. jejuni is the most prevalent species.

  8. Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus

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    Ge Beilei

    2010-02-01

    Full Text Available Abstract Background Vibrio parahaemolyticus is a leading cause of seafood-related bacterial gastroenteritis and outbreaks worldwide. Sensitive and specific detection methods are needed to better control V. parahaemolyticus infections. This study aimed at developing a highly specific and sensitive loop-mediated isothermal amplification (LAMP assay for detecting V. parahaemolyticus in oysters. A set of five LAMP primers, two outer, two inner, and one loop were designed based on the published V. parahaemolyticus toxR sequence. Specificity of the assay was evaluated using a panel of 36 V. parahaemolyticus and 39 other strains. The assay sensitivity was determined using serial dilutions of V. parahaemolyticus ATCC 27969 culture ranging from 108 CFU/ml to extinction. The assay was also tested in experimentally inoculated oyster samples. Results The toxR-based LAMP assay was able to specifically detect all of the 36 V. parahaemolyticus strains without amplification from 39 other strains. The detection limit was 47-470 cells per reaction in pure culture, up to 100-fold more sensitive than that of toxR-PCR. When applied in spiked oysters, the assay was able to detect 1.1 × 105 V. parahaemolyticus cells per gram of oyster without enrichment, up to 100-fold more sensitive than that of toxR-PCR. Standard curves generated for detecting V. parahaemolyticus in both pure culture and spiked oyster samples showed good linear relationship between cell numbers and the fluorescence or turbidity signals. Conclusions The toxR-based LAMP assay developed in this study was sensitive, specific, and quantitative, holding great potential for future field detection of V. parahaemolyticus in raw oysters.

  9. Rapid detection of newly isolated Tembusu-related Flavivirus by reverse-transcription loop-mediated isothermal amplification assay

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    Wang Youling

    2011-12-01

    Full Text Available Abstract Background From April 2010 to January 2011, a severe new viral disease had devastated most duck-farming regions in China. This disease affected not only laying ducks but also meat ducks, causing huge economic losses for the poultry industry. The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP assay for the detection of the new virus related to Tembusu-related Flavivirus. Results The RT-LAMP assay is very simple and rapid, and the amplification can be completed within 50 min under isothermal conditions at 63°C by a set of 6 primers targeting the E gene based on the sequences analysis of the newly isolated viruses and other closely related Flavivirus.The monitoring of gene amplification can also be visualized by using SYBR green I fluorescent dye. In addition, the RT-LAMP assay for newly isolated Tembusu-related Flavivirus showed higher sensitivity with an RNA detection-limit of 2 copies/μL compared with 190 copies/μL of the conventional RT-PCR method. The specificity was identified without cross reaction to other common avian pathogens. By screening a panel of clinical samples this method was more feasible in clinical settings and there was higher positive coincidence rate than conventional RT-PCR and virus isolation. Conclusion The RT-LAMP assay for newly isolated Tembusu-related Flavivirus is a valuable tool for the rapid and real-time detection not only in well-equipped laboratories but also in general conditions.

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

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    Kiddle Guy

    2012-04-01

    Full Text Available Abstract 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

  11. Development of a loop-mediated isothermal amplification method to rapidly detect porcine circovirus genotypes 2a and 2b

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    Qiu Xiaohuo

    2012-12-01

    Full Text Available Abstract Background Porcine circovirus type 2 (PCV2, is nowadays associated with a number of diseases known as porcine circovirus-associated diseases (PCVAD, especially postweaning multisystemic wasting syndrome (PMWS. The epidemiological investigation of PCV2 infection was usually conducted by PCR, nested PCR, PCR-RFLP, TaqMan-based assay and nucleotide sequencing. However, there is still no rapid, sensitive and practical method for detecting PCV2 genotypes. As a novel nucleic acid amplification method, the loop-mediated isothermal amplification method (LAMP has been used to detect a variety of pathogenic microorganisms. Results Herein, a LAMP method is developed to detect the genotypes of PCV2. The diagnostic sensitivity of LAMP is 1 copy/reaction for differentiating genotypes PCV2a and PCV2b. The reaction process was completed at 65°C for 1 hour in a water bath. Cross-reactivity assay shows that this method is specific for PCV2a and PCV2b and no reactive for PCV2c and other swine-origin viruses (i.e. CSFV, PRRSV, BVDV, TGEV and PEDV, etc. Identity between LAMP and nested PCR was 92.3% on 52 field clinical samples. Conclusions LAMP method provides a rapid, sensitive, reliable way to detect PCV2a and PCV2b, and a better means for the large scale investigation of PCV2a and PCV2b infection.

  12. An immunomagnetic separation/loop-mediated isothermal amplification method for rapid direct detection of thermotolerant Campylobacter spp. during poultry production.

    Science.gov (United States)

    Romero, M R; D'Agostino, M; Arias, A Pérez; Robles, S; Casado, C Fernández; Iturbe, L Orueta; Lerma, O Gurrutxaga; Andreou, M; Cook, N

    2016-02-01

    To develop a rapid test for thermotolerant Campylobacter in poultry faeces. The reported method is based on immunomagnetic separation and loop-mediated isothermal DNA amplification (IMS/LAMP). This LAMP assay is specific (demonstrated using 10 Campylobacter strains and 13 non-Campylobacter bacterial species) and sensitive (95% probability of detecting 22 genome copies). A competitive internal amplification control (IAC) has been incorporated to give unambiguous determination of negative results. Immunoseparation of Campylobacter allows direct LAMP detection from poultry boot swab samples in 90 min without enrichment or DNA purification (74% probability of detecting 10(4) CFU ml(-1) of a boot swab suspension). The analysis of 17 samples from commercial turkey farms showed 100% correlation with parallel results obtained by standard microbiological methods. A rapid test has been developed for direct detection of thermotolerant Campylobacter spp. in boot swab samples, thus bypassing culture enrichment or DNA extraction. The test has potential to be carried out by farm personnel on site. The method offers an inexpensive approach to monitor poultry infection in near real time, assisting flock management and controls to prevent introduction of Campylobacter into the food chain. © 2015 The Society for Applied Microbiology.

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

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    Preeti Singh

    2015-01-01

    Full Text Available 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.

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

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    Ya-Bing Duan

    Full Text Available 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.

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

    Science.gov (United States)

    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

  16. Rapid detection of viable Escherichia coli O157 by coupling propidium monoazide with loop-mediated isothermal amplification.

    Science.gov (United States)

    Zhao, Xihong; Wang, Jun; Forghani, Fereidoun; Park, Joong-Hyun; Park, Myoung-Su; Seo, Kun-Ho; Oh, Deog-Hwan

    2013-12-01

    Conventional molecular detection methods cannot distinguish between viable and dead Escherichia coli O157 cells. In this study, the loop-mediated isothermal amplification (LAMP) method combined with propidium monoazide (PMA) treatment was developed to selectively detect viable E. coli O157 cells. Four primers, including outer primers and inner primers, were specially designed for the recognition of six distinct sequences on the serogroups (O157) of the specific rfbE gene of the E. coli O157 genome. PMA selectively penetrated through the compromised cell membranes and intercalated into DNA. Amplification of DNA from dead cells was completely inhibited by 3.0 μg/ml PMA, whereas the DNA derived from viable cells was amplified remarkably within 1 h by PMA-LAMP. Exhibiting high sensitivity and specificity, PMA-LAMP is a suitable method for evaluating the inactivation efficacy of slightly acidic electrolyzed water in broth. PMA-LAMP can selectively detect viable E. coli O157 cells. This study offers a novel molecular detection method to distinguish between viable and dead E. coli O157 cells.

  17. Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.

    Science.gov (United States)

    Abbasi, Ibrahim; Kirstein, Oscar D; Hailu, Asrat; Warburg, Alon

    2016-10-01

    Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Evaluation of colorimetric loop-mediated isothermal amplification assay for visual detection of Streptococcus agalactiae and Streptococcus iniae in tilapia.

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    Suebsing, R; Kampeera, J; Tookdee, B; Withyachumnarnkul, B; Turner, W; Kiatpathomchai, W

    2013-10-01

    Streptococcus agalactiae and Strep. iniae are bacterial pathogens that cause streptococcosis in many fish species. An accelerated colorimetric loop-mediated isothermal amplification (LAMP) assay with pre-addition of calcein was established, and the transmission and detection of Strep. agalactiae and Strep. iniae in tilapia under natural aquatic environment were investigated. A positive reaction was observed by a colour change from orange to green through the naked eyes after completion at 63°C for 30 min with 10 times higher sensitivity than that of nested PCR assays and without cross-amplification with other fish bacterial pathogens. All sample types of Nile and red tilapia (broodstock, fertilized egg, fry) were Strep. agalactiae- and Strep. iniae positive by this new method, implying that they could be vertically transmitted. With its application for screening broodstock and fry before stocking and for monitoring fish health in grow-out ponds, the method would become very useful in fish farming industry. The application of colorimetric LAMP with pre-addition of calcein offers simple, rapid and sensitive technique with applicability for small field laboratories. This technique explored the possible vertical transmission mode of Strep. agalactiae and Strep. iniae under natural aquatic environment. It could be such preliminary data provided for the screening broodstock before breeding and/or the specific-pathogen-free production. © 2013 The Society for Applied Microbiology.

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

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    Joojin Jeong

    2015-09-01

    Full Text Available 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.

  20. Evaluation of a real-time PCR and a loop-mediated isothermal amplification for detection of Xanthomonas arboricola pv. pruni in plant tissue samples

    NARCIS (Netherlands)

    Palacio-Bielsa, Ana; López-Soriano, Pablo; Bühlmann, Andreas; Doorn, van Joop; Pham, Khanh; Cambra, Miguel A.; Berruete, Isabel M.; Pothier, Joël F.; Duffy, Brion; Olmos, Antonio; López, María M.

    2015-01-01

    Operational capacity of real-time PCR and loop-mediated isothermal amplification (LAMP) diagnostic assays for detection of Xanthomonas arboricola pv. pruni was established in a ring-test involving four laboratories. Symptomatic and healthy almond leaf samples with two methods of sample

  1. Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of Sugarcane mosaic virus and Sorghum mosaic virus in sugarcane

    Science.gov (United States)

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) in sugarcane. Six sets of four primers corresponding to the conserved coat protein gene were designed for each virus and their succ...

  2. Development of reverse transcription loop-mediated isothermal amplification assay for rapid detection of an emerging potyvirus: tomato necrotic stunt virus

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    Tomato necrotic stunt virus (ToNStV) is an emerging potyvirus that causes severe stunting to the infected tomato plants. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for a sensitive detection of ToNStV. The sensitivity of RT-LAMP was comparable to th...

  3. Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples

    NARCIS (Netherlands)

    Adams, Emily R.; Schoone, Gerard J.; Ageed, Al Farazdag; El Safi, Sayda; Schallig, Henk D. F. H.

    2010-01-01

    Here we describe a generic, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) assay, for the identification of Leishmania species from clinical samples. LAMP is an isothermal reaction recently developed as a point-of-care diagnostic tool. Primers were designed in the conserved

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

    Science.gov (United States)

    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/μl 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.

  5. Development of a loop-mediated isothermal amplification method for rapid detection of pigeon circovirus.

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    Tsai, Shinn Shyong; Chang, Yeng Ling; Huang, Yen Li; Liu, Hung Jen; Ke, Guan Ming; Chiou, Chwei Jang; Hsieh, Yao Ching; Chang, Tsung Chou; Cheng, Li Ting; Chuang, Kuo Pin

    2014-05-01

    There are no effective antiviral treatments for pigeon circovirus (PiCV); thus, rapid diagnosis is critical for effective control of the disease caused by this virus. The recent development of a novel LAMP technique that amplifies nucleic acids rapidly with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic-acid-based diagnostic tests. We established a LAMP method for rapid detection of PiCV using two pairs of primers that were designed from PiCV and compared its sensitivity and specificity with that of PCR. Amplification by LAMP was optimal at 63 °C for 60 min. The detection limit was nearly 0.5 pg of PiCV DNA, making it ten times more sensitive than PCR. There was no cross-reaction with porcine circovirus type 2 (PCV2), pigeon Trichomonas gallinae, or pigeon herpesvirus (PHV) under the same conditions. The assay also successfully detected the pathogen DNA in the tissues of infected pigeons. This is the first report indicating that LAMP is a valuable, rapid method of detecting PiCV with high sensitivity and specificity.

  6. The application of loop-mediated isothermal amplification (LAMP) in food testing for bacterial pathogens and fungal contaminants.

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

    2013-12-01

    Bacterial pathogens and toxicants, parasites as well as mycotoxin producing fungi are the major biotic factors influencing the safety of food. Moreover, viral infections and prions may be present as quasi biotic challenging factors. A vast array of culture dependent analytical methods and protocols for food safety testing has been developed during the past decades. Presently, protocols involving molecular biological techniques such as PCR-based nucleic acid amplification and hybridization have become available for many of the known pathogens with their major advantages being rapidness, high sensitivity and specificity. However, this type of assays is still quite labor- and cost intensive and mostly cannot be operated directly in the field. Recently, loop-mediated isothermal amplification (LAMP) of DNA has emerged as an alternative to the use of PCR-based methods not only in food safety testing but also in a wide array of application. Its advantages over PCR-based techniques are even shorter reaction time, no need for specific equipment, high sensitivity and specificity as well as comparably low susceptibility to inhibitors present in sample materials which enables detection of the pathogens in sample materials even without time consuming sample preparation. The present article presents a critical review of the application of LAMP-based methods and their usefulness in detecting and identifying food borne bacterial pathogens and toxicants as well as mycotoxin producing food borne fungi as compared to other methods. Moreover does it elaborate on new developments in the design and automation of LAMP-based assays and their implications for the future developments of food testing. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Development of a loop-mediated Isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus

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    Kawahara Ryuji

    2008-09-01

    Full Text Available Abstract Background Vibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH and TDH-related hemolysin (TRH are known as major virulence determinants of V. parahaemolyticus. Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH. Total V. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total V. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP assay for the sensitive and rapid detection of Vibrio parahaemolyticus. Results The assay provided markedly more sensitive and rapid detection of V. parahaemolyticus strains than conventional biochemical and PCR assays. The assay correctly identified 143 V. parahaemolyticus strains, but did not detect 33 non-parahaemolyticus Vibrio and 56 non-Vibrio strains. Sensitivity of the LAMP assay for direct detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples was 5.3 × 102 CFU per ml/g (2.0 CFU per reaction. The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13–22 min in a single colony on TCBS agar from each of 143 V. parahaemolyticus strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of V. parahaemolyticus required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination. Conclusion The LAMP assay is a sensitive, rapid and simple tool for the detection of V. parahaemolyticus and will facilitate the surveillance for control of contamination of V

  8. A simple and rapid identification method for newly emerged porcine Deltacoronavirus with loop-mediated isothermal amplification

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    Fanfan Zhang

    2017-10-01

    Full Text Available Abstract Background Porcine Deltacoronavirus (PDCoV is a newly emerged enteropathogenic coronavirus that causes diarrhea and mortality in neonatal piglets. PDCoV has spread to many countries around the world, leading to significant economic losses in the pork industry. Therefore, a rapid and sensitive method for detection of PDCoV in clinical samples is urgently needed. Results In this study, we developed a single-tube one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP assay specific for nucleocapsid gene to diagnose and monitor PDCoV infections. The detection limit of RT-LAMP assay was 1 × 101 copies of PDCoV, which was approximately 100-fold more sensitive than gel-based one-step reverse transcription polymerase chain reaction (RT-PCR. This assay could specifically amplify PDCoV and had no cross amplification with porcine epidemic diarrhea virus (PEDV, transmissible gastroenteritis virus (TGEV, porcine kobuvirus (PKoV, porcine astrovirus (PAstV, porcine reproductive and respiratory syndrome virus (PRRSV, classic swine fever virus (CSFV, and porcine circovirus type 2 (PCV2. By screening a panel of clinical specimens (N = 192, this method presented a similar sensitivity with nested RT-PCR and was 1–2 log more sensitive than conventional RT-PCR in detection of PDCoV. Conclusions The RT-LAMP assay established in this study is a potentially valuable tool, especially in low-resource laboratories and filed settings, for a rapid diagnosis, surveillance, and molecular epidemiology investigation of PDCoV infections. To the best of our knowledge, this is the first work for detection of newly emerged PDCoV with LAMP technology.

  9. Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death

    Science.gov (United States)

    Graham, Nicholas A; Tahmasian, Martik; Kohli, Bitika; Komisopoulou, Evangelia; Zhu, Maggie; Vivanco, Igor; Teitell, Michael A; Wu, Hong; Ribas, Antoni; Lo, Roger S; Mellinghoff, Ingo K; Mischel, Paul S; Graeber, Thomas G

    2012-01-01

    The altered metabolism of cancer can render cells dependent on the availability of metabolic substrates for viability. Investigating the signaling mechanisms underlying cell death in cells dependent upon glucose for survival, we demonstrate that glucose withdrawal rapidly induces supra-physiological levels of phospho-tyrosine signaling, even in cells expressing constitutively active tyrosine kinases. Using unbiased mass spectrometry-based phospho-proteomics, we show that glucose withdrawal initiates a unique signature of phospho-tyrosine activation that is associated with focal adhesions. Building upon this observation, we demonstrate that glucose withdrawal activates a positive feedback loop involving generation of reactive oxygen species (ROS) by NADPH oxidase and mitochondria, inhibition of protein tyrosine phosphatases by oxidation, and increased tyrosine kinase signaling. In cells dependent on glucose for survival, glucose withdrawal-induced ROS generation and tyrosine kinase signaling synergize to amplify ROS levels, ultimately resulting in ROS-mediated cell death. Taken together, these findings illustrate the systems-level cross-talk between metabolism and signaling in the maintenance of cancer cell homeostasis. PMID:22735335

  10. Autocatalytic sets in a partitioned biochemical network.

    Science.gov (United States)

    Smith, Joshua I; Steel, Mike; Hordijk, Wim

    2014-01-01

    In previous work, RAF theory has been developed as a tool for making theoretical progress on the origin of life question, providing insight into the structure and occurrence of self-sustaining and collectively autocatalytic sets within catalytic polymer networks. We present here an extension in which there are two "independent" polymer sets, where catalysis occurs within and between the sets, but there are no reactions combining polymers from both sets. Such an extension reflects the interaction between nucleic acids and peptides observed in modern cells and proposed forms of early life. We present theoretical work and simulations which suggest that the occurrence of autocatalytic sets is robust to the partitioned structure of the network. We also show that autocatalytic sets remain likely even when the molecules in the system are not polymers, and a low level of inhibition is present. Finally, we present a kinetic extension which assigns a rate to each reaction in the system, and show that identifying autocatalytic sets within such a system is an NP-complete problem. Recent experimental work has challenged the necessity of an RNA world by suggesting that peptide-nucleic acid interactions occurred early in chemical evolution. The present work indicates that such a peptide-RNA world could support the spontaneous development of autocatalytic sets and is thus a feasible alternative worthy of investigation.

  11. Rapid sex identification of papaya (Carica papaya) using multiplex loop-mediated isothermal amplification (mLAMP).

    Science.gov (United States)

    Hsu, Te-Hua; Gwo, Jin-Chywan; Lin, Kuan-Hung

    2012-10-01

    Papaya (Carica papaya L.) is established as a cash crop throughout the tropical and subtropical regions due to its easy adaptation to diverse agricultural conditions, high yields, and prompt returns. The sex types of papaya plants are hermaphrodite, male, and female. Among them, hermaphroditic plants are the major type in papaya production, because the fruit has commercial advantages over that of the other sexes. Sex inheritance in papaya is determined by the M and M(h) dominant alleles in males and hermaphrodites, respectively, and a recessive m allele in females. Currently, all hermaphrodite seeds are not available due to the lethality of dominant homozygosity. Therefore, in this study, six male-hermaphrodite-specific markers were developed for a rapid sex identification using multiplex loop-mediated isothermal amplification (mLAMP) to efficiently and precisely select hermaphroditic individuals in the seedling or early growth stage. The LM1-LAMP assay consisted of two sex-LAMP reactions for amplifying two male-specific markers (T12 and Cpsm90) in one reaction, and showed several advantages in terms of a rapid reaction time (papaya production.

  12. Multiplex reverse transcription loop-mediated isothermal amplification for the simultaneous detection of CVB and CSVd in chrysanthemum.

    Science.gov (United States)

    Liu, Xing-Liang; Zhao, Xi-Ting; Muhammad, Imtiaz; Ge, Bei-Bei; Hong, Bo

    2014-12-15

    A multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) assay was developed for the simultaneous detection of Chrysanthemum Virus B (CVB) and Chrysanthemum stunt viroid (CSVd), which are the major viral pathogens of chrysanthemum worldwide. Two sets of mRT-LAMP primers were designed for the coat protein gene of CVB and the complete nucleotide sequence of CSVd, and a restriction enzyme cleavage site was inserted into two pairs of species-specific primers. The mRT-LAMP assay was designed by combining these two sets for a total of eight primers. The mRT-LAMP method distinguished between CVB and CSVd due to the subsequent restriction enzyme analysis. The sensitivity of the mRT-LAMP method was 10(3) times higher than classical PCR regarding the detection limits for CVB and CSVd. No positive results were observed when RNA from other chrysanthemum pathogens were used as mRT-LAMP templates. The method was verified by testing chrysanthemum samples collected from Beijing and Henan Province and showed high reliability and sensitivity. The developed mRT-LAMP assay also offers an efficient, convenient, and rapid tool for screening chrysanthemum virus and viroid, especially CVB and CSVd, and can be diagnosed in a single reaction. These results suggest that the new mRT-LAMP method may be used routinely for virus and viroid surveys. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Rapid, Sensitive Detection of Bartonella quintana by Loop-Mediated Isothermal Amplification of the groEL Gene

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    Shoukui Hu

    2016-12-01

    Full Text Available Trench fever, caused by Bartonella quintana, is recognized as a re-emerging and neglected disease. Rapid and sensitive detection approaches are urgently required to monitor and help control B. quintana infections. Here, loop-mediated isothermal amplification (LAMP, which amplifies target DNA at a fixed temperature with high sensitivity, specificity and rapidity, was employed to detect B. quintana. Thirty-six strains, including 10 B. quintana, 13 other Bartonella spp., and 13 other common pathogens, were applied to verify and evaluate the LAMP assay. The specificity of the LAMP assay was 100%, and the limit of detection was 125 fg/reaction. The LAMP assay was compared with qPCR in the examination of 100 rhesus and 20 rhesus-feeder blood samples; the diagnostic accuracy was found to be 100% when LAMP was compared to qPCR, but the LAMP assay was significantly more sensitive (p < 0.05. Thus, LAMP methodology is a useful for diagnosis of trench fever in humans and primates, especially in low-resource settings, because of its rapid, sensitive detection that does not require sophisticated equipment.

  14. One-step reverse transcription loop-mediated isothermal amplification for the detection of Maize chlorotic mottle virus in maize.

    Science.gov (United States)

    Chen, Ling; Jiao, Zhiyuan; Liu, Dongmei; Liu, Xingliang; Xia, Zihao; Deng, Congliang; Zhou, Tao; Fan, Zaifeng

    2017-02-01

    Maize chlorotic mottle virus (MCMV) is spreading in many regions worldwide, causing maize lethal necrosis when co-infected with a potyvirid. In this study, one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect MCMV in maize. A set of four specific primers was designed based on the conserved coat protein gene sequences of MCMV. The RT-LAMP could be completed within 60min under isothermal condition at 63°C. The sensitivity test showed that the RT-LAMP was about 10-fold more sensitive than RT-PCR and no cross-reactivity was detected with other viral pathogens infecting maize in China. Moreover, the results of RT-LAMP could be visually inspected by SYBR Green I staining in a closed-tube, facilitating high-throughput application of MCMV detection. This method was further verified by testing field-collected samples. These results suggested that the developed MCMV RT-LAMP technique is a rapid, efficient and sensitive method which could be used as a routine screen for MCMV infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Field Evaluation of a High Throughput Loop Mediated Isothermal Amplification Test for the Detection of Asymptomatic Plasmodium Infections in Zanzibar.

    Science.gov (United States)

    Aydin-Schmidt, Berit; Morris, Ulrika; Ding, Xavier C; Jovel, Irina; Msellem, Mwinyi I; Bergman, Daniel; Islam, Atiqul; Ali, Abdullah S; Polley, Spencer; Gonzalez, Iveth J; Mårtensson, Andreas; Björkman, Anders

    2017-01-01

    New field applicable diagnostic tools are needed for highly sensitive detection of residual malaria infections in pre-elimination settings. Field performance of a high throughput DNA extraction system for loop mediated isothermal amplification (HTP-LAMP) was therefore evaluated for detecting malaria parasites among asymptomatic individuals in Zanzibar. HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a community-based survey in 2015. The PCR and HTP-LAMP determined malaria prevalences were 1.6% (95%CI 1.3-2.4) and 0.7% (95%CI 0.4-1.1), respectively. The sensitivity of HTP-LAMP compared to PCR was 40.8% (CI95% 27.0-55.8) and the specificity was 99.9% (CI95% 99.8-100). For the PCR positive samples, there was no statistically significant difference between the geometric mean parasite densities among the HTP-LAMP positive (2.5 p/μL, range 0.2-770) and HTP-LAMP negative (1.4 p/μL, range 0.1-7) samples (p = 0.088). Two lab technicians analysed up to 282 samples per day and the HTP-LAMP method was experienced as user friendly. Although field applicable, this high throughput format of LAMP as used here was not sensitive enough to be recommended for detection of asymptomatic low-density infections in areas like Zanzibar, approaching malaria elimination.

  16. Establishment and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Detection of Raccoon Dog in Meat Mixtures

    Directory of Open Access Journals (Sweden)

    Jinhua Liu

    2017-01-01

    Full Text Available Raccoon dog (Nyctereutes procyonoides is an economically important animal used for fur production, but consuming its meat is injurious to human health. Currently, no rapid and sensitive method for detecting raccoon dog meat in meat mixtures is available. In this study, we developed an easily applicable, rapid, and economically feasible method for identifying the presence of raccoon dog in meat mixtures based on loop-mediated isothermal amplification (LAMP. Four sets of LAMP primers were tested at different temperatures, and the primers that worked best at 62°C (set 2 were determined. In the LAMP assay, there was no cross-reactivity with the meat procured from other species of animals and the detection limit of DNA concentration was 0.1 pg·μL−1, slightly higher than TaqMan real-time PCR (0.01 pg·μL−1, but sensitivity of 0.1 pg·μL−1 complies with most requirements of routine analysis. Moreover, by the LAMP method, the meat mixtures containing more than 0.5% of the raccoon dog component were directly detected (without DNA extraction in the supernatant isolated from the meat mixtures after performing repeated cycles of thawing and freezing of minced meat mixtures. Our results show that LAMP assay is a valuable, straightforward, and sensitive detection tool for identification of raccoon dog meat in mixtures.

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

    Science.gov (United States)

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

    2014-12-01

    Aflatoxins represent a serious risk for human and animal health. They are mainly produced by Aspergillus flavus and Aspergillus parasiticus but also by Aspergillus nomius. Three species specific turbidimeter based real-time LAMP (loop-mediated isothermal amplification) assays were developed to quantify the three species individually in conidial solutions and to define contamination levels in samples of shelled Brazil nuts, maize, and peanuts. Standard curves relating spore numbers to time to threshold (Tt) values were set up for each of the species. Assays had detection limits of 10, 100 and 100 conidia per reaction of A. flavus, A. parasiticus, and A. nomius, respectively. Analysis of contaminated sample materials revealed that the A. nomius specific real-time LAMP assay detected a minimum of 10 conidia/g in Brazil nuts while assays specific for A. flavus and A. parasiticus had detection limits of 10(2) conidia/g and 10(5) conidia/g, respectively in peanut samples as well as 10(4) conidia/g and 10(4) conidia/g, respectively in samples of maize. The real-time LAMP assays developed here appear to be promising tools for the prediction of potential aflatoxigenic risk at an early stage and in all critical control points of the food and feed production chain. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Rapid detection of duck hepatitis A virus genotype C using reverse transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Li, Chuanfeng; Chen, Zongyan; Meng, Chunchun; Liu, Guangqing

    2014-02-01

    A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was used and optimized to develop a rapid and sensitive detection system for duck hepatitis A virus genotype C (DHAV-C) RNA. A set of four specific primers was designed against highly conserved sequences located within the 3D gene from DHAV (strain GX1201). Under optimal reaction conditions, the sensitivity of DHAV-C-specific RT-LAMP was 100-fold higher than that of reverse transcriptase-polymerase chain reaction (RT-PCR), with a detection limit of 0.3pg (6.59×10(4) copies) per reaction. No cross-reactivity was observed from the samples of other duck viruses, which is in good accordance with RT-PCR. Furthermore, a positive reaction can be visually inspected by observing turbidity or color change after the addition of SYBR green I dye. The DHAV-C-specific RT-LAMP assay was applied to the samples and compared with RT-PCR. The positive-sample ratios were 26.7% (12 of 45) by RT-LAMP and 20% (9 of 45) by RT-PCR. Therefore, the newly developed RT-LAMP assay is a rapid, specific, sensitive, and cost-effective method of DHAV-C detection. This assay has potential applications in both clinical diagnosis and field surveillance of DHAV-C infection. Copyright © 2013. Published by Elsevier B.V.

  19. Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran.

    Science.gov (United States)

    Ghasemian, Mehrdad; Gharavi, Mohammad Javad; Akhlaghi, Lame; Mohebali, Mehdi; Meamar, Ahmad Reza; Aryan, Ehsan; Oormazdi, Hormozd

    2014-03-01

    Parasitological methods for the diagnosis of Visceral leishmaniasis (VL) require invasive procedures, so serological and molecular approaches have been developed but are not generally applicable in the field. We evaluated a loop mediated isothermal amplification (LAMP) assay using blood from VL patients and compared it to nested PCR. Forty-seven subjects with clinical features (fever, hepatosplenomegaly and anemia) were confirmed positive for VL by the direct agglutination test (DAT) at titers >3200. Forty DAT negative individuals from non-endemic areas with no clinical signs or symptoms of VL served as controls. A LAMP assay was performed using a set of six primers targeting Leishmania infantum kinetoplast DNA (kDNA) minicircle gene under isothermal (64 °C) conditions. For nested PCR we used primers targeting the kDNA minicircle gene. The LAMP assay provided a detection limit of 1 parasite in 1 ml of peripheral blood and detected L. infantum DNA in 44 of 47 DAT-confirmed VL cases, with diagnostic sensitivity of 93.6% (95% CI). No L. infantum DNA was amplified in controls, indicating a specificity of 100%. The nested PCR yielded sensitivity of 96% (95% CI) and a specificity of 100% (95% CI). The LAMP assay gave results similar to those of nested PCR but in a shorter time. The LAMP method is simple; requires no sophisticated equipment; has a short reaction time; and results, indicated by turbidity of the reaction mixture, are observable with the naked eye.

  20. Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran.

    Directory of Open Access Journals (Sweden)

    Mehrdad Ghasemian

    2014-03-01

    Full Text Available Parasitological methods for the diagnosis of Visceral leishmaniasis (VL require invasive procedures, so serological and molecular approaches have been developed but are not generally applicable in the field. We evaluated a loop mediated isothermal amplification (LAMP assay using blood from VL patients and compared it to nested PCR.Forty-seven subjects with clinical features (fever, hepatosplenomegaly and anemia were confirmed positive for VL by the direct agglutination test (DAT at titers >3200. Forty DAT negative individuals from non-endemic areas with no clinical signs or symptoms of VL served as controls. A LAMP assay was performed using a set of six primers targeting Leishmania infantum kinetoplast DNA (kDNA minicircle gene under isothermal (64 °C conditions. For nested PCR we used primers targeting the kDNA minicircle gene.The LAMP assay provided a detection limit of 1 parasite in 1 ml of peripheral blood and detected L. infantum DNA in 44 of 47 DAT-confirmed VL cases, with diagnostic sensitivity of 93.6% (95% CI. No L. infantum DNA was amplified in controls, indicating a specificity of 100%. The nested PCR yielded sensitivity of 96% (95% CI and a specificity of 100% (95% CI.The LAMP assay gave results similar to those of nested PCR but in a shorter time. The LAMP method is simple; requires no sophisticated equipment; has a short reaction time; and results, indicated by turbidity of the reaction mixture, are observable with the naked eye.

  1. Loop mediated isothermal amplification (LAMP) for the detection and subtyping of human papillomaviruses (HPV) in oropharyngeal squamous cell carcinoma (OPSCC).

    Science.gov (United States)

    Livingstone, D M; Rohatensky, M; Mintchev, P; Nakoneshny, S C; Demetrick, D J; van Marle, G; Dort, J C

    2016-02-01

    Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) is a growing problem that presents a significant challenge to Otolaryngologist-Head and Neck Surgeons. Knowledge of HPV status yields critical prognostic information, with potential for treatment selection based on tumour HPV status. The current gold standard of diagnosis, PCR, is expensive, demanding and time consuming. Alternatives such as p16 immunohistochemistry are subjective and potentially inaccurate. Loop-mediated isothermal amplification (LAMP) is a rapid, robust and inexpensive molecular diagnostic technique. Our aim was to verify LAMP as a potential bedside diagnostic assay for subtyping of HPV in OPSCC. DNA from 72 formalin-fixed paraffin embedded (FFPE) OPSCC patient samples was tested. PCR and LAMP were then performed to specifically identify HPV 16, 18, 31, 33 and 35. For these high-risk subtypes, LAMP had an overall sensitivity of 99.4% and specificity of 93.2% relative to PCR. LAMP is a promising technology that can accurately diagnose high-risk HPV infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Rapid and sensitive detection of Plesiomonas shigelloides by loop-mediated isothermal amplification of the hugA gene.

    Directory of Open Access Journals (Sweden)

    Shuang Meng

    Full Text Available Plesiomonas shigelloides is one of the causative agents of human gastroenteritis, with increasing number of reports describing such infections in recent years. In this study, the hugA gene was chosen as the target to design loop-mediated isothermal amplification (LAMP assays for the rapid, specific, and sensitive detection of P. shigelloides. The performance of the assay with reference plasmids and spiked human stools as samples was evaluated and compared with those of quantitative PCR (qPCR. No false-positive results were observed for the 32 non-P. shigelloides strains used to evaluate assay specificity. The limit of detection for P. shigelloides was approximately 20 copies per reaction in reference plasmids and 5×10(3 CFU per gram in spiked human stool, which were more sensitive than the results of qPCR. When applied in human stool samples spiked with 2 low levels of P. shigelloides, the LAMP assays achieved accurate detection after 6-h enrichment. In conclusion, the LAMP assay developed in this study is a valuable method for rapid, cost-effective, and simple detection of P. shigelloides in basic clinical and field laboratories in the rural areas of China.

  3. Rapid detection of nusG and fadA in Fusobacterium nucleatum by loop-mediated isothermal amplification.

    Science.gov (United States)

    Huang, Simo; Yang, Zhan; Zou, Dayang; Dong, Derong; Liu, Anheng; Liu, Wei; Huang, Liuyu

    2016-08-01

    Fusobacterium nucleatum is associated with various human diseases such as periodontal disease and colorectal cancer (CRC); thus, F. nucleatum detection might serve as a novel diagnostic tool. Here, we describe the development of a sensitive and rapid molecular method for detecting two F. nucleatum genes: the highly conserved nusG and fadA, which encode a critical host colonization factor. Loop-mediated isothermal amplification (LAMP) primer sets for the rapid detection of nusG and fadA were designed and optimized. The nusG primers yielded consistent negative results for 20 non-F. nucleatum bacterial strains, confirming the high specificity of the primers. LAMP reaction primer sensitivity was determined, and its detection rate in comparison to conventional PCR was assessed using 57 clinical stool samples. The LAMP detection limit for nusG and fadA was 22.5 and 0.225 pg µl-1, respectively, indicating that the sensitivity of this method was 10-fold higher than that of conventional PCR. These results suggest that the LAMP technique is able to effectively identify F. nucleatum via nusG as well as detect its virulence factor. To the best of our knowledge, this study is the first to report the application of LAMP for the detection of nusG and fadA in F. nucleatum. The LAMP method constitutes a sensitive and specific visual assay for the rapid detection of the pathogen F. nucleatum.

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

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    Maina Ngotho

    2015-01-01

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Shah Mukim Uddin

    2015-03-01

    Full Text Available 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.

  6. Autocatalytic differentiation of epigenetic modifications within the Arabidopsis genome.

    Science.gov (United States)

    Inagaki, Soichi; Miura-Kamio, Asuka; Nakamura, Yasukazu; Lu, Falong; Cui, Xia; Cao, Xiaofeng; Kimura, Hiroshi; Saze, Hidetoshi; Kakutani, Tetsuji

    2010-10-20

    In diverse eukaryotes, constitutively silent sequences, such as transposons and repeats, are marked by methylation at histone H3 lysine 9 (H3K9me). Although selective H3K9me is critical for maintaining genome integrity, mechanisms to exclude H3K9me from active genes remain largely unexplored. Here, we show in Arabidopsis that the exclusion depends on a histone demethylase gene, IBM1 (increase in BONSAI methylation). Loss-of-function ibm1 mutation results in ectopic H3K9me and non-CG methylation in thousands of genes. The ibm1-induced genic H3K9me depends on both histone methylase KYP/SUVH4 and DNA methylase CMT3, suggesting interdependence of two epigenetic marks--H3K9me and non-CG methylation. Notably, IBM1 enhances loss of H3K9me in transcriptionally de-repressed sequences. Furthermore, disruption of transcription in genes induces ectopic non-CG methylation, which mimics the loss of IBM1 function. We propose that active chromatin is stabilized by an autocatalytic loop of transcription and H3K9 demethylation. This process counteracts a similarly autocatalytic accumulation of silent epigenetic marks, H3K9me and non-CG methylation.

  7. Microfluidic method for rapid turbidimetric detection of the DNA of Mycobacterium tuberculosis using loop-mediated isothermal amplification in capillary tubes

    International Nuclear Information System (INIS)

    Rafati, Adele; Gill, Pooria

    2015-01-01

    We describe a microfluidic method for rapid isothermal turbidimetric detection of the DNA of Mycobacterium tuberculosis. Loop-mediated isothermal amplification is accomplished in capillary tubes for amplifying DNA in less than 15 min, and sensitivity and specificity were compared to conventional loop-mediated isothermal amplification (LAMP). The method can detect as little as 1 pg mL −1 DNA in a sample. Results obtained with clinical specimens indicated 90 % sensitivity and 95 % specificity for microfluidic LAMP in comparison to culture methods. No interference occurred due to the presence of nonspecific DNAs. The findings demonstrate the power of the new microfluidic LAMP test for rapid molecular detection of microorganisms even when using bare eyes. (author)

  8. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

    OpenAIRE

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-01-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium b...

  9. Clinical value of IS6110-based loop-mediated isothermal amplification for detection of Mycobacterium tuberculosis complex in respiratory specimens.

    Science.gov (United States)

    Aryan, Ehsan; Makvandi, Manoochehr; Farajzadeh, Ahmad; Huygen, Kris; Alvandi, Amir-Hooshang; Gouya, Mohammad-Mehdi; Sadrizadeh, Ali; Romano, Marta

    2013-06-01

    A fundamental to global tuberculosis (TB) control is timely and accurate diagnosis of infectious cases of the disease. Among various methods, techniques based on nucleic acid amplification are the ones with promising prospects. The present study evaluates the diagnostic value of the recently developed IS6110-based loop-mediated isothermal amplification (LAMP) for detection of Mycobacterium tuberculosis complex (MTBC) in sputum specimens. In this cross-sectional study (2008-2009), IS6110-LAMP was evaluated on 101 sputum specimens from 93 highly suspected TB patients and compared to Amplicor MTB test and in-house IS6110-PCR and -nested PCR assays. Culture results or clinical recovery following anti-TB therapy was considered as a reference to prove the TB cases. The overall sensitivity of IS6110-LAMP, Amplicor, nPCR, and PCR were respectively 89.6% (69/77 specimens; 95% confidence interval [CI], 80.5-95.4%), 76.6% (59/77 specimens; CI, 65.6-85.5%), 79.2% (61/77 specimens; CI, 68.5-87.6%) and 59.7% (46/77 specimens; CI, 47.9-70.8%). The specificity and positive predictive value (PPV) were 100% for all the tests, and the negative predictive value (NPV) of IS6110-LAMP, Amplicor, nPCR, and PCR were respectively 75%, 57.1%, 60%, and 43.6%. There was an excellent overall agreement between LAMP and nPCR (k 0.828), and between LAMP and Amplicor (k 0.746), in addition to a better tolerance of IS6110-LAMP to inhibitors present in clinical specimens. The better diagnostic performance of IS6110-LAMP compared to Amplicor (p = 0.009), nPCR (p = 0.013) and PCR (p < 0.0001) besides its rapidity, simplicity, and cost-effectiveness makes it a valuable method for the detection of MTBC in clinical samples, particularly in resource-limited settings. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  10. Rapid detection of Puccinia triticina causing leaf rust of wheat by PCR and loop mediated isothermal amplification.

    Science.gov (United States)

    Manjunatha, C; Sharma, Sapna; Kulshreshtha, Deepika; Gupta, Sangeeta; Singh, Kartar; Bhardwaj, Subhash C; Aggarwal, Rashmi

    2018-01-01

    Leaf rust of wheat caused by Puccinia triticina has significant impact on wheat production worldwide. Effective and quick detection methodologies are required to mitigate yield loss and time constraints associated with monitoring and management of leaf rust of wheat. In the present study, detection of P. triticina has been simplified by developing a rapid, reliable, efficient and visual colorimetric method i.e., loop mediated isothermal amplification of DNA (LAMP). Based on in silico analysis of P. triticina genome, PTS68, a simple sequence repeat was found highly specific to leaf rust fungus. A marker (PtRA68) was developed and its specificity was validated through PCR technique which gave a unique and sharp band of 919 bp in P. triticina pathotypes only. A novel gene amplification method LAMP which enables visual detection of pathogen by naked eye was developed for leaf rust pathogen. A set of six primers was designed from specific region of P. triticina and conditions were optimised to complete the observation process in 60 minutes at 65o C. The assay developed in the study could detect presence of P. triticina on wheat at 24 hpi (pre-symptomatic stage) which was much earlier than PCR without requiring thermal cycler. Sensitivity of LAMP assay developed in the study was 100 fg which was more sensitive than conventional PCR (50 pg) and equivalent to qPCR (100 fg). The protocol developed in the study was utilized for detection of leaf rust infected samples collected from different wheat fields. LAMP based colorimetric detection assay showed sky blue color in positive reaction and violet color in negative reaction after addition of 120 μM hydroxyl napthol blue (HNB) solution to reaction mixture. Similarly, 0.6 mg Ethidium bromide/ml was added to LAMP products, placed on transilluminator to witness full brightness in positive reaction and no such brightness could be seen in negative reaction mixture. Further, LAMP products spread in a ladder like banding pattern in

  11. Development of a loop-mediated isothermal amplification assay for rapid, sensitive detection of Campylobacter jejuni in cattle farm samples.

    Science.gov (United States)

    Dong, Hee-Jin; Cho, Ae-Ri; Hahn, Tae-Wook; Cho, Seongbeom

    2014-09-01

    Campylobacter jejuni is a leading cause of bacterial foodborne disease worldwide. The detection of this organism in cattle and their environment is important for the control of C. jejuni transmission and the prevention of campylobacteriosis. Here, we describe the development of a rapid and sensitive method for the detection of C. jejuni in naturally contaminated cattle farm samples, based on real-time loop-mediated isothermal amplification (LAMP) of the hipO gene. The LAMP assay was specific (100% inclusivity and exclusivity for 84 C. jejuni and 41 non-C. jejuni strains, respectively), sensitive (detection limit of 100 fg/μl), and quantifiable (R(2) = 0.9133). The sensitivity of the LAMP assay was then evaluated for its application to the naturally contaminated cattle farm samples. C. jejuni strains were isolated from 51 (20.7%) of 246 cattle farm samples, and the presence of the hipO gene was tested using the LAMP assay. Amplification of the hipO gene by LAMP within 30 min (mean ~10.8 min) in all C. jejuni isolates (n = 51) demonstrated its rapidity and accuracy. Next, template DNA was prepared from a total of 186 enrichment broth cultures of cattle farm samples either by boiling or using a commercial kit, and the sensitivity of detection of C. jejuni was compared between the LAMP and PCR assays. In DNA samples prepared by boiling, the higher sensitivity of the LAMP assay (84.4%) compared with the PCR assay (35.5%) indicates that it is less susceptible to the existence of inhibitors in sample material. In DNA samples prepared using a commercial kit, both the LAMP and PCR assays showed 100% sensitivity. We anticipate that the use of this rapid, sensitive, and simple LAMP assay, which is the first of its kind for the identification and screening of C. jejuni in cattle farm samples, may play an important role in the prevention of C. jejuni contamination in the food chain, thereby reducing the risk of human campylobacteriosis.

  12. Development of loop-mediated isothermal amplification (LAMP) assays for the rapid detection of allergic peanut in processed food.

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    Sheu, Shyang-Chwen; Tsou, Po-Chuan; Lien, Yi-Yang; Lee, Meng-Shiou

    2018-08-15

    Peanut is a widely and common used in many cuisines around the world. However, peanut is also one of the most important food allergen for causing anaphylactic reaction. To prevent allergic reaction, the best way is to avoid the food allergen or food containing allergic ingredient such as peanut before food consuming. Thus, to efficient and precisely detect the allergic ingredient, peanut or related product, is essential and required for maintain consumer's health or their interest. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of allergic peanut using specifically designed primer sets. Two sets of the specific LAMP primers respectively targeted the internal transcribed sequence 1 (ITS1) of nuclear ribosomal DNA sequence regions and the ara h1 gene sequence of Arachia hypogeae (peanut) were used to address the application of LAMP for detecting peanut in processed food or diet. The results demonstrated that the identification of peanut using the newly designed primers for ITS 1 sequence is more sensitive rather than primers for sequence of Ara h1 gene when performing LAMP assay. Besides, the sensitivity of LAMP for detecting peanut is also higher than the traditional PCR method. These LAMP primers sets showed high specificity for the identification of the peanut and had no cross-reaction to other species of nut including walnut, hazelnut, almonds, cashew and macadamia nut. Moreover, when minimal 0.1% peanuts were mixed with other nuts ingredients at different ratios, no any cross-reactivity was evident during performing LAMP. Finally, genomic DNAs extracted from boiled and steamed peanut were used as templates; the detection of peanut by LAMP was not affected and reproducible. As to this established LAMP herein, not only can peanut ingredients be detected but commercial foods containing peanut can also be identified. This assay will be useful and potential for the rapid detection of peanut in practical food

  13. Field Evaluation of a High Throughput Loop Mediated Isothermal Amplification Test for the Detection of Asymptomatic Plasmodium Infections in Zanzibar.

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    Berit Aydin-Schmidt

    Full Text Available New field applicable diagnostic tools are needed for highly sensitive detection of residual malaria infections in pre-elimination settings. Field performance of a high throughput DNA extraction system for loop mediated isothermal amplification (HTP-LAMP was therefore evaluated for detecting malaria parasites among asymptomatic individuals in Zanzibar.HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a community-based survey in 2015.The PCR and HTP-LAMP determined malaria prevalences were 1.6% (95%CI 1.3-2.4 and 0.7% (95%CI 0.4-1.1, respectively. The sensitivity of HTP-LAMP compared to PCR was 40.8% (CI95% 27.0-55.8 and the specificity was 99.9% (CI95% 99.8-100. For the PCR positive samples, there was no statistically significant difference between the geometric mean parasite densities among the HTP-LAMP positive (2.5 p/μL, range 0.2-770 and HTP-LAMP negative (1.4 p/μL, range 0.1-7 samples (p = 0.088. Two lab technicians analysed up to 282 samples per day and the HTP-LAMP method was experienced as user friendly.Although field applicable, this high throughput format of LAMP as used here was not sensitive enough to be recommended for detection of asymptomatic low-density infections in areas like Zanzibar, approaching malaria elimination.

  14. Evaluation of a loop-mediated isothermal amplification method for the detection ofListeria monocytogenesin dairy food.

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    Tirloni, Erica; Bernardi, Cristian; Drago, Sandro; Stampone, Giuseppe; Pomilio, Francesco; Cattaneo, Patrizia; Stella, Simone

    2017-10-20

    Objective of the present study was to test the performances of a loop-mediated isothermal amplification (LAMP)-based method for the detection of Listeria monocytogenes , with particular focus on the dairy products. The specificity of the method was evaluated on 42 different Listeria spp. strains from collections, food and environmental samples. 100% (32 of 32) of the L. monocytogenes strains were correctly recognised, and none of other 10 Listeria spp. strains was misidentified. The sensitivity was evaluated on four L. monocytogenes strains from different sources. The instrument was able to detect 10-400 CFU/mL. The ability to detect low initial numbers of L. monocytogenes (0.3-0.7 Log CFU/g) was also evaluated, in duplicate, in pasteurised milk (whole and skimmed) and dairy samples (fresh ricotta, crescenza, mascarpone, mozzarella, cottage cheese, cream cheese, taleggio, gorgonzola). The analysis was performed after 18, 24 and 48 h of incubation, and was coupled with the count of L. monocytogenes in the broth. Microbial loads were insufficient to achieve a positive result after 18 and 24 h in most of the samples; after 48 h, all the products, except taleggio and one gorgonzola sample, were identified as positive; the sensitivity of the method when applied to contaminated dairy foods was about 5 Log CFU/g. The LAMP method tested can be considered a very useful tool, as it is a costeffective and easy-functioning method. The preliminary data obtained should be confirmed with a validation process taking into account different food typologies.

  15. Clinical evaluation of a loop-mediated isothermal amplification (LAMP assay for rapid detection of Neisseria meningitidis in cerebrospinal fluid.

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    DoKyung Lee

    Full Text Available 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.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.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.

  16. Loop-mediated isothermal amplification (LAMP) test for specific and rapid detection of Brucella abortus in cattle.

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    Karthik, K; Rathore, Rajesh; Thomas, Prasad; Arun, T R; Viswas, K N; Agarwal, R K; Manjunathachar, H V; Dhama, Kuldeep

    2014-01-01

    Brucella abortus, the major causative agent of abortion in cattle and a zoonotic pathogen, needs to be diagnosed at an early stage. Loop-mediated isothermal amplification (LAMP) test is easy to perform and also promising to be adapted at field level. To develop a LAMP assay for specific and rapid detection of B. abortus from clinical samples of cattle. LAMP primers were designed targeting BruAb2_0168 region using specific software tool and LAMP was optimized. The developed LAMP was tested for its specificity with 3 Brucella spp. and 11 other non-Brucella spp. Sensitivity of the developed LAMP was also carried out with known quantity of DNA. Cattle whole blood samples and aborted fetal stomach contents were collected and used for testing with developed LAMP assay and results were compared with polymerase chain reaction (PCR). The developed LAMP assay works at 61 °C for 60 min and the detection limit was observed to be 100-fold more than the conventional PCR that is commonly used for diagnosis of B. abortus. Clinical sensitivity and specificity of the developed LAMP assay was 100% when compared with Rose Bengal plate test and standard tube agglutination test. SYB® green dye I was used to visualize the result with naked eye. The novelty of the developed LAMP assay for specifically detecting B. abortus infection in cattle along with its inherent rapidness and high sensitivity can be employed for detecting this economically important pathogen of cattle at field level as well be exploited for screening of human infections.

  17. Rapid detection and subtyping of human papillomaviruses in condyloma acuminatum using loop-mediated isothermal amplification with hydroxynaphthol blue dye.

    Science.gov (United States)

    Zhong, Q; Li, K; Chen, D; Wang, H; Lin, Q; Liu, W

    2018-03-14

    Objective Condyloma acuminatum (CA) is a common, viral, sexually transmitted disease worldwide. Human papillomavirus (HPV) genotyping has important clinical implications for the treatment of CA. We developed a loop-mediated isothermal amplification (LAMP) method for the detection of HPV. Methods We collected 294 cervical scrape samples, including 30 HPV-6-positive, 30 HPV-11-positive, 22 HPV-16-positive, 20 HPV-42-positve, 30 HPV-43-positive, 20 HPV-44-positive and 142 HPV-negative samples. Tissues from 40 patients with a pathological diagnosis of CA were paraffin-embedded and analyzed by LAMP and Luminex. Hydroxynaphthol blue (HNB) and electrophoresis were used to detect the results of LAMP. Results LAMP and Luminex systems were compared in detecting six subtypes of HPV. LAMP reactions were specific for each subtype. The sensitivity of LAMP for HPV-6, as determined by the HNB indicator assay, was 1000 copies/tube. The kappa value between the two methods was 0.98 (HPV-6), 0.94 (HPV-11), 0.89 (HPV-43), 0.87 (HPV-42) 0.79 (HPV-16) and 0.68 (HPV-44). Among the 142 HPV-negative samples determined by the Luminex assay, HPV-6 was detected in eight and HPV-11 in one by LAMP. Among the 40 CA samples, the results of LAMP and Luminex were in agreement in 38 (95%). Conclusion The results of this study indicated that the LAMP assay with HNB is superior to the Luminex method in terms of sensitivity and specificity. The specificity of LAMP was 100% and the sensitivity of LAMP was 1000 copies/tube using HNB. LAMP is therefore a useful, quick and accurate method for the clinical diagnosis of HPV subtypes.

  18. Evaluation of a loop-mediated isothermal amplification method for the detection of Listeria monocytogenes in dairy food

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    Erica Tirloni

    2017-12-01

    Full Text Available Objective of the present study was to test the performances of a loop-mediated isothermal amplification (LAMP-based method for the detection of Listeria monocytogenes, with particular focus on the dairy products. The specificity of the method was evaluated on 42 different Listeria spp. strains from collections, food and environmental samples. 100% (32 of 32 of the L. monocytogenes strains were correctly recognised, and none of other 10 Listeria spp. strains was misidentified. The sensitivity was evaluated on four L. monocytogenes strains from different sources. The instrument was able to detect 10-400 CFU/mL. The ability to detect low initial numbers of L. monocytogenes (0.3- 0.7 Log CFU/g was also evaluated, in duplicate, in pasteurised milk (whole and skimmed and dairy samples (fresh ricotta, crescenza, mascarpone, mozzarella, cottage cheese, cream cheese, taleggio, gorgonzola. The analysis was performed after 18, 24 and 48 h of incubation, and was coupled with the count of L. monocytogenes in the broth. Microbial loads were insufficient to achieve a positive result after 18 and 24 h in most of the samples; after 48 h, all the products, except taleggio and one gorgonzola sample, were identified as positive; the sensitivity of the method when applied to contaminated dairy foods was about 5 Log CFU/g. The LAMP method tested can be considered a very useful tool, as it is a costeffective and easy-functioning method. The preliminary data obtained should be confirmed with a validation process taking into account different food typologies.

  19. Development of loop-mediated isothermal amplification to detect Streptococcus suis and its application to retail pork meat in Japan.

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    Arai, Sakura; Tohya, Mari; Yamada, Ryoko; Osawa, Ro; Nomoto, Ryohei; Kawamura, Yoshiaki; Sekizaki, Tsutomu

    2015-09-02

    We here developed a novel loop-mediated isothermal amplification (LAMP) method to detect Streptococcus suis in raw pork meat. This method, designated LAMPSS, targeted the recombination/repair protein (recN) gene of S. suis and detected all serotypes of S. suis, except those taxonomically removed from authentic S. suis, i.e., serotypes 20, 22, 26, 32, 33, and 34. The specificity of LAMPSS was confirmed and its detection limit was 5.4cfu/reaction. Among the 966 raw pork meat samples examined, including sliced pork, minced pork, and the liver, tongue, heart, and small intestine, 255 samples tested positive with LAMPSS. The rate of contamination was higher in the organs than in pork. No significant difference was observed in the total bacterial count between LAMPSS-positive and -negative samples. The number of shops that provided LAMPSS-positive pork was slightly higher in those that sold swine organs and pork than in those that sold only pork, suggesting that cross contamination occurred from the organs to pork. Among the 255 which tested positive for LAMPSS, only 47 samples tested positive for the previously described LAMP specific for S. suis serotype 2. Two isolates of S. suis serotype 2, belonging to sequence type 28, which is potentially hazardous to humans, as well as those of some other serotypes were obtained from 19 out of 47 samples by combining LAMP with a replica plating method. These results suggest that LAMPSS will be a useful tool for the surveillance of raw pork meat in the retail market. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. [Rapid detection of Macrobrachium rosenbergii nodavirus isolated in China by a reverse-transcription loop-mediated isothermal amplification assay combined with a lateral flow dipstick method].

    Science.gov (United States)

    Lin, Feng; Liu, Li; Hao, Gui-Jie; Cao, Zheng; Sheng, Peng-Cheng; Wu, Ying-Lei; Shen, Jin-Yu

    2014-09-01

    White coloration of the muscle of the giant river prawn (Macrobrachium rosenbergii) is a serious problem in China. The Macrobrachium rosenbergii Nodavirus (MrNV) has been confirmed to be the pathogen that causes this disorder. To develop a rapid, sensitive and specific technology for the detection of Macrobrachium rosenbergii Nodavirus isolated from China (MrNV-China), a reverse-transcription loop- mediated isothermal amplification assay combined with a lateral flow dipstick (RT-LAMP-LFD) assay method is described. A set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the MrNV RNA2 gene. Results showed the sensitivity of the RT-LAMP-LFD assay was ten-times higher than the reverse-transcription loop-mediated isothermal amplification assay (RT-LAMP) with agarose gel electrophoresis. The assay was conducted with one-step amplification at 61°C in a single tube within 45 min. No product was generated from shrimps infected with other viruses, including DNA viruses (infectious hypodermal and hematopoietic necrosis virus (IHHNV); white spot syndrome virus (WSSV)) and RNA viruses (Taura syndrome virus (TSV); infectious myonecrosis virus (IMNV); yellow head virus (YHV)). Results were visualized by the LFD method. Therefore, the described rapid and sensitive assay is potentially useful for MrNV detection.

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

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    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

  2. Real-time sequence-validated loop-mediated isothermal amplification assays for detection of Middle East respiratory syndrome coronavirus (MERS-CoV.

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    Sanchita Bhadra

    Full Text Available 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 (ORF1a 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.

  3. Evaluation of the loop mediated isothermal DNA amplification (LAMP kit for malaria diagnosis in P. vivax endemic settings of Colombia.

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    Andrés F Vallejo

    2015-01-01

    Full Text Available Most commonly used malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections which are frequent in low transmission settings. Molecular methods such as polymerase chain reaction (PCR are highly sensitive but remain too laborious for field deployment. In this study, the applicability of a malaria diagnosis kit based on loop-mediated isothermal amplification (mLAMP was assessed in malaria endemic areas of Colombia with Plasmodium vivax predominance.First, a passive case detection (PCD study on 278 febrile patients recruited in Tierralta (department of Cordoba was conducted to assess the diagnostic performance of the mLAMP method. Second, an active case detection (ACD study on 980 volunteers was conducted in 10 sentinel sites with different epidemiological profiles. Whole blood samples were processed for microscopic and mLAMP diagnosis. Additionally RT-PCR and nested RT-PCR were used as reference tests. In the PCD study, P. falciparum accounted for 23.9% and P. vivax for 76.1% of the infections and no cases of mixed-infections were identified. Microscopy sensitivity for P. falciparum and P. vivax were 100% and 86.1%, respectively. mLAMP sensitivity for P. falciparum and P. vivax was 100% and 91.4%, respectively. In the ACD study, mLAMP detected 65 times more cases than microscopy. A high proportion (98.0% of the infections detected by mLAMP was from volunteers without symptoms.mLAMP sensitivity and specificity were comparable to RT-PCR. LAMP was significantly superior to microscopy and in P. vivax low-endemicity settings and under minimum infrastructure conditions, it displayed sensitivity and specificity similar to that of single-well RT-PCR for detection of both P. falciparum and P. vivax infections. Here, the dramatically increased detection of asymptomatic malaria infections by mLAMP demonstrates the usefulness of this new tool for diagnosis, surveillance, and screening in

  4. Rapid, simple and sensitive detection of Q fever by loop-mediated isothermal amplification of the htpAB gene.

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    Lei Pan

    Full Text Available BACKGROUND: Q fever is the most widespread zoonosis, and domestic animals are the most common sources of transmission. It is not only difficult to distinguish from other febrile diseases because of the lack of specific clinical manifestations in humans, but it is also difficult to identify the disease in C. burnetii-carrying animals because of the lack of identifiable features. Conventional serodiagnosis requires sera from the acute and convalescent stages of infection, which are unavailable at early diagnosis. Nested PCR and real-time PCR require equipment. In this study, we developed a Loop-Mediated Isothermal Amplification (LAMP assay to identify C. burnetii rapidly and sensitively. METHODS: A universal LAMP primer set was designed to detect the repeated sequence IS1111a of the htpAB gene of C. burnetii using PrimerExplorer V4 software. The sensitivity of the LAMP assay was evaluated using known quantities of recombined reference plasmids containing the targeted genes. The specificity of the developed LAMP assay was determined using 26 members of order Rickettsiae and 18 other common pathogens. The utility of the LAMP assay was further compared with real time PCR by the examination 24 blood samples including 6 confirmed and 18 probable Q fever cases, which diagnosed by IFA serological assessment and real time PCR. In addition, 126 animal samples from 4 provinces including 97 goats, 7 cattle, 18 horses, 3 marmots and 1 deer were compared by these two methods. RESULTS: The limits of detection of the LAMP assay for the htpAB gene were 1 copy per reaction. The specificity of the LAMP assay was 100%, and no cross-reaction was observed among the bacteria used in the study. The positive rate of unknown febrile patients was 33.3%(95%CI 30.2%-36.4% for the LAMP assay and 8.3%(95%CI 7.4%-9.2% for the real time PCR(P<0.05. Similarly, the total positive rate of animals was 7.9%(95%CI 7.1%-8.7% for the LAMP assay and 0.8%(95%CI 0.7%-0.9%for the real time

  5. [Investigation on Toxoplasma gondii by polymerase chain reaction and loop-mediated isothermal amplification in water samples from Giresun, Turkey].

    Science.gov (United States)

    Demirel, Elif; Kolören, Zeynep; Karaman, Ulkü; Ayaz, Emine

    2014-10-01

    Toxoplasmosis is generally asymptomatic in immunocompetent subjects, however serious manifestations of the disease may develop in immunocompromised patients and in pregnant women. The mean Toxoplasma gondii seroprevalence which is approximately 40% in Turkish population, indicates the high risk for the development of acute toxoplasmosis in those cases. One of the transmission ways of T.gondii is the consumption of contaminated water. The aim of this study was to detect the presence of T.gondii in the environmental and drinking water samples by using standard polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) methods. A total of 96 water samples, of them 76 were environmental water samples collected from creeks in Giresun province center and its various districts (Piraziz, Bulancak, Keşap, Espiye) and 20 from drinking water samples, were included in the study. The samples were precipitated by the aluminium sulfate method and DNAs were isolated from the pellets formed with the sucrose gradient method. PCR and LAMP were applied to the isolated DNAs, by the use of primers F3 and B3 specific to B1 gene of the parasite. In our study, T.gondii was detected in none of the drinking water samples by PCR and LAMP, however T.gondii DNAs were positive in 13.2% (10/76) of the environmental water samples. Both of the methods yielded the same results in those samples. The stations that were positive for T.gondii were Aksu creek in Giresun province center; Gelivera creek in Espiye; Yolağzı, Keşap, Keşap Login Bridge creeks in Keşap; Bulancak, Karadere and İncivez creeks in Bulancak; Piraziz and Çayırağzı creeks in Piraziz counties. Resistance of T.gondii to chlorination and the inadequacy of the filtration processes, create a serious threat among people in contact with rivers, sea and drinking waters particularly in areas with high humidity. As far as the national literature was considered, no report about the water-borne T

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

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    Marcus Beissner

    2015-11-01

    Full Text Available 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.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.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

  7. Sensitive and less invasive confirmatory diagnosis of visceral leishmaniasis in Sudan using loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Mukhtar, Maowia; Ali, Sababil S; Boshara, Salah A; Albertini, Audrey; Monnerat, Séverine; Bessell, Paul; Mori, Yasuyoshi; Kubota, Yutaka; Ndung'u, Joseph M; Cruz, Israel

    2018-02-01

    Confirmatory diagnosis of visceral leishmaniasis (VL), as well as diagnosis of relapses and test of cure, usually requires examination by microscopy of samples collected by invasive means, such as splenic, bone marrow or lymph node aspirates. This causes discomfort to patients, with risks of bleeding and iatrogenic infections, and requires technical expertise. Molecular tests have great potential for diagnosis of VL using peripheral blood, but require well-equipped facilities and trained personnel. More user-friendly, and field-amenable options are therefore needed. One method that could meet these requirements is loop-mediated isothermal amplification (LAMP) using the Loopamp Leishmania Detection Kit, which comes as dried down reagents that can be stored at room temperature, and allows simple visualization of results. The Loopamp Leishmania Detection Kit (Eiken Chemical Co., Japan), was evaluated in the diagnosis of VL in Sudan. A total of 198 VL suspects were tested by microscopy of lymph node aspirates (the reference test), direct agglutination test-DAT (in house production) and rK28 antigen-based rapid diagnostic test (OnSite Leishmania rK39-Plus, CTK Biotech, USA). LAMP was performed on peripheral blood (whole blood and buffy coat) previously processed by: i) a direct boil and spin method, and ii) the QIAamp DNA Mini Kit (QIAgen). Ninety seven of the VL suspects were confirmed as cases by microscopy of lymph node aspirates. The sensitivity and specificity for each of the tests were: rK28 RDT 98.81% and 100%; DAT 88.10% and 78.22%; LAMP-boil and spin 97.65% and 99.01%; LAMP-QIAgen 100% and 99.01%. Due to its simplicity and high sensitivity, rK28 RDT can be used first in the diagnostic algorithm for primary VL diagnosis, the excellent performance of LAMP using peripheral blood indicates that it can be also included in the algorithm for diagnosis of VL as a simple test when parasitological confirmatory diagnosis is required in settings that are lower than the

  8. Development of reverse transcription loop-mediated isothermal amplification assay as a simple detection method of Chrysanthemum stem necrosis virus in chrysanthemum and tomato.

    Science.gov (United States)

    Suzuki, Ryoji; Fukuta, Shiro; Matsumoto, Yuho; Hasegawa, Toru; Kojima, Hiroko; Hotta, Makiko; Miyake, Noriyuki

    2016-10-01

    For a simple and rapid detection of Chrysanthemum stem necrosis virus (CSNV) from chrysanthemum and tomato, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed. A primer set designed to the genome sequences of CSNV worked most efficiently at 63°C and could detect CSNV RNA within 12min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. The result of a specificity test using seven other viruses and one viroid-infectable chrysanthemum or tomato showed that the assay could amplify CSNV specifically, and a sensitivity comparison showed that the RT-LAMP assay was as sensitive as the reverse transcriptase polymerase chain reaction. The RT-LAMP assay using crude RNA, extracted simply, could detect CSNV. Overall, the RT-LAMP assay was found to be a simple, specific, convenient, and time-saving method for CSNV detection. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Biochip for Real-Time Monitoring of Hepatitis B Virus (HBV) by Combined Loop-Mediated Isothermal Amplification and Solution-Phase Electrochemical Detection

    Science.gov (United States)

    Tien, Bui Quang; Ngoc, Nguyen Thy; Loc, Nguyen Thai; Thu, Vu Thi; Lam, Tran Dai

    2017-06-01

    Accurate in situ diagnostic tests play a key role in patient management and control of most infectious diseases. To achieve this, use of handheld biochips that implement sample handling, sample analysis, and result readout together is an ideal approach. We present herein a fluid-handling biochip for real-time electrochemical monitoring of nucleic acid amplification based on loop-mediated isothermal amplification and real-time electrochemical detection on a microfluidic platform. Intercalation between amplifying DNA and free redox probe in solution phase was used to monitor the number of DNA copies. The whole diagnostic process is completed within 70 min. Our platform offers a fast and easy tool for quantification of viral pathogens in shorter time and with limited risk of all potential forms of cross-contamination. Such diagnostic tools have potential to make a huge difference to the lives of millions of people worldwide.

  10. Development of rapid and highly sensitive detection of Bean common mosaic necrosis virus in leguminous crops using loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Lee, Siwon; Kim, Heejung; Lee, Jin-Young; Rho, Jae-Young

    2017-11-01

    Bean common mosaic necrosis virus (BCMNV) is a plant pathogenic virus that can infect leguminous crops such as kidney beans, sunn hemp, red beans, and mung beans. BCMNV has not been reported in Korea and is classified as a quarantine plant virus. Currently, the standard diagnostic method for diagnosis of BCMNV is reverse transcription (RT)-nested PCR system. However a more rapid monitoring system is needed to enable the testing of more samples. The use of highly efficient loop-mediated isothermal amplification (LAMP) assay for its detection has not yet been reported, and development of LAMP for detecting BCMNV in this study. In addition, confirmation of LAMP amplification can be achieved using restriction enzyme Mse I (T/TAA). The developed technique could be used for more rapid, specific and sensitive monitoring of BCMNV in leguminous crops than conventional nested RT-PCR. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Rapid, Sensitive, and Carryover Contamination-Free Loop-Mediated Isothermal Amplification-Coupled Visual Detection Method for 'Candidatus Liberibacter asiaticus'.

    Science.gov (United States)

    Qian, Wenjuan; Meng, Youqing; Lu, Ying; Wu, Cui; Wang, Rui; Wang, Liu; Qian, Cheng; Ye, Zunzhong; Wu, Jian; Ying, Yibin

    2017-09-27

    Huanglongbing is a devastating citrus disease, and 'Candidatus Liberibacter asiaticus' (Las) is the most prevalent huanglongbing-associated bacterium. Its field detection remains challenging. In this work, a visual, rapid, sensitive, and carryover contamination-free method was developed for field detection of Las. Leaf samples were treated with 500 μL of 0.5 M sodium hydroxide solution for 3 min, and 50-fold dilutions were directly amplified by loop-mediated isothermal amplification. Then, a novel SYTO-9-based visual detection method was used to evaluate amplification results without uncapping operation. Negative samples remained colorless, while positive samples generated obvious green fluorescence, which could be easily distinguished by the naked eye with a mini-fluorescent-emission cartridge developed originally. The proposed detection method could be accomplished within 40 min and is about 100 times more sensitive than conventional TaqMan polymerase chain reaction. The reliability of this method was also verified by analyzing practical samples.

  12. Application of a Real-time Reverse Transcription Loop Mediated Amplification Method to the Detection of Rabies Virus in Arctic Foxes in Greenland

    DEFF Research Database (Denmark)

    Wakeley, Philip; Johnson, Nicholas; Rasmussen, Thomas Bruun

    Reverse transcription loop mediated amplification (RT-LAMP) offers a rapid, isothermal method for amplification of virus RNA. In this study a panel of positive rabies virus samples originally prepared from arctic fox brain tissue was assessed for the presence of rabies viral RNA using a real time...... RT-LAMP. The method had previously been shown to work with samples from Ghana which clustered with cosmopolitan lineage rabies viruses but the assay had not been assessed using samples from animals infected with rabies from the arctic region. The assay is designed to amplify both cosmopolitan strains...... virus of arctic origin virus can be detected using RT-LAMP and the method reported is more rapid than the real-time RT-PCR. Further arctic fox samples are under analysis in order to confirm these findings....

  13. Loop-Mediated Isothermal Amplification Using a Lab-on-a-Disc Device with Thin-film Phase Change Material.

    Science.gov (United States)

    Ko, Junguk; Yoo, Jae-Chern

    2018-03-05

    The design and fabrication of temperature measurement systems that facilitate successful realization of DNA amplification using a lab-on-a-disc (LOD) device are a highly challenging task. The major challenge lies in the fact that such a system must be directly attached to a heating chamber in a way that enables the accurate measurement of temperature of the chamber while allowing the LOD to rotate. This paper presents a temperature control system for implementing isothermal amplification of DNA samples using an LOD device. The proposed system utilizes a thin-film phase change material and non-contact heating system to remotely measure the actual temperature of the chamber and, if required, rapidly heat it to the desired temperature. The results of the experiments performed in this study demonstrate that the proposed system provides an automated platform for molecular amplification and exhibits an operational performance comparable to that of traditional microcentrifuge tube-based isothermal amplification systems.

  14. The heads and tails of buoyant autocatalytic balls

    Science.gov (United States)

    Rogers, Michael C.; Morris, Stephen W.

    2012-09-01

    Buoyancy produced by autocatalytic reaction fronts can produce fluid flows that advect the front position, giving rise to interesting feedback between chemical and hydrodynamic effects. In this paper, we numerically investigate the evolution of autocatalytic iodate-arsenous acid reaction fronts initialized in spherical configurations. Deformation of these "autocatalytic balls" is driven by buoyancy produced by the reaction. In our simulations, we have found that depending on the initial ball radius, the reaction front will develop in one of three different ways. In an intermediate range of ball size, the flow can evolve much like an autocatalytic plume: the ball develops a reacting head and tail that is akin to the head and conduit of an autocatalytic plume. In the limit of large autocatalytic balls, however, growth of a reacting tail is suppressed and the resemblance to plumes disappears. Conversely, very small balls of product solution fail to initiate sustained fronts and eventually disappear.

  15. A loop-mediated isothermal amplification method for a differential identification of Taenia tapeworms from human: application to a field survey.

    Science.gov (United States)

    Nkouawa, Agathe; Sako, Yasuhito; Li, Tiaoying; Chen, Xingwang; Nakao, Minoru; Yanagida, Tetsuya; Okamoto, Munehiro; Giraudoux, Patrick; Raoul, Francis; Nakaya, Kazuhiro; Xiao, Ning; Qiu, Jiamin; Qiu, Dongchuan; Craig, Philip S; Ito, Akira

    2012-12-01

    In this study, we applied a loop-mediated isothermal amplification method for identification of human Taenia tapeworms in Tibetan communities in Sichuan, China. Out of 51 proglottids recovered from 35 carriers, 9, 1, and 41 samples were identified as Taenia solium, Taenia asiatica and Taenia saginata, respectively. Same results were obtained afterwards in the laboratory, except one sample. These results demonstrated that the LAMP method enabled rapid identification of parasites in the field surveys, which suggested that this method would contribute to the control of Taenia infections in endemic areas. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  16. Evaluation of a real-time PCR and a loop-mediated isothermal amplification for detection of Xanthomonas arboricola pv. pruni in plant tissue samples.

    Science.gov (United States)

    Palacio-Bielsa, Ana; López-Soriano, Pablo; Bühlmann, Andreas; van Doorn, Joop; Pham, Khanh; Cambra, Miguel A; Berruete, Isabel M; Pothier, Joël F; Duffy, Brion; Olmos, Antonio; López, María M

    2015-05-01

    Operational capacity of real-time PCR and loop-mediated isothermal amplification (LAMP) diagnostic assays for detection of Xanthomonas arboricola pv. pruni was established in a ring-test involving four laboratories. Symptomatic and healthy almond leaf samples with two methods of sample preparation were analyzed. Kappa coefficient, sensitivity, specificity, likelihood ratios and post-test probability of detection were estimated to manage the risk associated with the use of the two methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) Using Simultaneous Detection of mecA, nuc, and femB by Loop-Mediated Isothermal Amplification (LAMP).

    Science.gov (United States)

    Chen, Changguo; Zhao, Qiangyuan; Guo, Jianwei; Li, Yanjun; Chen, Qiuyuan

    2017-08-01

    The aim of this study was to develop a rapid detection assay to identify methicillin-resistant Staphylococcus aureus by simultaneous testing for the mecA, nuc, and femB genes using the loop-mediated isothermal amplification (LAMP) method. LAMP primers were designed using online bio-software ( http://primerexplorer.jp/e/ ), and amplification reactions were performed in an isothermal temperature bath. The products were then examined using 2% agarose gel electrophoresis. MecA, nuc, and femB were confirmed by triplex TaqMan real-time PCR. For better naked-eye inspection of the reaction result, hydroxy naphthol blue (HNB) was added to the amplification system. Within 60 min, LAMP successfully amplified the genes of interest under isothermal conditions at 63 °C. The results of 2% gel electrophoresis indicated that when the Mg 2+ concentration in the reaction system was 6 μmol, the amplification of the mecA gene was relatively good, while the amplification of the nuc and femB genes was better at an Mg 2+ concentration of 8 μmol. Obvious color differences were observed by adding 1 μL (3.75 mM) of HNB into 25 μL reaction system. The LAMP assay was applied to 128 isolates cases of methicillin-resistant Staphylococcus aureus, which were separated from the daily specimens and identified by Vitek microbial identification instruments. The results were identical for both LAMP and PCR. LAMP offers an alternative detection assay for mecA, nuc, and femB and is faster than other methods.

  18. The Sugar Model: Autocatalytic Activity of the Triose Ammonia Reaction

    Science.gov (United States)

    Weber, Arthur L.

    2007-04-01

    Reaction of triose sugars with ammonia under anaerobic conditions yielded autocatalytic products. The autocatalytic behavior of the products was examined by measuring the effect of the crude triose ammonia reaction product on the kinetics of a second identical triose ammonia reaction. The reaction product showed autocatalytic activity by increasing both the rate of disappearance of triose and the rate of formation of pyruvaldehyde, the product of triose dehydration. This synthetic process is considered a reasonable model of origin-of-life chemistry because it uses plausible prebiotic substrates, and resembles modern biosynthesis by employing the energized carbon groups of sugars to drive the synthesis of autocatalytic molecules.

  19. Direct bacterial loop-mediated isothermal amplification detection on the pathogenic features of the nosocomial pathogen - Methicillin resistant Staphylococcus aureus strains with respiratory origins.

    Science.gov (United States)

    Lin, Qun; Xu, Pusheng; Li, Jiaowu; Chen, Yin; Feng, Jieyi

    2017-08-01

    Loop-mediated isothermal amplification based detection assays using bacterial culture or colony for direct detection of methicillin resistant Staphylococcus aureus(MRSA) had been developed and evaluated, followed by its extensive application on a large scale of clinical MRSA isolated from respiratory origins, including nasal swabs and sputums. Six primers, including outer primers, inner primers and loop primers, were specifically designed for recognizing eight distinct sequences on four targets: 16SrRNA, femA, mecA and orfX. Twenty-seven reference strains were used to develop, evaluate and optimize this assay. Then, a total of 532 clinical MRSA isolates were employed for each detected targets. And the results were determined through both visual observation of the color change by naked eye and electrophoresis. The specific of each primer had been confirmed, and the optimal amplification was obtained under 65 °C for 40 min. The limit of detections (LOD) of bacteria culture LAMP assays were determined to be 10 4  CFU/ml for 16S rRNA, femA, as well as orfX and 10 5  CFU/ml for mecA, respectively. The established novel assays on MRSA detection may provide new strategies for rapid detection of foodborne pathogens. Copyright © 2017. Published by Elsevier Ltd.

  20. Artemisinin induces A549 cell apoptosis dominantly via a reactive oxygen species-mediated amplification activation loop among caspase-9, -8 and -3.

    Science.gov (United States)

    Gao, Weijie; Xiao, Fenglian; Wang, Xiaoping; Chen, Tongsheng

    2013-10-01

    This report is designed to explore the roles of caspase-8, -9 and -3 in artemisinin (ARTE)-induced apoptosis in non-small cell lung cancer cells (A549 cells). ARTE induced reactive oxygen species (ROS)-mediated apoptosis in dose- and time-dependent fashion. Although ARTE treatment did not induce Bid cleavage and significant loss of mitochondrial membrane potential, it induced release of Smac and AIF but not cytochrome c from mitochondria, and silencing of Bak but not Bax significantly prevented ARTE-induced cytotoxicity. Moreover, ARTE treatment induced ROS-dependent activation of caspase-9, -8 and -3. Of the utmost importance, silencing or inhibiting any one of caspase-8, -9 and -3 almost completely prevented ARTE-induced activation of all the three caspases and remarkably abrogated the cytotoxicity of ARTE, suggesting that ARTE triggered an amplification activation loop among caspase-9, -8 and -3. Collectively, our data demonstrate that ARTE induces a ROS-mediated amplification activation loop among caspase-9, -8 and -3 to dominantly mediate the apoptosis of A549 cells.

  1. Development and application of loop-mediated isothermal amplification methods targeting the seM gene for detection of Streptococcus equi subsp. equi.

    Science.gov (United States)

    Hobo, Seiji; Niwa, Hidekazu; Oku, Kazuomi

    2012-03-01

    Loop-mediated isothermal amplification (LAMP) constitutes a potentially valuable diagnostic tool for rapid diagnosis of contagious diseases. In this study, we developed a novel LAMP method (seM-LAMP) to detect the seM gene of Streptococcus equi subsp. equi (S. equi), the causative agent of strangles in equids. The seM-LAMP successfully amplified the target sequence of the seM gene at 63°C within 60 min. The sensitivity of the seM-LAMP was slightly lower than the 2nd reaction of the seM semi-nested PCR. To evaluate the species specificity of the seM-LAMP, we tested 100 S. equi and 189 non-S. equi strains. Significant amplification of the DNA originating from S. equi was observed within 60 min incubation, but no amplification of non-S. equi DNA occurred. The results were identical to those of seM semi-nested PCR. To investigate the clinical usefulness of the methods, the seM-LAMP and the seM semi-nested PCR were used to screen 590 nasal swabs obtained during an outbreak of strangles. Both methods showed that 79 and 511 swabs were S. equi positive and negative, respectively, and the results were identical to those of the culture examination. These results indicate that the seM-LAMP is potentially useful for the reliable routine diagnosis of Streptococcus equi subsp. equi infections.

  2. Visual detection of West Nile virus using reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip

    Directory of Open Access Journals (Sweden)

    Zengguo eCao

    2016-04-01

    Full Text Available West Nile virus (WNV causes a severe zoonosis, which can lead to a large number of casualties and considerable economic losses. A rapid and accurate identification methodfor WNV for use in field laboratories is urgently needed. Here, a method utilizing reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip (RT-LAMP-VF was developed to detect the envelope (E gene of WNV. The RT-LAMP-VF assay could detect 102 copies/μl ofan WNV RNA standard using a 40 min amplification reaction followed by a 2 min incubationof the amplification product on the visualization strip, and no cross-reaction with other closely related members of theFlavivirus genus was observed. The assay was further evaluated using cells and mouse brain tissues infected with a recombinant rabies virus expressing the E protein of WNV.The assay produced sensitivities of 101.5TCID50/ml and 101.33 TCID50/ml for detection of the recombinant virus in the cells and brain tissues, respectively. Overall, the RT-LAMP-VF assay developed in this study is rapid, simple and effective, and it is therefore suitable for clinical application in the field.

  3. CFD modeling of passive autocatalytic recombiners*

    Directory of Open Access Journals (Sweden)

    Orszulik Magdalena

    2015-06-01

    Full Text Available This study deals with numerical modeling of passive autocatalytic hydrogen recombiners (PARs. Such devices are installed within containments of many nuclear reactors in order to remove hydrogen and convert it to steam. The main purpose of this work is to develop a numerical model of passive autocatalytic recombiner (PAR using the commercial computational fluid dynamics (CFD software ANSYS-FLUENT and tuning the model using experimental results. The REKO 3 experiment was used for this purpose. Experiment was made in the Institute for Safety Research and Reactor Technology in Julich (Germany. It has been performed for different hydrogen concentrations, different flow rates, the presence of steam, and different initial temperatures of the inlet mixture. The model of this experimental recombiner was elaborated within the framework of this work. The influence of mesh, gas thermal conductivity coefficient, mass diffusivity coefficients, and turbulence model was investigated. The best results with a good agreement with REKO 3 data were received for k-ɛ model of turbulence, gas thermal conductivity dependent on the temperature and mass diffusivity coefficients taken from CHEMKIN program. The validated model of the PAR was next implemented into simple two-dimensional simulations of hydrogen behavior within a subcompartment of a containment building.

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

    Science.gov (United States)

    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.

  5. Development of a loop-mediated isothermal amplification assay for rapid detection of Nocardia salmonicida, the causative agent of nocardiosis in fish.

    Science.gov (United States)

    Xia, Liqun; Zhang, Honglian; Lu, Yishan; Cai, Jia; Wang, Bei; Jian, Jichang

    2015-03-01

    Nocardia salmonicida is one of the main pathogens of fish nocardiosis. The purpose of this study was to build a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of N. salmonicida. A set of four primers were designed from the 16S-23S rRNA intergenic spacer region of N. salmonicida, and conditions for LAMP were optimized as incubating all the reagents for 60 min at 64°C. LAMP products were judged with agar gel electrophoresis as well as with the naked eye after the addition of SYBR Green I. Results showed the sensitivity of the LAMP assay was 1.68 × 10(3) CFU/ml (16.8 CFU per reaction) and 10-fold higher than that of PCR. The LAMP method was also effectively applied to detect N. salmonicida in diseased fish samples, and it may potentially facilitate the surveillance and early diagnosis of fish nocardiosis.

  6. Loop-mediated isothermal amplification (LAMP) assay-A rapid detection tool for identifying red fox (Vulpes vulpes) DNA in the carcasses of harbour porpoises (Phocoena phocoena).

    Science.gov (United States)

    Heers, Teresa; van Neer, Abbo; Becker, André; Grilo, Miguel Luca; Siebert, Ursula; Abdulmawjood, Amir

    2017-01-01

    Carcasses of wild animals are often visited by different scavengers. However, determining which scavenger caused certain types of bite marks is particularly difficult and knowledge thereof is lacking. Therefore, a loop-mediated isothermal amplification (LAMP) assay (target sequence cytochrome b) was developed to detect red fox DNA in carcasses of harbour porpoises. The MSwab™ method for direct testing without prior DNA isolation was validated. As a detection device, the portable real-time fluorometer Genie® II was used, which yields rapid results and can be used in field studies without huge laboratory equipment. In addition to in vitro evaluation and validation, a stranded and scavenged harbour porpoise carcass was successfully examined for red fox DNA residues. The developed LAMP method is a valuable diagnostic tool for confirming presumable red fox bite wounds in harbour porpoises without further DNA isolation steps.

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

    Directory of Open Access Journals (Sweden)

    Deguo Wang

    2015-05-01

    Full Text Available 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.

  8. A lab-on-a-chip system with integrated sample preparation and loop-mediated isothermal amplification for rapid and quantitative detection of Salmonella spp. in food samples

    DEFF Research Database (Denmark)

    Sun, Yi; Than Linh, Quyen; Hung, Tran Quang

    2015-01-01

    Foodborne disease is a major public health threat worldwide. Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture or molecular-based methods are time consuming...... and usually take a few hours to days to complete. In response to the demand for rapid on line or at site detection of pathogens, in this study, we describe for the first time an eight-chamber lab-on-a-chip (LOC) system with integrated magnetic beads-based sample preparation and loop-mediated isothermal...... was capable to detect Salmonella at concentration of 50 cells per test within 40 min. The simple design, together with high level of integration, isothermal amplification, and quantitative analysis of multiple samples in short time will greatly enhance the practical applicability of the LOC system for rapid...

  9. Detection of Coconut cadang-cadang viroid (CCCVd) in oil palm by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

    Science.gov (United States)

    Thanarajoo, Sathis Sri; Kong, Lih Ling; Kadir, Jugah; Lau, Wei Hongi; Vadamalai, Ganesan

    2014-06-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60 min at 60 °C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Evaluation of two loop-mediated isothermal amplification methods for the detection of Salmonella Enteritidis and Listeria monocytogenes in artificially contaminated ready-to-eat fresh products

    Directory of Open Access Journals (Sweden)

    Angeliki Birmpa

    2015-08-01

    Full Text Available In the present study, the effectiveness of two loop-mediated isothermal amplification (LAMP assays was evaluated. Samples of romaine lettuce, strawberries, cherry tomatoes, green onions and sour berries were inoculated with known dilutions (100-108 CFU/g of produce of S. Enteritidis and L. monocytogenes. With LAMP assay, pathogens can be detected in less than 60 min. The limits of detection of S. Enteritidis and L. monocytogenes depended on the food sample tested and on the presence of enrichment step. After enrichment steps, all food samples were found positive even at low initial pathogen levels. The developed LAMP, assays, are expected to become a valuable, robust, innovative, powerful, cheap and fast monitoring tool, which can be extensively used for routine analysis, and screening of contaminated foods by the food industry and the Public Food Health Authorities.

  11. On-site detection of Xylella fastidiosa in host plants and in “spy insects” using the real-time loop-mediated isothermal amplification method

    Directory of Open Access Journals (Sweden)

    Thaer YASEEN

    2015-12-01

    Full Text Available A recent severe outbreak of Xylella fastidiosa associated with ‘olive quick decline syndrome’ (OQDS was reported in Apulia (Southern Italy. In this study an on-site real-time loop-mediated isothermal amplification (real-time LAMP was developed for detecting X. fastidiosa in host plants and insects. A marked simplification of the DNA extraction procedure was obtained by heating the samples in a portable Smart-Dart device and using an optimized enhancer reaction buffer. The connection to a tablet or Smartphone allowed to visualize the results of the reaction in real time. Compared to PCR and ELISA, with which it showed comparable results in terms of sensitivity and reliability in the X. fastidiosa detection, this simplified real-time LAMP procedure proved to be “user friendly”, displaying the advantages to be an on-site detection method of easy handling, rapid execution and low cost.

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

    Science.gov (United States)

    Wang, Deguo; Liu, Yanhong

    2015-05-26

    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.

  13. Clues on chemical mechanisms from renormalizability: The example of a noisy cubic autocatalytic model

    Science.gov (United States)

    Gagnon, Jean-Sébastien; Pérez-Mercader, Juan

    2017-08-01

    We study the effect of external power-law noise on the renormalizability of a specific reaction-diffusion system of equations describing a cubic autocatalytic chemical reaction. We show that changing the noise exponent modifies the divergence structure of loop integrals and thus the renormalizability of the model. The effects of noise-generated higher order interactions are discussed. We show how noise induces new interaction terms that can be interpreted as a manifestation of some (internal) ;chemical mechanism;. We also show how ideas of effective field theory can be applied to construct a more fundamental chemical model for this system.

  14. Combined multiplex loop-mediated isothermal amplification with lateral flow assay to detect sea and seb genes of enterotoxic Staphylococcus aureus.

    Science.gov (United States)

    Yin, H Y; Fang, T J; Wen, H W

    2016-07-01

    Staphylococcal enterotoxins (SEs) are the most common cause of food poisoning worldwide and can induce symptoms, such as diarrhoea, vomiting and abdominal cramping. Thus, the aim of this study is to develop a multiplex loop-mediated isothermal amplification combined with a lateral flow assay (m-LAMP/LFA) to simultaneously detect the sea and seb genes of Staphylococcus aureus. The amplicons of the sea gene were labelled with digoxigenin (Dig) and biotin while those of seb gene were labelled with fluorescein isothiocyanate (FITC) and biotin. These amplicons were detected using a multiplex LFA with NeutrAvidin-tagged gold nanoparticles as the detection reagent. After optimization, the detection limit of this assay was 10(2)  CFU ml(-1) Staph. aureus, which was one tenth that of a multiplex PCR. This assay did not exhibit any cross-reactivity in detecting other enterotoxic Staph. aureus strains or other food pathogens. After 6 h of enrichment, this developed assay detected 1 CFU ml(-1) of Staph. aureus in milk, apple juice, cheese and rice. The developed m-LAMP/LFA method does not require expensive equipment and can be completely implemented within an 8-h workday. Therefore, this method can provide an effective means of quickly screening staphylococcal enterotoxin A- and/or staphylococcal enterotoxin B-producing Staph. aureus in food samples. Staphylococcus aureus is one of the major foodborne pathogens worldwide, and its staphylococcal enterotoxin A and B are strongly associated with food poisoning. This work developed a multiplex loop-mediated isothermal amplification combined with a lateral flow assay (m-LAMP/LFA) to simultaneously detect the sea and seb genes of Staph. aureus in food samples. The assay has good specificity and sensitivity with ease-of-use features, making it ideal for on-site detection. © 2016 The Society for Applied Microbiology.

  15. Colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (NINA-LAMP.

    Directory of Open Access Journals (Sweden)

    Catherine B Poole

    Full Text Available Accurate detection of filarial parasites in humans is essential for the implementation and evaluation of mass drug administration programs to control onchocerciasis and lymphatic filariasis. Determining the infection levels in vector populations is also important for assessing transmission, deciding when drug treatments may be terminated and for monitoring recrudescence. Immunological methods to detect infection in humans are available, however, cross-reactivity issues have been reported. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity, and can be used to detect infection in both humans and vectors. In this study we developed loop-mediated isothermal amplification (LAMP tests to detect three different filarial DNAs in human and insect samples using pH sensitive dyes for enhanced visual detection of amplification. Furthermore, reactions were performed in a portable, non-instrumented nucleic acid amplification (NINA device that provides a stable heat source for LAMP. The efficacy of several strand displacing DNA polymerases were evaluated in combination with neutral red or phenol red dyes. Colorimetric NINA-LAMP assays targeting Brugia Hha I repeat, Onchocerca volvulus GST1a and Wuchereria bancrofti LDR each exhibit species-specificity and are also highly sensitive, detecting DNA equivalent to 1/10-1/5000th of one microfilaria. Reaction times varied depending on whether a single copy gene (70 minutes, O. volvulus or repetitive DNA (40 min, B. malayi and W. bancrofti was employed as a biomarker. The NINA heater can be used to detect multiple infections simultaneously. The accuracy, simplicity and versatility of the technology suggests that colorimetric NINA-LAMP assays are ideally suited for monitoring the success of filariasis control programs.

  16. Time Course of Detection of Human Male DNA from Stained Blood Sample on Various Surfaces by Loop Mediated Isothermal Amplification and Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Panan Kanchanaphum

    2018-01-01

    Full Text Available This study explores determining the sex of humans from blood stains taken from different surfaces and compares the time course of detection with the conventional PCR, Conventional Loop Mediated Isothermal Amplification (LAMP, and LAMP-Lateral Flow Dipstick (LFD. For the DNA templates, 7 male and 7 female blood stained samples were extracted and added to LAMP and PCR reaction solution to amplify the SRY gene. The DNA samples were extracted from the following blood stained materials: cloth, wood, clay, and tile. Then, the samples were stored at room temperature for 1, 7, 30, and 60 day(s. After the DNA amplification, the gel electrophoresis process was applied to detect LAMP product. The LFD was combined with the LAMP to detect LAMP product on the male cloth samples. For the male samples, the time course of detection on the first and seventh days indicated positive for both LAMP and PCR products on all the surfaces while no DNA amplification was found on any of the female samples. On day 30, positive LAMP product was still found on all the male samples. However, it had faded on the tiles. Moreover, all the male samples, which had tested positive for PCR product, were blurred and unclear. On day 60, LAMP product was still found on all the male samples. Conversely, the PCR method resulted in no bands showing for any of the male samples. However, the LAMP-LFD method detected product on all the male samples of cloth. The results show that the LAMP is an effective, practical, and reliable molecular-biological method. Moreover, the LFD can increase the efficiency and sensitivity of the LAMP, making it more suitable for field studies because gel electrophoresis apparatus is not required.

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

    Science.gov (United States)

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

    2013-01-02

    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.

  18. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection ofCylindrocladium scopariumon Eucalyptus.

    Science.gov (United States)

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-10-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium , has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium . The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

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

    Science.gov (United States)

    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.

  20. Loop-Mediated Isothermal Amplification untuk Mendeteksi Gen blaTEM sebagai Penyandi Extended-Spectrum Beta-Lactamase pada Isolat Enterobacteriaceae

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    Bayu A. P. Wilopo

    2015-12-01

    Full Text Available Extended-spectrum beta-lactamase (ESBL is a beta-lactamase enzyme that is capable of hydrolyzing penicillin, cephalosporin, and monobactam, and can be inhibited by clavulanic acid. This enzyme is encoded by multiple genes, one of them is blaTEM. Polymerase chain reaction (PCR is one of the DNA amplification methods that are frequently used; however, there are other methods that can be used including, among others, loop-mediated isothermal amplification (LAMP. LAMP requires simple equipment with quicker and easy-to-read results compared to PCR. This study was a diagnostic test to explore the sensitivity and specificity of LAMP method compared to PCR in detecting blaTEM gene. Furthermore, the concordance between LAMP and PCR methods was assessed. A total of 92 Enterobacteriaceae isolates were examined by PCR and LAMP methods and compared. The result showed that the LAMP method had a sensitivity of 91.4% and a specificity of 91.2% with a concordance value (kappa of 85.4%. In conclusion, LAMP method has a good validity and a very good conformity compared to the PCR method. Therefore, LAMP method can be used as an alternative diagnostic test, especially in limited settings.

  1. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

    Directory of Open Access Journals (Sweden)

    Tian-Min Qiao

    2016-10-01

    Full Text Available Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP were developed for detection of C. scoparium based on factor 1-alpha (tef1 and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

  2. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

    Science.gov (United States)

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-01-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products. PMID:27721691

  3. Direct duplex real-time loop mediated isothermal amplification assay for the simultaneous detection of cow and goat species origin of milk and yogurt products for field use.

    Science.gov (United States)

    Kim, Mi-Ju; Kim, Hae-Yeong

    2018-04-25

    A multiple loop-mediated isothermal amplification (LAMP) method was developed to detect cow and goat milk in the field using a portable fluorescence device. For rapid on-site detection, this duplex LAMP assay was used in combination with direct amplification, without DNA extraction. The cow- and goat-specific LAMP primer sets were designed based on the mitochondrial cytochrome b gene, and showed specificity against 13 other animal species in the reactions. The sensitivity of the duplex LAMP assay for cow and goat was 0.1 and 1 pg, respectively. The detection limit for both target species in milk mixtures was 2%. This assay successfully amplified and identified the two target species in 24 samples of commercial milk and yogurt products, with 30 min sampling-to-result analysis time. Therefore, this direct duplex real-time LAMP assay is useful for on-site simultaneous detection of cow and goat milk in commercial products, a capability needed to confirm accurate labeling. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. 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.

    Science.gov (United States)

    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 μl. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    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.

  6. Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loop-mediated isothermal amplification (LAMP.

    Directory of Open Access Journals (Sweden)

    Aongart Mahittikorn

    Full Text Available 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.

  7. Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

    2016-06-01

    For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. © The American Society of Tropical Medicine and Hygiene.

  8. Development of a Reverse Transcription Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Subtype H7N9 Avian Influenza Virus

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    Hongmei Bao

    2014-01-01

    Full Text Available A novel influenza A (H7N9 virus has emerged in China. To rapidly detect this virus from clinical samples, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP method for the detection of the H7N9 virus. The minimum detection limit of the RT-LAMP assay was 0.01 PFU H7N9 virus, making this method 100-fold more sensitive to the detection of the H7N9 virus than conventional RT-PCR. The H7N9 virus RT-LAMP assays can efficiently detect different sources of H7N9 influenza virus RNA (from chickens, pigeons, the environment, and humans. No cross-reactive amplification with the RNA of other subtype influenza viruses or of other avian respiratory viruses was observed. The assays can effectively detect H7N9 influenza virus RNA in drinking water, soil, cloacal swab, and tracheal swab samples that were collected from live poultry markets, as well as human H7N9 virus, in less than 30 min. These results suggest that the H7N9 virus RT-LAMP assays were efficient, practical, and rapid diagnostic methods for the epidemiological surveillance and diagnosis of influenza A (H7N9 virus from different resource samples.

  9. A rapid and sensitive loop-mediated isothermal amplification procedure (LAMP) for Mycoplasma hyopneumoniae detection based on the p36 gene.

    Science.gov (United States)

    Liu, M J; Du, G M; Bai, F F; Wu, Y Z; Xiong, Q Y; Feng, Z X; Li, B; Shao, G Q

    2015-05-04

    The aim of this study was to establish a method for sensitive and rapid diagnosis of Mycoplasma hyopneumoniae in clinical specimens. To this effect, we employed three sets of primers specifically designed for amplification of nucleic acids under isothermal conditions. After optimization of reaction conditions, M. hyopneumoniae could be successfully detected at 63°C in 45 min through use of the loop-mediated isothermal amplification (LAMP) assay. A positive reaction was identified visually as white precipitate and confirmed by gel electrophoresis. The detection limit for this assay was 10 copies/μL, as observed by electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease digestion as well as by direct sequencing of the amplified product. This method can specifically detect M. hyopneumoniae; other species with high homology and other bacterial and virus strains gave negative results. To test the utility of this procedure, the LAMP assay was applied to 40 clinical samples collected from swine lung tissues experimentally challenged with M. hyopneumoniae isolates, and compared to the results from a real-time polymerase chain reaction (PCR) assay. A concordance of 100% was observed between the two assays. In conclusion, the results from our study demonstrated that the LAMP assay provided a rapid reaction and was inexpensive to perform, with no need of complex instruments or systems such as Geneamp PCR. The LAMP assay may therefore be applied in routine diagnosis in the clinical laboratory and for in-field detection of M. hyopneumoniae infection.

  10. [Development and evaluation of novel loop-mediated isothermal amplification for rapid detection of bla(IMP-1) and bla(VIM-2) genes].

    Science.gov (United States)

    Kojima, Tadashi; Shibata, Naohiro; Ikedo, Masanari; Arakawa, Yoshichika

    2006-07-01

    Loop-mediated isothermal amplification (LAMP) amplifies a target gene with high specificity and rapidity under isothermal conditions. LAMP assays were developed for the rapid detection of metallo-beta-lactamase (MBL) genes such as bla(IMP-1)) and bla(VIM-2). We initially designed specific primers to detect MBL genes for LAMP assays and evaluated the specificity and sensitivity of these assays. LAMP assays amplified MBL genes under a constant temperature of 63 degrees C within 1 hour, and were compared to PCR in MBL-producing strains. The results of MBL genes typing by LAMP assays agree completely with PCR results. The lower detection limits of bla(IMP-1)- and bla(VIM-2)-LAMP assays using real-time turbidimeters were 30cfu/test and 3cfu/test. After amplification, products were directly observed by the naked eye with a fluorescent detection reagent. In conclusion, LAMP assays are convenient, rapid, and fully feasible for detecting MBL genes in ordinary clinical microbiology laboratories without special apparatus.

  11. Rapid detection of Piper yellow mottle virus and Cucumber mosaic virus infecting black pepper (Piper nigrum) by loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Bhat, A I; Siljo, A; Deeshma, K P

    2013-10-01

    The loop-mediated isothermal amplification (LAMP) assay for Piper yellow mottle virus and the reverse transcription (RT) LAMP assay for Cucumber mosaic virus each consisted of a set of five primers designed against the conserved sequences in the viral genome. Both RNA and DNA isolated from black pepper were used as a template for the assay. The results were assessed visually by checking turbidity, green fluorescence and pellet formation in the reaction tube and also by gel electrophoresis. The assay successfully detected both viruses in infected plants whereas no cross-reactions were recorded with healthy plants. Optimum conditions for successful amplification were determined in terms of the concentrations of magnesium sulphate and betaine, temperature, and duration. The detection limit for both LAMP and RT-LAMP was up to 100 times that for conventional PCR and up to one-hundredth of that for real-time PCR. The optimal conditions arrived at were validated by testing field samples of infected vines of three species from different regions. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens.

    Science.gov (United States)

    Higgins, Owen; Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert; Smith, Terry J

    2018-02-09

    Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae , Neisseria meningitidis and Haemophilus influenzae . Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae , N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology.

  13. Rapid and Sensitive Detection of the Main Contaminating Fungus Penicillium restrictum in Jet Fuel using Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick

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    Xiong Yun

    2016-06-01

    Full Text Available We report a new contaminating fungus of jet fuel, Penicillium restrictum, which accounted for nearly 17% of the total sequence identified from five jet fuel samples as determined by the application of Illumina MiSeq sequencing-by-synthesis. We also report the development and validation of a new loop-mediated isothermal amplification (LAMP assay combined with a lateral flow dipstick (LFD for the repaid detection of P. restrictum. The optimal reaction conditions and primer set for LAMP were determined using a real-time turbidimeter. The LAMP-LFD assay was 1000-fold more sensitive than traditional PCR. P. restrictum could be detected specifically using the LAMP-LFD assay, and no amplification was observed when genomic DNA from another seven fungi found in jet fuel was tested. Eleven jet fuel samples from the field were tested using the LAMP-LFD assay we developed. Seven of them were positive for the presence of P. restrictum. These results were verified by traditional microbiological detection methods. Our results indicate that the LAMP-LFD assay is a rapid, accurate and sensitive tool for the detection of P. restrictum and could represent a new template for the detection of contaminating fungi in jet fuel.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Visual detection of human enterovirus 71 subgenotype C4 and Coxsackievirus A16 by reverse transcription loop-mediated isothermal amplification with the hydroxynaphthol blue dye.

    Science.gov (United States)

    Nie, Kai; Zhang, Yong; Luo, Le; Yang, Meng-Jie; Hu, Xiu-Mei; Wang, Miao; Zhu, Shuang-Li; Han, Feng; Xu, Wen-Bo; Ma, Xue-Jun

    2011-08-01

    A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of human enterovirus 71 subgenotype C4 (EV71-C4) and Coxsackievirus A16 (CVA16) infection, respectively. The reaction was performed in one step in a single tube at 65°C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limits of the RT-LAMP assay were 0.33 and 1.58 of a 50% tissue culture infective dose (TCID(50)) per reaction based on 10-fold dilutions of a titrated EV71 or CVA16 strain, respectively. No cross-reaction was observed with Coxsackievirus A (CVA) viruses (CVA2, 4, 5, 7, 9, 10, 14, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3, 6, 11, and 19). The assay was further evaluated with 47 clinical stool specimens diagnosed previously with EV71, CVA16 or other human enterovirus infections. Virus isolates from stool samples were confirmed by virus neutralization testing and sequencing. RT-LAMP with HNB dye was demonstrated to be a sensitive and cost-effective assay for rapid visual detection of human EV71-C4 and CVA16. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP Assay for the Detection of Bacterial Meningitis Pathogens

    Directory of Open Access Journals (Sweden)

    Owen Higgins

    2018-02-01

    Full Text Available Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology.

  17. CFD Analysis of Passive Autocatalytic Recombiner

    Directory of Open Access Journals (Sweden)

    B. Gera

    2011-01-01

    Full Text Available In water-cooled nuclear power reactors, significant quantities of hydrogen could be produced following a postulated loss-of-coolant accident (LOCA along with nonavailability of emergency core cooling system (ECCS. Passive autocatalytic recombiners (PAR are implemented in the containment of water-cooled power reactors to mitigate the risk of hydrogen combustion. In the presence of hydrogen with available oxygen, a catalytic reaction occurs spontaneously at the catalyst surfaces below conventional ignition concentration limits and temperature and even in presence of steam. Heat of reaction produces natural convection flow through the enclosure and promotes mixing in the containment. For the assessment of the PAR performance in terms of maximum temperature of catalyst surface and outlet hydrogen concentration an in-house 3D CFD model has been developed. The code has been used to study the mechanism of catalytic recombination and has been tested for two literature-quoted experiments.

  18. Autocatalytic water dissociation on Cu(110) at near ambient conditions

    Energy Technology Data Exchange (ETDEWEB)

    Mulleregan, Alice; Andersson, Klas; Ketteler, Guido; Bluhm, Hendrik; Yamamoto, Susumu; Ogasawara, Hirohito; Pettersson, Lars G.M.; Salmeron, Miquel; Nilsson, Anders

    2007-05-16

    Autocatalytic dissociation of water on the Cu(110) metal surface is demonstrated based on X-ray photoelectron spectroscopy studies carried out in-situ under near ambient conditions of water vapor pressure (1 Torr) and temperature (275-520 K). The autocatalytic reaction is explained as the result of the strong hydrogen-bond in the H{sub 2}O-OH complex of the dissociated final state, which lowers the water dissociation barrier according to the Broensted-Evans-Polanyi relations. A simple chemical bonding picture is presented which predicts autocatalytic water dissociation to be a general phenomenon on metal surfaces.

  19. Novel system uses probasin-based promoter, transcriptional silencers and amplification loop to induce high-level prostate expression

    Directory of Open Access Journals (Sweden)

    Yu Hong

    2007-02-01

    Full Text Available Abstract Background Despite several effective treatment options available for prostate cancer, it remains the second leading cause of cancer death in American men. Thus, there is a great need for new treatments to improve outcomes. One such strategy is to eliminate cancer through the expression of cytotoxic genes specifically in prostate cells by gene therapy vectored delivery. To prevent systemic toxicity, tissue- and/or cancer-specific gene expression is required. However, the use of tissue- or cancer-specific promoters to target transgene expression has been hampered by their weak activity. Results To address this issue, we have developed a regulation strategy that includes feedback amplification of gene expression along with a differentially suppressible tetracycline regulated expression system (DiSTRES. By differentially suppressing expression of the tetracycline-regulated transcriptional activator (tTA and silencer (tTS genes based on the cell origin, this leads to the activation and silencing of the TRE promoter, respectively. In vitro transduction of LNCaP cells with Ad/GFPDiSTRES lead to GFP expression levels that were over 30-fold higher than Ad/CMV-GFP. Furthermore, Ad/FasL-GFPDiSTRES demonstrated cytotoxic effects in prostate cancer cells known to be resistant to Fas-mediated apoptosis. Conclusion Prostate-specific regulation from the DiSTRES system, therefore, serves as a promising new regulation strategy for future applications in the field of cancer gene therapy and gene therapy as a whole.

  20. Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9.

    Science.gov (United States)

    Kanitkar, Yogendra H; Stedtfeld, Robert D; Steffan, Robert J; Hashsham, Syed A; Cupples, Alison M

    2016-01-08

    Real-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA are commonly used to quantify Dehalococcoides spp. in groundwater from chlorinated solvent-contaminated sites. In this study, loop-mediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes (vcrA, bvcA, and tceA) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both contain Dehalococcoides species). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas only vcrA was quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly used Dehalococcoides species-containing cultures. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

    Science.gov (United States)

    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

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

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    Gregory L. Damhorst

    2015-09-01

    Full Text Available Viral load measurements are an essential tool for the long-term clinical care of human 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.

  3. Development of Loop-Mediated Isothermal Amplification (LAMP) assay for rapid detection of Fusarium oxysporum f. sp. ciceris - wilt pathogen of chickpea.

    Science.gov (United States)

    Ghosh, Raju; Nagavardhini, Avuthu; Sengupta, Anindita; Sharma, Mamta

    2015-02-11

    Fusarium oxysporum f. sp. ciceris (Foc), the causal agent of Fusarium wilt is a devastating pathogen of chickpea. In chickpea, various soil borne pathogens produce (s) similar symptoms, therefore cannot be distinguished easily at field level. There is real need for a rapid, inexpensive, and easy to operate and maintain genotyping tool to facilitate accurate disease diagnosis and surveillance for better management of Fusarium wilt outbreaks. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay targeting the elongation factor 1 alpha gene sequence for visual detection of Foc. The LAMP reaction was optimal at 63°C for 60 min. When hydroxynaphthol blue (HNB) was added before amplification, samples with Foc DNA developed a characteristic sky blue colour but those without DNA or with the DNA of six other plant pathogenic fungi did not. Results obtained with LAMP and HNB were confirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for Foc was 10 fg of genomic DNA per reaction, while that of conventional PCR was 100 pg. In conclusion, it was found that a LAMP assay combined with HNB is simple, rapid, sensitive, and specific. The LAMP assay does not require specialized equipment, hence can be used in the field for the rapid detection of Foc. This is the first report of the use of LAMP assay for the detection of Foc. The presented LAMP method provides a specific, sensitive and rapid diagnostic tool for the distinction of Foc, with the potential to be standardized as a detection method for Foc in endemic areas and will be very useful for monitoring the disease complex in the field further suggesting the management strategies.

  4. Application of a loop-mediated isothermal amplification (LAMP) assay targeting cox1 gene for the detection of Clonorchis sinensis in human fecal samples.

    Science.gov (United States)

    Rahman, S M Mazidur; Song, Hyun Beom; Jin, Yan; Oh, Jin-Kyoung; Lim, Min Kyung; Hong, Sung-Tae; Choi, Min-Ho

    2017-10-01

    Clonorchiasis is prevalent in the Far East, and a major health problem in endemic areas. Infected persons may experience, if not treated, serious complications such as bile stone formation, pyogenic cholangitis, and even cholangiocarcinoma. Early diagnosis and treatment are important to prevent serious complications and, therefore, the simple and reliable diagnostic method is necessary to control clonorchiasis in endemic areas, where resources for the diagnosis are limited. The loop-mediated isothermal amplification (LAMP) assay has been applied for the detection of Clonorchis sinensis DNA. Six primers targeting eight locations on the cytochrome c oxidase subunit 1 gene of C. sinensis were designed for species-specific amplification using the LAMP assay. The LAMP assay was sensitive enough to detect as little as 100 fg of C. sinensis genomic DNA and the detection limit in 100 mg of stool was as low as one egg. The assay was highly specific because no cross-reactivity was observed with the DNA of other helminths, protozoa or Escherichia coli. Then, LAMP assay was applied to human fecal samples collected from an endemic area of clonorchiasis in Korea. Using samples showing consistent results by both Kato-Katz method and real-time PCR as reference standards, the LAMP assay showed 97.1% (95% CI, 90.1-99.2) of sensitivity and 100% (95% CI, 92.9-100) of specificity. In stool samples with more than 100 eggs per gram of feces, the sensitivity achieved 100%. To detect C. sinensis in human fecal samples, the LAMP assay was applied and achieved high sensitivity and specificity. The LAMP assay can be utilized in field laboratories as a powerful tool for diagnosis and epidemiological survey of clonorchiasis.

  5. Application of a loop-mediated isothermal amplification (LAMP assay targeting cox1 gene for the detection of Clonorchis sinensis in human fecal samples.

    Directory of Open Access Journals (Sweden)

    S M Mazidur Rahman

    2017-10-01

    Full Text Available Clonorchiasis is prevalent in the Far East, and a major health problem in endemic areas. Infected persons may experience, if not treated, serious complications such as bile stone formation, pyogenic cholangitis, and even cholangiocarcinoma. Early diagnosis and treatment are important to prevent serious complications and, therefore, the simple and reliable diagnostic method is necessary to control clonorchiasis in endemic areas, where resources for the diagnosis are limited.The loop-mediated isothermal amplification (LAMP assay has been applied for the detection of Clonorchis sinensis DNA. Six primers targeting eight locations on the cytochrome c oxidase subunit 1 gene of C. sinensis were designed for species-specific amplification using the LAMP assay. The LAMP assay was sensitive enough to detect as little as 100 fg of C. sinensis genomic DNA and the detection limit in 100 mg of stool was as low as one egg. The assay was highly specific because no cross-reactivity was observed with the DNA of other helminths, protozoa or Escherichia coli. Then, LAMP assay was applied to human fecal samples collected from an endemic area of clonorchiasis in Korea. Using samples showing consistent results by both Kato-Katz method and real-time PCR as reference standards, the LAMP assay showed 97.1% (95% CI, 90.1-99.2 of sensitivity and 100% (95% CI, 92.9-100 of specificity. In stool samples with more than 100 eggs per gram of feces, the sensitivity achieved 100%.To detect C. sinensis in human fecal samples, the LAMP assay was applied and achieved high sensitivity and specificity. The LAMP assay can be utilized in field laboratories as a powerful tool for diagnosis and epidemiological survey of clonorchiasis.

  6. Lateral Flow Loop-Mediated Isothermal Amplification Test with Stem Primers: Detection of Cryptosporidium Species in Kenyan Children Presenting with Diarrhea

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    Timothy S. Mamba

    2018-01-01

    Full Text Available Background. Cryptosporidium is a protozoan parasite and a major cause of diarrhea in children and immunocompromised patients. Current diagnostic methods for cryptosporidiosis such as microscopy have low sensitivity while techniques such as PCR indicate higher sensitivity levels but are seldom used in developing countries due to their associated cost. A loop-mediated isothermal amplification (LAMP technique, a method with shorter time to result and with equal or higher sensitivity compared to PCR, has been developed and applied in the detection of Cryptosporidium species. The test has a detection limit of 10 pg/µl (~100 oocysts/ml indicating a need for more sensitive diagnostic tools. This study developed a more sensitive lateral flow dipstick (LFD LAMP test based on SAM-1 gene and with the addition of a second set of reaction accelerating primers (stem primers. Results. The stem LFD LAMP test showed analytical sensitivity of 10 oocysts/ml compared to 100 oocysts/ml (10 pg/ul for each of the SAM-1 LAMP test and nested PCR. The stem LFD LAMP and nested PCR detected 29/39 and 25/39 positive samples of previously identified C. parvum and C. hominis DNA, respectively. The SAM-1 LAMP detected 27/39. On detection of Cryptosporidium DNA in 67 clinical samples, the stem LFD LAMP detected 16 samples and SAM-2 LAMP 14 and nested PCR identified 11. Preheating the templates increased detection by stem LFD LAMP to 19 samples. Time to results from master mix preparation step took ~80 minutes. The test was specific, and no cross-amplification was recorded with nontarget DNA. Conclusion. The developed stem LFD LAMP test is an appropriate method for the detection of C. hominis, C. parvum, and C. meleagridis DNA in human stool samples. It can be used in algorithm with other diagnostic tests and may offer promise as an effective diagnostic tool in the control of cryptosporidiosis.

  7. A loop-mediated isothermal amplification (LAMP assay for early detection of Schistosoma mansoni in stool samples: a diagnostic approach in a murine model.

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    Pedro Fernández-Soto

    2014-09-01

    Full Text Available Human schistosomiasis, mainly due to Schistosoma mansoni species, is one of the most prevalent parasitic diseases worldwide. To overcome the drawbacks of classical parasitological and serological methods in detecting S. mansoni infections, especially in acute stage of the disease, development of cost-effective, simple and rapid molecular methods is still needed for the diagnosis of schistosomiasis. A promising approach is the loop-mediated isothermal amplification (LAMP technology. Compared to PCR-based assays, LAMP has the advantages of reaction simplicity, rapidity, specificity, cost-effectiveness and higher amplification efficiency. Additionally, as results can be inspected by the naked eye, the technique has great potential for use in low-income countries.A sequence corresponding to a mitochondrial S. mansoni minisatellite DNA region was selected as a target for designing a LAMP-based method to detect S. mansoni DNA in stool samples. We used a S. mansoni murine model to obtain well defined stool and sera samples from infected mice with S. mansoni cercariae. Samples were taken weekly from week 0 to 8 post-infection and the Kato-Katz and ELISA techniques were used for monitoring the infection. Primer set designed were tested using a commercial reaction mixture for LAMP assay and an in house mixture to compare results. Specificity of LAMP was tested using 16 DNA samples from different parasites, including several Schistosoma species, and no cross-reactions were found. The detection limit of our LAMP assay (SmMIT-LAMP was 1 fg of S. mansoni DNA. When testing stool samples from infected mice the SmMIT-LAMP detected S. mansoni DNA as soon as 1 week post-infection.We have developed, for the first time, a cost-effective, easy to perform, specific and sensitive LAMP assay for early detection of S. mansoni in stool samples. The method is potentially and readily adaptable for field diagnosis and disease surveillance in schistosomiasis-endemic areas.

  8. Detection of Panton-Valentine Leukocidin DNA from methicillin-resistant Staphylococcus aureus by resistive pulse sensing and loop-mediated isothermal amplification with gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Alice Kar Lai, E-mail: s0907465@cuhk.mail.serv.edu.hk [Program of Biochemistry, School of Life Sciences, The Chinese University of Hong Kong (Hong Kong); Lu, Haifei, E-mail: hflu@ee.cuhk.edu.hk [Center for Advanced Research in Photonics, Department of Electronic Engineering, The Chinese University of Hong Kong (Hong Kong); Wu, Shu Yuen, E-mail: sywu@ee.cuhk.edu.hk [Center for Advanced Research in Photonics, Department of Electronic Engineering, The Chinese University of Hong Kong (Hong Kong); Kwok, Ho Chin, E-mail: hckwock@ee.cuhk.edu.hk [Center for Advanced Research in Photonics, Department of Electronic Engineering, The Chinese University of Hong Kong (Hong Kong); Ho, Ho Pui, E-mail: hpho@ee.cuhk.edu.hk [Center for Advanced Research in Photonics, Department of Electronic Engineering, The Chinese University of Hong Kong (Hong Kong); Yu, Samuel, E-mail: samscyu@gmail.com [The MacDiarmid Institute for Advanced Materials and Nanotechnology, Christchurch (New Zealand); Izon Science, PO Box 39-168, Harewood, Christchurch 8545 (New Zealand); Cheung, Anthony Ka Lun, E-mail: kalun2004@hotmail.com [Program of Biochemistry, School of Life Sciences, The Chinese University of Hong Kong (Hong Kong); Kong, Siu Kai, E-mail: skkong@cuhk.edu.hk [Program of Biochemistry, School of Life Sciences, The Chinese University of Hong Kong (Hong Kong)

    2013-06-11

    Graphical abstract: -- Highlights: •A novel diagnostic assay is developed to detect the MRSA's Panton-Valentine Leukocidin toxin. •Detection is based on target DNA amplification at one single temperature at 65 °C by LAMP. •Amplicons are then hybridized with 2 Au-nanoparticles with specific DNA probes for sensing. •The supra-assemblies are subsequently sensed by resistive pulse sensing. •Detection limit: ∼200 copies of DNA; time for detection: completed within 2 h. -- Abstract: This report describes a novel diagnostic assay for rapid detection of the Panton-Valentine Leukocidin (PVL) toxin of methicillin-resistant Staphylococcus aureus (MRSA) utilizing resistive pulse sensing (RPS), loop-mediated isothermal DNA amplification (LAMP) in combination with gold nanoparticles (AuNPs). The PVL DNA from MRSA was specifically amplified by LAMP using four primers at one temperature (65 °C). The DNA products with biotin were then conjugated to a first AuNP1 (55 ± 2 nm) through biotin–avidin binding. A second AuNP2 (30 ± 1.5 nm) coated with a specific DNA probe hybridized with the LAMP DNA products at the loop region to enhance assay sensitivity and specificity, to generate supra-AuNP1-DNA-AuNP2 assemblies. Scanning electron microscopy confirmed the presence of these supra-assemblies. Using RPS, detection and quantitation of the agglomerated AuNPs were performed by a tunable fluidic nanopore sensor. The results demonstrate that the LAMP-based RPS sensor is sensitive and rapid for detecting the PVL DNA. This technique could achieve a limit of detection (LOD) up to about 500 copies of genomic DNA from the bacteria MRSA MW2 and the detection can be completed within two hours with a straightforward signal-to-readout setup. It is anticipated that this LAMP-based AuNP RPS may become an effective tool for MRSA detection and a potential platform in clinical laboratory to report the presence or absence of other types of infectious agents.

  9. Development of a loop-mediated isothermal amplification technique and comparison with quantitative real-time PCR for the rapid visual detection of canine neosporosis.

    Science.gov (United States)

    Mahittikorn, Aongart; Thammasonthijarern, Nipa; Roobthaisong, Amonrattana; Udonsom, Ruenruetai; Popruk, Supaluk; Siri, Sukhontha; Mori, Hirotake; Sukthana, Yaowalark

    2017-08-23

    Dogs are the definitive hosts of Neospora caninum and play an important role in the transmission of the parasite. Despite the high sensitivity of existing molecular tools such as quantitative real-time PCR (qPCR), these techniques are not suitable for use in many countries because of equipment costs and difficulties in implementing them for field diagnostics. Therefore, we developed a simplified technique, loop-mediated isothermal amplification (LAMP), for the rapid visual detection of N. caninum. LAMP specificity was evaluated using a panel containing DNA from a range of different organisms. Sensitivity was evaluated by preparing 10-fold serial dilutions of N. caninum tachyzoites and comparing the results with those obtained using qPCR. Assessment of the LAMP results was determined by recognition of a colour change after amplification. The usefulness of the LAMP assay in the field was tested on 396 blood and 115 faecal samples from dogs, and one placenta from a heifer collected in Lopburi, Nakhon Pathom, Sa Kaeo, and Ratchaburi provinces, Thailand. Specificity of the LAMP technique was shown by its inability to amplify DNA from non-target pathogens or healthy dogs. The detection limit was the equivalent of one genome for both LAMP and qPCR. LAMP and qPCR detected positive N. caninum infection in 15 of 396 (3.8%) blood samples; LAMP detected 9/115 (7.8%) positive faecal samples, while qPCR detected 5/115 (4.3%) positive faecal samples. The placental tissue was shown to be positive by both techniques. Agreement between LAMP and qPCR was perfect in blood samples (kappa value, 1.00) and substantial in faecal samples (kappa value, 0.697). This is the first known LAMP assay developed for the amplification of N. caninum. The technique effectively and rapidly detected the parasite with high sensitivity and specificity and was cost-effective. This assay could be used in the field to confirm the diagnosis of canine or bovine neosporosis.

  10. Application of wearable optical coherence tomography (OCT) and loop-mediated isothermal amplification (LAMP) techniques for in situ real-time field inspection of apple Marssonina blotch disease

    Science.gov (United States)

    Wijesinghe, Ruchire Eranga; Lee, Seung-Yeol; Ravichandran, Naresh Kumar; Shirazi, Muhammad Faizan; Han, Sangyeop; Jeong, Hyosang; Kim, Pilun; Jung, Hee-Young; Jeon, Mansik; Kim, Jeehyun

    2017-04-01

    Here we describe the possible application of optical coherence tomography (OCT) to inspect Marssonina coronaria infected apple blotch disease of in situ apple leaves. To fulfill the in situ field inspection requirement, we developed a compact wearable OCT system. For the confirmation of OCT results, simultaneous experiment was performed in realtime using loop-mediated isothermal amplification (LAMP), which is frequently used in agriculture. LAMP method was developed as an alternative approach for the inspection of disease. We performed field inspection for 30 consecutive days, and all the acquired results from both OCT and lamp were compared to confirm the correlation. A clear identification between healthy specimens, apparently healthy but infected specimens, and infected specimens could be obtained through the real-time OCT images, and the correlation between OCT and lamp results was confirmed through the obtained realtime lamp results. Based on this feasibility study, we conclude that the combination of both these diagnosing modalities can be effective for various novel agricultural discoveries.

  11. Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for the Detection ofTreponema pallidumDNA in the Peripheral Blood of Secondary Syphilis Patients.

    Science.gov (United States)

    Xiao, Yongjian; Xie, Yafeng; Xu, Man; Liu, Shuangquan; Jiang, Chuanhao; Zhao, Feijun; Zeng, Tiebing; Liu, Zhuoran; Yu, Jian; Wu, Yimou

    2017-12-01

    Secondary syphilis (SS) has always been puzzling for the clinicians because of the similarity of the appearance of skin rashes with other dermatoses. Serological assays are useful, but less sensitive at an early stage of SS or when patients are immunodeficient. Therefore, there is an urgent need to develop a rapid and effective tool for the diagnosis of SS, which may play an important role in the control of epidemic syphilis outbreaks. In this study, we evaluated a loop-mediated isothermal amplification (LAMP) assay, targeting gene encoding the basic membrane protein of Treponema pallidum , to detect the presence of circulating T. pallidum DNA in the blood of SS patients. The specificity of LAMP was validated using three strains of Spirochaetales and six common clinical bacteria. The clinical applicability of LAMP assay was assessed using 642 blood samples from clinically suspected SS patients and 80 samples from healthy blood donors, showing a sensitivity of 82.1% and a specificity of 100.0% in the diagnosis of SS. Thus, our results indicate that the LAMP can be used as a supplementary method for the diagnosis of SS.

  12. Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe.

    Science.gov (United States)

    Watthanapanpituck, Khanistha; Kiatpathomchai, Wansika; Chu, Eric; Panvisavas, Nathinee

    2014-11-01

    A DNA test based on loop-mediated isothermal amplification (LAMP) and colorimetric gold nanoparticle (AuNP) hybridization probe to detect the presence of human DNA in forensic evidence was developed. The LAMP primer set targeted eight regions of the human cytochrome b, and its specificity was verified against the DNA of 11 animal species, which included animals closely related to humans, such as chimpanzee and orangutan. By using the AuNP probe, sequence-specific LAMP product could be detected and the test result could be visualized through the change in color. The limit of detection was demonstrated with reproducibility to be as low as 718 fg of genomic DNA, which is equivalent to approximately 100 plasmid DNA copies containing the cytochrome b DNA target region. A simple DNA extraction method for the commonly found forensic biological samples was also devised to streamline the test process. This LAMP-AuNP human DNA test showed to be a robust, specific, and cost-effective tool for the forensic identification of human specimens without requiring sophisticated laboratory instruments.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Utility of loop-mediated isothermal amplification assay, polymerase chain reaction, and elisa for diagnosis of leptospirosis in South Indian patients

    Directory of Open Access Journals (Sweden)

    Mallika Sengupta

    2017-01-01

    Full Text Available Background: Leptospirosis is a zoonotic disease which requires laboratory diagnosis for confirmation. Materials and Methods: In this study serum samples from adults with acute undifferentiated fever (duration ≤15 days were tested for IgM antibodies to Leptospira by ELISA, PCR for rrs gene and loop-mediated isothermal amplification (LAMP assay for LipL32 and LipL41. Results: Among the 150 sera tested, three were positive by PCR, LAMP and IgM ELISA/modified Faines' criteria, two by only PCR; seven only by LAMP assay and forty fulfilled modified Faine's criteria (illness clinically compatible and IgM ELISA positive for leptospirosis. Clinical correlation revealed renal compromise, low platelet count and severe jaundice were significantly related to leptospirosis (P < 0.05. Conclusion: This study suggests that LAMP assay could be useful for diagnosis of leptospirosis during the 1st week of illness whereas IgM ELISA forms the mainstay of diagnosis from the 2nd week onward. Further studies especially community based, comparing ELISA, PCR, LAMP, culture and microscopic agglutination test are required to evaluate the veracity of these findings.

  15. Loop-mediated isothermal amplification (LAMP) for point-of-care detection of asymptomatic low-density malaria parasite carriers in Zanzibar.

    Science.gov (United States)

    Cook, Jackie; Aydin-Schmidt, Berit; González, Iveth J; Bell, David; Edlund, Elin; Nassor, Majda H; Msellem, Mwinyi; Ali, Abdullah; Abass, Ali K; Mårtensson, Andreas; Björkman, Anders

    2015-01-28

    Asymptomatic, low parasite density malaria infections are difficult to detect with currently available point-of-care diagnostics. This study piloted a loop-mediated isothermal amplification (LAMP) kit for field-friendly, high-throughput detection of asymptomatic malaria infections during mass screening and treatment (MSAT) in Zanzibar, a malaria pre-elimination setting. Screening took place in three known hotspot areas prior to the short rains in November. Finger-prick blood was taken for screening by rapid diagnostic test (RDT) and LAMP and collected on filter paper for subsequent polymerase chain reaction (PCR) analyses. LAMP results were compared to RDT and to PCR using McNemar's test. Approximately 1,000 people were screened. RDT detected ten infections (1.0% (95% CI 0.3-1.6)) whilst both LAMP and PCR detected 18 (1.8% (95% CI 0.9-2.6)) infections. However, PCR identified three infections that LAMP did not detect and vice versa. LAMP testing was easy to scale-up in field conditions requiring minimal training and equipment, with results ready one to three hours after screening. Despite lower than expected prevalence, LAMP detected a higher number of infections than the currently used diagnostic, RDT. LAMP is a field-friendly, sensitive diagnostic test that could be useful for MSAT malaria campaigns which require quick results to enable prompt treatment.

  16. An optimized protocol for DNA extraction from wheat seeds and Loop-Mediated Isothermal Amplification (LAMP) to detect Fusarium graminearum contamination of wheat grain.

    Science.gov (United States)

    Abd-Elsalam, Kamel; Bahkali, Ali; Moslem, Mohamed; Amin, Osama E; Niessen, Ludwig

    2011-01-01

    A simple, rapid, and efficient method for isolating genomic DNA from germinated seeds of wheat that is free from polysaccharides and polyphenols is reported. DNA was extracted, treated with RNase, measured and tested for completeness using agarose gel electrophoresis. DNA purification from wheat grains yielded abundant, amplifiable DNA with yields typically between 100 and 200 ng DNA/mg. The effectiveness and reliability of the method was tested by assessing quantity and quality of the isolated DNA using three PCR-based markers. Inter-simple sequence repeats (ISSRs) were used to assess the genetic diversity between different wheat varieties. Specific PCR primer pair Tox5-1/Tox5-2 and a loop-mediated isothermal amplification (LAMP) procedure were used to detect genomic DNA of Fusarium graminearum in contaminated wheat seeds. In this method there is no need to use liquid nitrogen for crushing germinated seedlings. The protocol takes approximately one hour to prepare high quality DNA. In combination with the LAMP assay it is a fast and cost-effective alternative to traditional diagnostic methods for the early detection of toxigenic fusaria in cereals.

  17. Loop-mediated isothermal amplification assay for detection and discrimination of Toxocara canis and Toxocara cati eggs directly from sand samples.

    Science.gov (United States)

    Macuhova, K; Kumagai, T; Akao, N; Ohta, N

    2010-12-01

    We developed a novel and simple method, using loop-mediated isothermal amplification (LAMP), for the detection and discrimination of Toxocara canis and Toxocara cati eggs. The new method employs 4 steps: (1) concentration of Toxocara eggs in a small amount of sand; (2) dissolution of the proteinaceous membrane of eggs and simultaneously separation of them from the sand using NaClO treatment; (3) extraction of DNA using NaOH treatment; and (4) detection of T. canis / T. cati DNA using a LAMP assay. All these steps are fast, easy to perform, and do not require expensive equipment or reagents. The novel method was tested both experimentally and in a field study. In the laboratory, we could reliably detect as few as 3 T. canis eggs in artificially contaminated sand, if the experiment was repeated twice. In the field trial, we were able to detect T. cati DNA from 4 natural sandpits having moderate to heavy contamination, although not in a single lightly contaminated sandpit. All of the examined sandpits were found to be contaminated with eggs of T. cati, but none appeared to contain T. canis. Our new method could extract DNA from T. canis and T. cati eggs directly from sand samples as well as detect and distinguish these 2 species in a few easy steps, with markedly reduced time and expense.

  18. Determining putative vectors of the Bogia Coconut Syndrome phytoplasma using loop-mediated isothermal amplification of single-insect feeding media

    Science.gov (United States)

    Lu, Hengyu; Wilson, Bree A. L.; Ash, Gavin J.; Woruba, Sharon B.; Fletcher, Murray J.; You, Minsheng; Yang, Guang; Gurr, Geoff M.

    2016-01-01

    Phytoplasmas are insect vectored mollicutes responsible for disease in many economically important crops. Determining which insect species are vectors of a given phytoplasma is important for managing disease but is methodologically challenging because disease-free plants need to be exposed to large numbers of insects, often over many months. A relatively new method to detect likely transmission involves molecular testing for phytoplasma DNA in sucrose solution that insects have fed upon. In this study we combined this feeding medium method with a loop-mediated isothermal amplification (LAMP) assay to study 627 insect specimens of 11 Hemiptera taxa sampled from sites in Papua New Guinea affected by Bogia coconut syndrome (BCS). The LAMP assay detected phytoplasma DNA from the feeding solution and head tissue of insects from six taxa belonging to four families: Derbidae, Lophopidae, Flatidae and Ricaniidae. Two other taxa yielded positives only from the heads and the remainder tested negative. These results demonstrate the utility of combining single-insect feeding medium tests with LAMP assays to identify putative vectors that can be the subject of transmission tests and to better understand phytoplasma pathosystems. PMID:27786249

  19. [Establish and optimization of real-time fluorescent reverse transcription loop-mediated isothermal amplification for detection of avian influenza H5 hemagglutinin gene].

    Science.gov (United States)

    Liu, Yun; Tang, Jin-ming; Tao, Hong; Sun, Jie; Lu, Ti-kang; Liao, Li-shan; Liu, Jian-li; Zeng, Shao-ling; Cao, Chen-fu; Zhang, Cai-hong; Ruan, Zhou-xi; Lv, Jian-qiang; Yang, Jun-xing; Hua, Qun-yi; Chen, Zheng-li; Qin, Zhi-feng

    2013-09-01

    H5 subtype avian influenza (AIV-H5) is a major causative agent of animalloimia a rapid and sensitive molecular biological diagnosis is crucial to the control program of AIV-H5. AIV-H5 real-time fluorescent reverse transcription loop-mediated isothermal amplification (qRT-LAMP) was established by means of heat treatment of the samples. The sensitivity, specificity and repeatability of this method were assessed and the performance of Calcein,SYBR Green I,HNB,SYTO 81 in colorimetric detection was comparatively analyzed to screen the optimum dye. The results showed the sensitivity of this method was 100 times higher than that of standard real-time fluorescent RT-PCR, and the detection limit was one copy of the gene per reaction. This method had no cross-reactivity with other common avian respiratory tract infectious disease-related pathogens such as IBV and NDV. The present study suggested Calcein was the optimum dye. Small-scale tests suggested this method was reliable for survey monitoring of AIV-H5 on the spot, indicating its potential applications in field investigation.

  20. Development and evaluation of a loop-mediated isothermal amplification assay for the rapid detection of porcine cytomegalovirus under field conditions

    Directory of Open Access Journals (Sweden)

    Yang Jin-Long

    2012-12-01

    Full Text Available Abstract Background Porcine cytomegalovirus (PCMV induces silent infection in adult pigs but more frequently causes fatal, generalized infection in newborn piglets. This study aimed to develop a new loop-mediated isothermal amplification (LAMP method for the sensitive, rapid, and inexpensive detection of PCMV under field conditions. Methods Tissue obtained from nine-week-old PCMV-free Landrace pigs or pig samples from postmortem examinations were analyzed. The samples were found to have clinical signs and lesions consistent with inclusion body rhinitis. Six specific primers were designed by targeting the PCMV DNA polymerase (DPOL DNA. The LAMP reaction was optimized in a water bath. The sensitivity and specificity of LAMP and polymerase chain reaction (PCR were compared. Results PCMV DNA was amplified at 65°C, and the result could be detected as early as 30 min into the reaction. Positive reactions could be visualized by the naked eye as a color change brought on by the addition of SYBR Green. The sensitivity and specificity of LAMP were found to be similar to those of the PCR. Conclusions LAMP is a high-throughput technique for the detection of PCMV and has a high specificity, sensitivity and simplicity; these factors make it suitable for detection of PCMV under field conditions.

  1. Development of a Loop-Mediated Isothermal Amplification Assay for Rapid and Specific Identification of ACT Producing Alternaria alternata, the Agent of Brown Spot Disease in Tangerine.

    Science.gov (United States)

    Moghimi, Hamid; Moradi, Amir; Hamedi, Javad; Basiri, Mina

    2016-03-01

    Rapid, accurate, and easy identification of pathogenic agents has always been important in medicine, veterinary, and agriculture. The brown spot infection is among the most common diseases in tangerine caused by Alternaria alternata. Due to the existence of seven various pathotypes of A. alternata species, it is challenging and time consuming to detect a pathotype responsible for citrus brown spot. In this study, we were seeking a rapid and specific approach to identify the tangerine pathotype within the A. alternata-pathogenic species, using the loop-mediated isothermal amplification (LAMP) method and actts2 gene as a marker molecule. Nine pathogenic samples were obtained from the region of Ramsar, Iran, and certified as A. alternata-pathogenic isolates. Specific primers were designed for regions coding for Alternaria citri toxin (ACT), and the PCR and LAMP reactions were performed. Our data showed that the primers designed for the tangerine pathotype of A. alternata were specific, and in both reactions, positive results were only observed in desired pathotypes. In the other pathotypes of this species as well as other standard fungal samples as negative controls, no positive result was observed. Therefore, our results suggest the possibility to detect the tangerine-specific A. alternata pathotype from other related species with a high accuracy and in early stages of the disease.

  2. Colorimetric Method of Loop-Mediated Isothermal Amplification with the Pre-Addition of Calcein for Detecting Flavobacterium columnare and its Assessment in Tilapia Farms.

    Science.gov (United States)

    Suebsing, Rungkarn; Kampeera, Jantana; Sirithammajak, Sarawut; Withyachumnarnkul, Boonsirm; Turner, Warren; Kiatpathomchai, Wansika

    2015-03-01

    Flavobacterium columnare, the causative agent of columnaris disease in fish, affects many economically important freshwater fish species. A colorimetric method of loop-mediated isothermal amplification with the pre-addition of calcein (LAMP-calcein) was developed and used to detect the presence of F. columnare in farmed tilapia (Nile Tilapia Oreochromis niloticus and red tilapia [Nile Tilapia × Mozambique Tilapia O. mossambicus]) and rearing water. The detection method, based on a change in color from orange to green, could be performed within 45 min at 63°C. The method was highly specific, as it had no cross-detections with 14 other bacterial species, including other fish pathogens and two Flavobacterium species. The method has a minimum detection limit of 2.2 × 10(2) F. columnare CFU; thus, it is about 10 times more sensitive than conventional PCR. With this method, F. columnare was detected in gonad, gill, and blood samples from apparently healthy tilapia broodstock as well as in samples of fertilized eggs, newly hatched fry, and rearing water. The bacteria isolated from the blood were further characterized biochemically and found to be phenotypically identical to F. columnare. The amplified products from the LAMP-calcein method had 97% homology with the DNA sequence of F. columnare.

  3. Loop-Mediated Isothermal Amplification Assay for Detection of Generic and Verocytotoxin-Producing Escherichia coli among Indigenous Individuals in Malaysia

    Directory of Open Access Journals (Sweden)

    Cindy Shuan Ju Teh

    2014-01-01

    Full Text Available We have successfully developed a Loop-mediated isothermal amplification (LAMP assay that could specifically detect generic Escherichia coli (E. coli. This assay was tested on 85 bacterial strains and successfully identified 54 E. coli strains (average threshold time, Tt = 21.26. The sensitivity of this assay was evaluated on serial dilutions of bacterial cultures and spiked faeces. The assay could detect 102 CFU/mL for bacterial culture with Tt = 33.30 while the detection limit for spiked faeces was 103 CFU/mL (Tt = 31.12. We have also detected 46 generic E. coli from 50 faecal samples obtained from indigenous individuals with 16% of the positive samples being verocytotoxin-producing E. coli (VTEC positive. VT1/VT2 allele was present in one faecal sample while the ratio of VT1 to VT2 was 6 : 1. Overall, our study had demonstrated high risk of VTEC infection among the indigenous community and most of the asymptomatic infection occurred among those aged below 15 years. The role of asymptomatic human carriers as a source of dissemination should not be underestimated. Large scale screening of the VTEC infection among indigenous populations and the potential contamination sources will be possible and easy with the aid of this newly developed rapid and simple LAMP assay.

  4. A highly specific and sensitive loop-mediated isothermal amplification method for the detection of Escherichia coli O157:H7.

    Science.gov (United States)

    Ravan, Hadi; Amandadi, Mojdeh; Sanadgol, Nima

    2016-02-01

    E. coli O157:H7 is one of the most important foodborne pathogen that causes some human illnesses such as bloody diarrhea, hemolytic-uremic syndrome, and kidney failure. We developed a loop-mediated isothermal amplification (LAMP) assay with six special primers that target a highly specific 299-bp region of the Z3276 gene for the detection of E. coli O157:H7. Among 117 bacterial strains tested in this study, positive results were only obtained from E. coli O157:H7 strains. The sensitivity level of the Z3276-LAMP assay was determined to be 5 CFU/reaction tube in pure bacterial culture. Moreover, the LAMP assay was successfully applied to artificially contaminated ground beef with a sensitivity level of 10(3) CFU/mL without pre-enrichment and 10 CFU/mL after a 4-h pre-enrichment. In conclusion, the present LAMP assay would be a useful and powerful tool for the rapid, sensitive, and specific diagnosis of E. coli O157:H7 strains in resource limited laboratories. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. An integrated rotary microfluidic system with DNA extraction, loop-mediated isothermal amplification, and lateral flow strip based detection for point-of-care pathogen diagnostics.

    Science.gov (United States)

    Park, Byung Hyun; Oh, Seung Jun; Jung, Jae Hwan; Choi, Goro; Seo, Ji Hyun; Kim, Do Hyun; Lee, Eun Yeol; Seo, Tae Seok

    2017-05-15

    Point-of-care (POC) molecular diagnostics plays a pivotal role for the prevention and treatment of infectious diseases. In spite of recent advancement in microfluidic based POC devices, there are still rooms for development to realize rapid, automatic and cost-effective sample-to-result genetic analysis. In this study, we propose an integrated rotary microfluidic system that is capable of performing glass microbead based DNA extraction, loop mediated isothermal amplification (LAMP), and colorimetric lateral flow strip based detection in a sequential manner with an optimized microfluidic design and a rotational speed control. Rotation direction-dependent coriolis force and siphon valving structures enable us to perform the fluidic control and metering, and the use of the lateral flow strip as a detection method renders all the analytical processes for nucleic acid test simplified and integrated without the need of expensive instruments or human intervention. As a proof of concept for point-of-care DNA diagnostics, we identified the food-borne bacterial pathogen which was contaminated in water or milk. Not only monoplex Salmonella Typhimurium but also multiplex Salmonella Typhimurium and Vibrio parahaemolyticus were analysed on the integrated rotary genetic analysis microsystem with a limit of detection of 50 CFU in 80min. In addition, three multiple samples were simultaneously analysed on a single device. The sample-to-result capability of the proposed microdevice provides great usefulness in the fields of clinical diagnostics, food safety and environment monitoring. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Loop-Mediated Isothermal Amplification Label-Based Gold Nanoparticles Lateral Flow Biosensor for Detection ofEnterococcus faecalisandStaphylococcus aureus.

    Science.gov (United States)

    Wang, Yi; Li, Hui; Wang, Yan; Zhang, Lu; Xu, Jianguo; Ye, Changyun

    2017-01-01

    The report describes a simple, rapid and sensitive assay for visual and multiplex detection of Enterococcus faecalis and Staphylococcus aureus based on multiple loop-mediated isothermal amplification (mLAMP) and lateral flow biosensor (LFB). Detection and differentiation of the Ef0027 gene ( E. faecalis -specific gene) and nuc gene ( S. aureus -specific gene) were determined using fluorescein (FITC)-and digoxin-modified primers in the mLAMP process. In the presence of biotin- and FITC-/digoxin-modified primers, the mLAMP yielded numerous biotin- and FITC-/digoxin-attached duplex products, which were detected by LFB through biotin/streptavidin interaction (biotin on the duplex and streptavidin on the gold nanoparticle) and immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the LFB test line). The accumulation of gold nanoparticles generated a characteristic red line, enabling visual and multiplex detection of target pathogens without instrumentation. The limit of detection (LoD), analytical specificity and feasibility of LAMP-LFB technique were successfully examined in pure culture and blood samples. The entire procedure, including specimen (blood samples) processing (30 min), isothermal reaction (40 min) and result reporting (within 2 min), could be completed within 75 min. Thus, this assay offers a simple, rapid, sensitive and specific test for multiplex detection of E. faecalis and S. aureus strains. Furthermore, the LAMP-LFB strategy is a universal technique, which can be extended to detect various target sequences by re-designing the specific LAMP primers.

  7. The evaluation of loop-mediated isothermal amplification-quartz crystal microbalance (LAMP-QCM) biosensor as a real-time measurement of HPV16 DNA.

    Science.gov (United States)

    Jearanaikoon, Patcharee; Prakrankamanant, Preeda; Leelayuwat, Chanvit; Wanram, Surasak; Limpaiboon, Temduang; Promptmas, Chamras

    2016-03-01

    We have previously developed quartz crystal microbalance biosensor integrated with loop-mediated isothermal amplification (LAMP-QCM) for human papillomavirus (HPV) type58 DNA detection. Infection with HPV, particularly HPV16, remains a serious health problem due to its major risk factor contributing to cervical cancer. In the present study, LAMP-QCM biosensor was evaluated in terms of a quantitative assay for copy number of HPV16 DNA in cervical samples compared to quantitative PCR using TaqMan assay (TaqMan-qPCR). The detection limit of LAMP-QCM was found to be 10 fold more sensitive than TaqMan-qPCR with 100% specificity and 7.6% imprecision. Different plot of HPV16 DNA copy number using Bland-Altman analysis revealed 94% correlation between LAMP-QCM and qPCR. We therefore concluded that the developed LAMP-QCM biosensor provides a possible rapid and sensitive assay for HPV16 DNA quantification in a routine laboratory. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Sensitive and rapid detection of campylobacter species from stools of children with diarrhea in Japan by the loop-mediated isothermal amplification method.

    Science.gov (United States)

    Ushijima, Hiroshi; Nishimura, Shuichi; Thongprachum, Aksara; Shimizu-Onda, Yuko; Tran, Dinh Nguyen; Pham, Ngan Thi Kim; Takanashi, Sayaka; Dey, Shuvra Kanti; Okitsu, Shoko; Yamazaki, Wataru; Mizuguchi, Masashi; Hayakawa, Satoshi

    2014-01-01

    We detected Campylobacter spp. in 5% (20/380) of diarrheal stool samples collected at an outpatient clinic in Kyoto using a commercial loop-mediated isothermal amplification (LAMP) kit with a fluorescent detection reagent after DNA extraction. The sensitivity and specificity were 100% in comparison with those of semi-nested PCR for the differentiation of Campylobacter jejuni and Campylobacter coli. Fourteen of the 20 samples were already determined as C. jejuni by the culture method. All 20 samples were also positive for C. jejuni by the PCR method. Among the 58 cultured samples, the sensitivity of the culture method against the LAMP method was 93.3% (14/15) and the specificity was 100% (43/43). The detection rate of Campylobacter spp. from the heated supernatants by the LAMP method was lower than that from the supernatant after DNA extraction. In total, 25% (5/20) of the Campylobacter-positive samples by the LAMP method were co-infected with norovirus (3/20), rotavirus (1/20), and human parechovirus (1/20), although no other bacterial co-infection was identified by the culture method. C. jejuni was mostly detected in children aged >5 years throughout the year. Based on these results, we concluded that care should be taken while diagnosing Campylobacter infection in children. Our newly modified LAMP method is a rapid, easy, and useful method for this diagnosis.

  9. The divergence and natural selection of autocatalytic primordial metabolic systems.

    Science.gov (United States)

    Marakushev, Sergey A; Belonogova, Ol'ga V

    2013-06-01

    The diversity of the central metabolism of modern organisms is caused by the existence of a few metabolic modules, combination of which produces multiple metabolic pathways. This paper analyzes biomimetically reconstructed coupled autocatalytic cycles as the basis of ancestral metabolic systems. The mechanism for natural selection and evolution in autocatalytic chemical systems may be affected by natural homeostatic parameters such as ambient chemical potentials, temperature, and pressure. Competition between separate parts of an autocatalytic network with positive-plus-negative feedback resulted in the formation of primordial autotrophic, mixotrophic, and heterotrophic metabolic systems. This work examined the last common ancestor of a set of coupled metabolic cycles in a population of protocells. Physical-chemical properties of these cycles determined the main principles of natural selection for the ancestral Bacteria and Archaea taxa.

  10. The Divergence and Natural Selection of Autocatalytic Primordial Metabolic Systems

    Science.gov (United States)

    Marakushev, Sergey A.; Belonogova, Ol'ga V.

    2013-06-01

    The diversity of the central metabolism of modern organisms is caused by the existence of a few metabolic modules, combination of which produces multiple metabolic pathways. This paper analyzes biomimetically reconstructed coupled autocatalytic cycles as the basis of ancestral metabolic systems. The mechanism for natural selection and evolution in autocatalytic chemical systems may be affected by natural homeostatic parameters such as ambient chemical potentials, temperature, and pressure. Competition between separate parts of an autocatalytic network with positive-plus-negative feedback resulted in the formation of primordial autotrophic, mixotrophic, and heterotrophic metabolic systems. This work examined the last common ancestor of a set of coupled metabolic cycles in a population of protocells. Physical-chemical properties of these cycles determined the main principles of natural selection for the ancestral Bacteria and Archaea taxa.

  11. Detection of influenza viruses by coupling multiplex reverse-transcription loop-mediated isothermal amplification with cascade invasive reaction using nanoparticles as a sensor

    Directory of Open Access Journals (Sweden)

    Ge Y

    2017-04-01

    Full Text Available Yiyue Ge,1 Qiang Zhou,2 Kangchen Zhao,1 Ying Chi,1 Bin Liu,3 Xiaoyan Min,4 Zhiyang Shi,1 Bingjie Zou,2 Lunbiao Cui1 1Institute of Pathogenic Microbiology, Key Laboratories of Enteric Pathogenic Microbiology (Ministry of Health, Jiangsu Provincial Center for Disease Control and Prevention, 2Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, 3Department of Biomedical Engineering, Nanjing Medical University, 4Department of Geriatrics, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of China Abstract: Influenza virus infections represent a worldwide public health and economic problem due to the significant morbidity and mortality caused by seasonal epidemics and pandemics. Sensitive and convenient methodologies for detection of influenza viruses are essential for further disease control. Loop-mediated isothermal amplification (LAMP is the most commonly used method of nucleic acid isothermal amplification. However, with regard to multiplex LAMP, differentiating the ladder-like LAMP products derived from multiple targets is still challenging today. The requirement of specialized instruments has further hindered the on-site application of multiplex LAMP. We have developed an integrated assay coupling multiplex reverse transcription LAMP with cascade invasive reaction using nanoparticles (mRT-LAMP-CIRN as a sensor for the detection of three subtypes of influenza viruses: A/H1N1pdm09, A/H3 and influenza B. The analytic sensitivities of the mRT-LAMP-CIRN assay were 101 copies of RNA for both A/H1N1pdm09 and A/H3, and 102 copies of RNA for influenza B. This assay demonstrated highly specific detection of target viruses and could differentiate them from other genetically or clinically related viruses. Clinical specimen analysis showed the mRT-LAMP-CIRN assay had an overall sensitivity and specificity of 98.3% and 100%, respectively. In summary, the mRT-LAMP-CIRN assay is

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

    Science.gov (United States)

    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

  13. Analytical Performance of a Loop-Mediated Isothermal Amplification Assay forLeishmaniaDNA Detection in Sandflies and Direct Smears of Patients with Cutaneous Leishmaniasis.

    Science.gov (United States)

    León, Cielo M; Muñoz, Marina; Tabares, Juan H; Hernandez, Carolina; Florez, Carolina; Ayala, Martha S; Ramírez, Juan David

    2018-03-12

    Loop-mediated isothermal amplification (LAMP) is ideal for the detection of Leishmania DNA as it is a quick and easy-to-perform test that does not require complex or sophisticated equipment or infrastructure. However, the application of this technique in the detection of Leishmania DNA has not been comprehensively analyzed to date (analytical validation). Our objective was to evaluate the sensitivity and analytical specificity (anticipated reportable range [ARR], the limit of detection [LoD], and accuracy) of LAMP targeting the 18S rRNA gene in the diagnosis of six New World Leishmania species. We then applied the validated LAMP assay across 50 samples of sandflies and 50 direct smears from a recent outbreak of cutaneous leishmaniasis in Colombia to determine its diagnostic performance. The LAMP assay exclusively amplified the DNA of Leishmania spp., and an ARR of between 1 × 10 4 and 1 × 10 -2 equivalent parasites/mL was determined. An LoD of 1 × 10 -2 equivalent parasites/mL was established and there was no statistically significant variation in terms of accuracy. Finally, a sensitivity of 100% in direct smears and sandflies samples was calculated and a specificity of 90.9% for direct smears using microscopy as reference and 96.8% for sandflies using real-time polymerase chain reaction as reference were determined. To our knowledge, this is the first attempt to analytically validate a LAMP test to detect Leishmania DNA, which showed good diagnostic potential from sandflies and direct smear samples.

  14. Development of loop-mediated isothermal amplification-based diagnostic assays for detection of Pasteurella multocida and hemorrhagic septicemia-associated P multocida serotype B:2.

    Science.gov (United States)

    Moustafa, Ahmed M; Bennett, Mark D

    2017-02-01

    OBJECTIVE To develop 2 rapid loop-mediated isothermal amplification (LAMP) assays for detection of Pasteurella multocida DNA (Pm-LAMP assay) and P multocida DNA from strains associated with hemorrhagic septicemia (HS) in cattle and buffalo (HS-LAMP assay). SAMPLE Solutions containing 16 P multocida strains and 9 other bacterial species at various concentrations. PROCEDURES Optimal conditions were determined for running the Pm-LAMP and HS-LAMP assays. The assays were then used to detect DNA of the test organisms. Results of LAMP assays were validated against conventional PCR assays designed for specific detection of P multocida and the B:2 serotype of HS-associated strains. RESULTS Following incubation of sample reaction mixtures for 27 minutes, specificity and sensitivity of the HS-LAMP assay at template DNA amounts as low as 5 pg were 93% and 97%, respectively. When duplicates of each sample were incubated for 28 minutes (a positive result defined as positive results for both reactions of a given sample), specificity and sensitivity of the HS-LAMP assay in the same conditions increased to 100%. The best specificity and sensitivity of Pm-LAMP single (93% and 91%) and duplicate (97% and 98%) reactions at template DNA amounts as low as 10 pg were achieved at 33 and 34 minutes, respectively. CONCLUSIONS AND CLINICAL RELEVANCE These preliminary findings suggested the developed HS-LAMP assay had high sensitivity and specificity for detection of HS-associated P multocida. Additional research is needed to determine the accuracy of the assay for use on clinical specimens obtained in HS-endemic countries such as Pakistan and Thailand.

  15. Field deployment of loop-mediated isothermal amplification for centralized mass-screening of asymptomatic malaria in Zanzibar: a pre-elimination setting.

    Science.gov (United States)

    Morris, Ulrika; Khamis, Mwinyi; Aydin-Schmidt, Berit; Abass, Ali K; Msellem, Mwinyi I; Nassor, Majda H; González, Iveth J; Mårtensson, Andreas; Ali, Abdullah S; Björkman, Anders; Cook, Jackie

    2015-05-17

    Molecular tools for detection of low-density asymptomatic Plasmodium infections are needed in malaria elimination efforts. This study reports results from the hitherto largest implementation of loop-mediated isothermal amplification (LAMP) for centralized mass screening of asymptomatic malaria in Zanzibar. Healthy individuals present and willing to participate in randomly selected households in 60 villages throughout Zanzibar were screened for malaria by rapid diagnostic tests (RDT). In 50% of the study households, participants were asked to provide 60 μL of finger-prick blood for additional LAMP screening. LAMP was conducted in two centralized laboratories in Zanzibar, by trained technicians with limited or no previous experience of molecular methods. The LAMP assay was performed with Loopamp(TM) MALARIA Pan/Pf Detection Kit (Eiken Chemical Company, Japan). Samples positive for Plasmodium genus (Pan)-LAMP were re-tested using Plasmodium falciparum-specific LAMP kits. Paired RDT and LAMP samples were available from 3983 individuals. The prevalence of asymptomatic malaria was 0.5% (CI 95% 0.1-0.8) and 1.6% (CI 95% 1.1-2.2) by RDT and Pan-LAMP, respectively. LAMP detected 3.4 (CI 95% 2.2-5.2) times more Plasmodium positive samples than RDT. DNA contamination was experienced, but solved by repetitive decontamination of all equipment and reagents. LAMP is a simple and sensitive molecular tool, and has potential in active surveillance and mass-screening programmes for detection of low-density asymptomatic malaria in pre-elimination settings. However, in order to deploy LAMP more effectively in field settings, protocols may need to be adapted for processing larger numbers of samples. A higher throughput, affordable closed system would be ideal to avoid contamination.

  16. Rapid detection of Salmonella in food and feed by coupling loop-mediated isothermal amplification with bioluminescent assay in real-time.

    Science.gov (United States)

    Yang, Qianru; Domesle, Kelly J; Wang, Fei; Ge, Beilei

    2016-06-17

    Salmonella is among the most significant pathogens causing food and feed safety concerns. This study examined the rapid detection of Salmonella in various types of food and feed samples by coupling loop-mediated isothermal amplification (LAMP) with a novel reporter, bioluminescent assay in real-time (BART). Performance of the LAMP-BART assay was compared to a conventional LAMP and the commercially available 3M Molecular Detection Assay (MDA) Salmonella. The LAMP-BART assay was 100 % specific among 178 strains (151 Salmonella and 27 non-Salmonella) tested. The detection limits were 36 cells per reaction in pure culture and 10(4) to 10(6) CFU per 25 g in spiked food and feed samples without enrichment, which were comparable to those of the conventional LAMP and 3M MDA Salmonella but 5-10 min faster. Ground turkey showed a strong inhibition on 3M MDA Salmonella, requiring at least 10(8) CFU per 25 g for detection. The correlation between Salmonella cell numbers and LAMP-BART signals was high (R (2) = 0.941-0.962), suggesting good quantification capability. After 24 h enrichment, all three assays accurately detected 1 to 3 CFU per 25 g of Salmonella among five types of food (cantaloupe, ground beef, ground turkey, shell eggs, and tomato) and three types of feed (cattle feed, chicken feed, and dry dog food) examined. However, 10(1) CFU per 25 g was required for cattle feed when tested by 3M MDA Salmonella. The Salmonella LAMP-BART assay was rapid, specific, sensitive, quantitative, and robust. Upon further validation, it may become a valuable tool for routine screening of Salmonella in various types of food and feed samples.

  17. Rapid and sensitive detection of Bartonella bacilliformis in experimentally infected sand flies by loop-mediated isothermal amplification (LAMP of the Pap31 gene.

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    Nasikarn Angkasekwinai

    2014-12-01

    Full Text Available 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.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.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.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Diagnostic accuracy of loop-mediated isothermal amplification (LAMP for screening patients with imported malaria in a non-endemic setting

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    Ponce Camille

    2017-01-01

    Full Text Available Background: Sensitive and easy-to-perform methods for the diagnosis of malaria are not yet available. Improving the limit of detection and following the requirements for certification are issues to be addressed in both endemic and non-endemic settings. The aim of this study was to test whether loop-mediated isothermal amplification of DNA (LAMP may be an alternative to microscopy or real-time PCR for the screening of imported malaria cases in non-endemic area. Results: 310 blood samples associated with 829 suspected cases of imported malaria were tested during a one year period. Microscopy (thin and thick stained blood slides, reference standard was used for the diagnosis. Real-time PCR was used as a standard of truth, and LAMP (Meridian Malaria Plus was used as an index test in a prospective study conducted following the Standards for Reporting Diagnosis Accuracy Studies. In the 83 positive samples, species identification was P. falciparum (n = 66, P. ovale (n = 9, P. vivax (n = 3 P. malariae (n = 3 and 2 co-infections with P. falciparum + P.malariae. Using LAMP methods, 93 samples gave positive results, including 4 false-positives. Sensitivity, specificity, positive predictive value and negative predictive value for LAMP tests were 100%, 98.13%, 95.51%, and 100% compared to PCR. Conclusion: High negative predictive value, and limit of detection suggest that LAMP can be used for screening of imported malaria cases in non-endemic countries when expert microscopists are not immediately available. However, the rare occurrence of non-valid results and the need for species identification and quantification of positive samples preclude the use of LAMP as a single reference method.

  20. A loop-mediated isothermal amplification method for rapid direct detection and differentiation of nonpathogenic and verocytotoxigenic Escherichia coli in beef and bovine faeces.

    Science.gov (United States)

    Stratakos, A Ch; Linton, M; Millington, S; Grant, I R

    2017-03-01

    To develop a multiplex loop-mediated isothermal amplification (LAMP) assay capable of quantifying Escherichia coli and differentiating verocytotoxigenic E. coli (VTEC). Primer sets were selected to amplify the phoA gene (all E. coli strains) and stx1 and/or stx2 genes (VTEC strains only). LAMP calibration curves demonstrated good quantification capability compared with conventional culture. The limits of detection 50% (LOD 50 ) of the multiplex LAMP assay were 2·8 (95% CI 2·4-3·3), 3·2 (95% CI 2·5-3·9) and 2·8-3·2 (95% CI 2·1-3·5) log CFU per g for the phoA, stx1 and stx2 genes, respectively. When validated by testing retail beef and bovine faeces samples, good correlation between E. coli counts indicated by the LAMP assay and culture was observed; however, false-negative LAMP assay results were obtained for 12·5-14·7% of samples. A rapid, multiplex LAMP assay for direct quantification of E. coli and specific detection of VTEC in beef and faeces was successfully developed. Further optimisation of the assay would be needed to improve detection sensitivity. The multiplex LAMP assay represents a rapid alternative to culture for monitoring E. coli levels on beef for hygiene monitoring purposes, and, potentially, a method for detection of VTEC in beef and faeces. © 2016 The Society for Applied Microbiology.

  1. Development of real-time PCR and loop-mediated isothermal amplification (LAMP assays for the differential detection of digital dermatitis associated treponemes.

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    Kelly Anklam

    Full Text Available Bovine digital dermatitis (DD is a severe infectious cause of lameness in cattle worldwide, with important economic and welfare consequences. There are three treponeme phylogroups (T. pedis, T. phagedenis, and T. medium that are implicated in playing an important causative role in DD. This study was conducted to develop real-time PCR and loop-mediated isothermal amplification (LAMP assays for the detection and differentiation of the three treponeme phylogroups associated with DD. The real-time PCR treponeme phylogroup assays targeted the 16S-23S rDNA intergenic space (ITS for T. pedis and T. phagedenis, and the flagellin gene (flaB2 for T. medium. The 3 treponeme phylogroup LAMP assays targeted the flagellin gene (flaB2 and the 16S rRNA was targeted for the Treponeme ssp. LAMP assay. The real-time PCR and LAMP assays correctly detected the target sequence of all control strains examined, and no cross-reactions were observed, representing 100% specificity. The limit of detection for each of the three treponeme phylogroup real-time PCR and LAMP assays was ≤ 70 fg/μl. The detection limit for the Treponema spp. LAMP assay ranged from 7-690 fg/μl depending on phylogroup. Treponemes were isolated from 40 DD lesion biopsies using an immunomagnetic separation culture method. The treponeme isolation samples were then subjected to the real-time PCR and LAMP assays for analysis. The treponeme phylogroup real-time PCR and LAMP assay results had 100% agreement, matching on all isolation samples. These results indicate that the developed assays are a sensitive and specific test for the detection and differentiation of the three main treponeme phylogroups implicated in DD.

  2. The development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of Babesia spp. infective to sheep and goats in China.

    Science.gov (United States)

    Guan, Guiquan; Chauvin, Alain; Luo, Jianxun; Inoue, Noboru; Moreau, Emmanuelle; Liu, Zhijie; Gao, Jinliang; Thekisoe, Oriel M M; Ma, Miling; Liu, Aihong; Dang, Zhisheng; Liu, Junlong; Ren, Qiaoyun; Jin, Yurong; Sugimoto, Chihiro; Yin, Hong

    2008-09-01

    The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies with high sensitivity, efficiency, and rapidity, deoxyribonucleic acid (DNA) under isothermal condition in simple incubators. Two primer sets for the LAMP method were designed using the nucleotide sequences of 18S rRNA gene of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 isolated in China. The primers were used to detect parasite DNA extracted from infected blood and purified parasites by LAMP. The specific ladder bands were amplified from the autologous genomic DNA of two Babesia species, respectively, and did not cross-react with the genomic DNA of Theileria sp. China 1, Theileria sp. China 2, B. bovis, Theileria sp. (Japan) and sheep. The LAMP was sensitive enough to detect 0.02 pg and 0.2 pg genomic DNA of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005, respectively, from 10-fold serially diluted samples corresponding to the amount of DNA present in 50 microl of 0.000002% and 0.00002% parasitemic erythrocytes. Furthermore, DNA extracted from blood of intact (non-splenectomized) sheep experimentally infected with Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 was amplified by the LAMP from week 1 to 9 and week 2 and 3 post-infection, respectively, demonstrating the high sensitivity of these primers. Of 365 samples collected from Gansu province, 14.3% (52/365) were positively detected by the LAMP. Of 145 samples collected on filter papers (Whatman) from the grazing sheep in Xinjiang province, 3.5% (5/145) were positive. These results show that the LAMP could be an alternative diagnostic tool for the detection of babesial infection in sheep and goats.

  3. Loop-mediated isothermal DNA amplification for asymptomatic malaria detection in challenging field settings: Technical performance and pilot implementation in the Peruvian Amazon.

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    Elisa Serra-Casas

    Full Text Available Loop-mediated isothermal DNA amplification (LAMP methodology offers an opportunity for point-of-care (POC molecular detection of asymptomatic malaria infections. However, there is still little evidence on the feasibility of implementing this technique for population screenings in isolated field settings.Overall, we recruited 1167 individuals from terrestrial ('road' and hydric ('riverine' communities of the Peruvian Amazon for a cross-sectional survey to detect asymptomatic malaria infections. The technical performance of LAMP was evaluated in a subgroup of 503 samples, using real-time Polymerase Chain Reaction (qPCR as reference standard. The operational feasibility of introducing LAMP testing in the mobile screening campaigns was assessed based on field-suitability parameters, along with a pilot POC-LAMP assay in a riverine community without laboratory infrastructure.LAMP had a sensitivity of 91.8% (87.7-94.9 and specificity of 91.9% (87.8-95.0, and the overall accuracy was significantly better among samples collected during road screenings than riverine communities (p≤0.004. LAMP-based diagnostic strategy was successfully implemented within the field-team logistics and the POC-LAMP pilot in the riverine community allowed for a reduction in the turnaround time for case management, from 12-24 hours to less than 5 hours. Specimens with haemolytic appearance were regularly observed in riverine screenings and could help explaining the hindered performance/interpretation of the LAMP reaction in these communities.LAMP-based molecular malaria diagnosis can be deployed outside of reference laboratories, providing similar performance as qPCR. However, scale-up in remote field settings such as riverine communities needs to consider a number of logistical challenges (e.g. environmental conditions, labour-intensiveness in large population screenings that can influence its optimal implementation.

  4. Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by loop-mediated isothermal amplification (LAMP.

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    Dennis J Grab

    2011-08-01

    Full Text Available The loop-mediated isothermal amplification (LAMP assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per µL or 10(3 per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay.For proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite. The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 µL of CSF as the source of template was at best 10(3 parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards.This simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the developing world.

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

    Science.gov (United States)

    Lisulo, Malimba; Sugimoto, Chihiro; Kajino, Kiichi; Hayashida, Kyouko; Mudenda, Macarthy; Moonga, Ladslav; Ndebe, Joseph; Nzala, Selestine; Namangala, Boniface

    2014-01-10

    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. 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. 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. 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

  6. Using Detergent to Enhance Detection Sensitivity of African Trypanosomes in Human CSF and Blood by Loop-Mediated Isothermal Amplification (LAMP)

    Science.gov (United States)

    Grab, Dennis J.; Nikolskaia, Olga V.; Inoue, Noboru; Thekisoe, Oriel M. M.; Morrison, Liam J.; Gibson, Wendy; Dumler, J. Stephen

    2011-01-01

    Background The loop-mediated isothermal amplification (LAMP) assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per µL or 103 per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay. Methodology/Principal Findings For proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite). The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF) or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 µL of CSF as the source of template was at best 103 parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards. Conclusions/Significance This simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    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. - Highlights: • We develop multiplex LAMP label-based lateral flow dipstick biosensor for detection of pathogenic Leptospira. • We design primers for multiplex LAMP targeting the conserved LipL32 gene of pathogenic Leptospira and LAMP internal

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

    Energy Technology Data Exchange (ETDEWEB)

    Nurul Najian, A.B.; Engku Nur Syafirah, E.A.R.; Ismail, Nabilah [Department of Medical Microbiology & Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Mohamed, Maizan [Faculty of Veterinary Medicine, Universiti Malaysia Kelantan, City Campus, Pengkalan Chepa, Locked Bag 36, 16100 Kota Bharu, Kelantan (Malaysia); Yean, Chan Yean, E-mail: yeancyn@yahoo.com [Department of Medical Microbiology & Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia)

    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{sup −1} genomic equivalent ml{sup −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. - Highlights: • We develop multiplex LAMP label-based lateral flow dipstick biosensor for detection of pathogenic Leptospira. • We design primers for multiplex LAMP targeting the conserved LipL32 gene of pathogenic Leptospira and LAMP

  9. Simple and rapid method for the detection of Filobasidiella neoformans in a probiotic dairy product by using loop-mediated isothermal amplification.

    Science.gov (United States)

    Ishikawa, Hiroshi; Kasahara, Kohei; Sato, Sumie; Shimakawa, Yasuhisa; Watanabe, Koichi

    2014-05-16

    Yeast contamination is a serious problem in the food industry and a major cause of food spoilage. Several yeasts, such as Filobasidiella neoformans, which cause cryptococcosis in humans, are also opportunistic pathogens, so a simple and rapid method for monitoring yeast contamination in food is essential. Here, we developed a simple and rapid method that utilizes loop-mediated isothermal amplification (LAMP) for the detection of F. neoformans. A set of five specific LAMP primers was designed that targeted the 5.8S-26S rDNA internal transcribed spacer 2 region of F. neoformans, and the primer set's specificity was confirmed. In a pure culture of F. neoformans, the LAMP assay had a lower sensitivity threshold of 10(2)cells/mL at a runtime of 60min. In a probiotic dairy product artificially contaminated with F. neoformans, the LAMP assay also had a lower sensitivity threshold of 10(2)cells/mL, which was comparable to the sensitivity of a quantitative PCR (qPCR) assay. We also developed a simple two-step method for the extraction of DNA from a probiotic dairy product that can be performed within 15min. This method involves initial protease treatment of the test sample at 45°C for 3min followed by boiling at 100°C for 5min under alkaline conditions. In a probiotic dairy product artificially contaminated with F. neoformans, analysis by means of our novel DNA extraction method followed by LAMP with our specific primer set had a lower sensitivity threshold of 10(3)cells/mL at a runtime of 60min. In contrast, use of our novel method of DNA extraction followed by qPCR assay had a lower sensitivity threshold of only 10(5)cells/mL at a runtime of 3 to 4h. Therefore, unlike the PCR assay, our LAMP assay can be used to quickly evaluate yeast contamination and is sensitive even for crude samples containing bacteria or background impurities. Our study provides a powerful tool for the primary screening of large numbers of food samples for yeast contamination. Copyright © 2014

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

    Science.gov (United States)

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

    2014-12-01

    Extended-spectrum β-lactamases (ESBLs) produced by Enterobacteriaceae are one of the resistance mechanisms to most β-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. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

  11. Development of a simple and rapid reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for sensitive detection of Citrus tristeza virus.

    Science.gov (United States)

    Warghane, Ashish; Misra, Pragati; Bhose, Sumit; Biswas, Kajal Kumar; Sharma, Ashwani Kumar; Reddy, M Krishna; Ghosh, Dilip Kumar

    2017-12-01

    Tristeza is a devastating disease of citrus and reported to be present in almost all countries where it is cultivated as a commercial crop. The etiological agent of this disease is Citrus tristeza virus (CTV), a member of the genus Closterovirus with in the family Closteroviridae. The pathogen is restricted to the phloem tissue of the infected citrus plant and has a monopartite ss (+) RNA genome of ∼20kb size. Till date, there is no effective control measure available for this virus. Management of tristeza depends on destruction of CTV infected field plants, production of virus-free planting material for new orchard establishment and controlling viruliferous aphid vectors responsible for field spread of the pathogen. Availability of rapid diagnostic assay is essential for rapid and efficient detection of the pathogen. In the present investigation, RT-LAMP (reverse transcription-loop mediated isothermal amplification), a highly sensitive, robust and low cost assay has been developed for rapid detection of CTV in infected citrus plant samples. Based on conserved nucleotide sequences available in GenBank and specific to p25 gene (major coat protein gene) of predominant CTV isolates of India, four primer sets (CTV-F3, CTV-B3, CTV-FIP and CTV-BIP) ware designed and custom synthesized. The amplified LAMP products obtained after maintaining isothermal condition of 65°C for 60min duration could be visible easily with necked eyes in presence of SYBR Green I (100X). Subsequently, LAMP products were verified by electrophoresis run in 1.5% agarose gel. The RT-LAMP results obtained with known CTV isolates maintained in screen house of CCRI, Nagpur were validated using field samples and thereafter it was further confirmed by conventional RT-PCR (reveres transcription-polymerase chain reaction) assay. The sensitivity of CTV-RT-LAMP protocol standardized in the present study was 100 times more than conventional one step RT-PCR assay. It also has maximum detection limit up to 0

  12. Unbounded autocatalytic growth on diffusive substrate: The extinction transition

    International Nuclear Information System (INIS)

    Moalem, Sasi; Shnerb, Nadav M.

    2007-01-01

    The effect of diffusively correlated spatial fluctuations on the proliferation-extinction transition of autocatalytic agents is investigated numerically. Reactants adaptation to spatio-temporal active regions is shown to lead to proliferation even if the mean field rate equations predict extinction, in agreement with previous theoretical predictions. While in the proliferation phase the system admits a typical time scale that dictates the exponential growth, the extinction times distribution obeys a power law at the parameter region considered

  13. Autocatalytic, bistable, oscillatory networks of biologically relevant organic reactions

    Science.gov (United States)

    Semenov, Sergey N.; Kraft, Lewis J.; Ainla, Alar; Zhao, Mengxia; Baghbanzadeh, Mostafa; Campbell, Victoria E.; Kang, Kyungtae; Fox, Jerome M.; Whitesides, George M.

    2016-09-01

    Networks of organic chemical reactions are important in life and probably played a central part in its origin. Network dynamics regulate cell division, circadian rhythms, nerve impulses and chemotaxis, and guide the development of organisms. Although out-of-equilibrium networks of chemical reactions have the potential to display emergent network dynamics such as spontaneous pattern formation, bistability and periodic oscillations, the principles that enable networks of organic reactions to develop complex behaviours are incompletely understood. Here we describe a network of biologically relevant organic reactions (amide formation, thiolate-thioester exchange, thiolate-disulfide interchange and conjugate addition) that displays bistability and oscillations in the concentrations of organic thiols and amides. Oscillations arise from the interaction between three subcomponents of the network: an autocatalytic cycle that generates thiols and amides from thioesters and dialkyl disulfides; a trigger that controls autocatalytic growth; and inhibitory processes that remove activating thiol species that are produced during the autocatalytic cycle. In contrast to previous studies that have demonstrated oscillations and bistability using highly evolved biomolecules (enzymes and DNA) or inorganic molecules of questionable biochemical relevance (for example, those used in Belousov-Zhabotinskii-type reactions), the organic molecules we use are relevant to metabolism and similar to those that might have existed on the early Earth. By using small organic molecules to build a network of organic reactions with autocatalytic, bistable and oscillatory behaviour, we identify principles that explain the ways in which dynamic networks relevant to life could have developed. Modifications of this network will clarify the influence of molecular structure on the dynamics of reaction networks, and may enable the design of biomimetic networks and of synthetic self-regulating and evolving

  14. Evolution of Autocatalytic Sets in Computational Models of Chemical Reaction Networks.

    Science.gov (United States)

    Hordijk, Wim

    2016-06-01

    Several computational models of chemical reaction networks have been presented in the literature in the past, showing the appearance and (potential) evolution of autocatalytic sets. However, the notion of autocatalytic sets has been defined differently in different modeling contexts, each one having some shortcoming or limitation. Here, we review four such models and definitions, and then formally describe and analyze them in the context of a mathematical framework for studying autocatalytic sets known as RAF theory. The main results are that: (1) RAF theory can capture the various previous definitions of autocatalytic sets and is therefore more complete and general, (2) the formal framework can be used to efficiently detect and analyze autocatalytic sets in all of these different computational models, (3) autocatalytic (RAF) sets are indeed likely to appear and evolve in such models, and (4) this could have important implications for a possible metabolism-first scenario for the origin of life.

  15. A novel method for the purification of DNA by capturing nucleic acid and magnesium complexes on non-woven fabric filters under alkaline conditions for the gene diagnosis of tuberculosis by loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Fukasawa, Tadashi; Oda, Naozumi; Wada, Yasunao; Tamaru, Aki; Fukushima, Yukari; Nakajima, Chie; Suzuki, Yasuhiko

    2010-07-01

    A novel method for purifying DNA from clinical samples based on the complex formation of DNA and magnesium ion (Mg(2+)) was developed for the detection of Mycobacterium tuberculosis. The formation of a DNA-Mg(2+) complex under alkaline conditions was observed by analyzing electrophoretic mobility reduction of DNA on agarose gel. The DNA-Mg(2+) complex increases the efficacy of DNA recovery from the sample solution on polyethylene terephthalate non-woven fabric (PNWF) filters. Among the various divalent metal cations, only Mg(2+) was associated with this effect. The applicability of DNA recovered on the PNWF filter was examined for the gene amplification method; loop-mediated isothermal amplification (LAMP). DNA on the PNWF filter could be used for the amplification of specific DNA fragments without elution from the filter. Using this method, DNA was directly purified from M. tuberculosis spiked sputum and examined by LAMP assay, showing a high sensitivity in comparison to the commercially available DNA extraction kit. These results indicated that the method developed in this study is useful for rapid gene diagnosis of tuberculosis.

  16. Highly sensitive detection of gluten-containing cereals in food samples by real-time Loop-mediated isothermal AMPlification (qLAMP) and real-time polymerase chain reaction (qPCR).

    Science.gov (United States)

    Garrido-Maestu, Alejandro; Azinheiro, Sarah; Fuciños, Pablo; Carvalho, Joana; Prado, Marta

    2018-04-25

    The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. The Sugar Model: Autocatalytic Activity of the Triose-Ammonia Reaction

    Science.gov (United States)

    Weber, Arthur L.

    2006-01-01

    Reaction of triose sugars with ammonia under anaerobic conditions yielded autocatalytic products. The autocatalytic behavior of the products was examined by measuring the effect of the crude triose-ammonia reaction product on the kinetics of a second identical triose-ammonia reaction. The reaction product showed autocatalytic activity by increasing both the rate of disappearance of triose and the rate formation of pyruvaldehyde, the product of triose dehydration. This synthetic process is considered a reasonable model of origin-of-life chemistry because it uses plausible prebiotic substrates, and resembles modern biosynthesis by employing the energized carbon groups of sugars to drive the synthesis of autocatalytic molecules.

  18. On-chip electrical detection of parallel loop-mediated isothermal amplification with DG-BioFETs for the detection of foodborne bacterial pathogens

    Science.gov (United States)

    The use of field effect transistors (FETs) as the transduction element for the detection of DNA amplification reactions will enable portable and inexpensive nucleic acid analysis. Transistors used as biological sensors,or BioFETs, minimize the cost and size of detection platforms by leveraging fabri...

  19. An autocatalytic kinetic model for describing microbial growth during fermentation.

    Science.gov (United States)

    Ibarz, Albert; Augusto, Pedro E D

    2015-01-01

    The mathematical modelling of the behaviour of microbial growth is widely desired in order to control, predict and design food and bioproduct processing, stability and safety. This work develops and proposes a new semi-empirical mathematical model, based on an autocatalytic kinetic, to describe the microbial growth through its biomass concentration. The proposed model was successfully validated using 15 microbial growth patterns, covering the three most important types of microorganisms in food and biotechnological processing (bacteria, yeasts and moulds). Its main advantages and limitations are discussed, as well as the interpretation of its parameters. It is shown that the new model can be used to describe the behaviour of microbial growth.

  20. Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples.

    OpenAIRE

    Susana A Besuschio; Mónica Llano Murcia; Alejandro F Benatar; Severine Monnerat; Israel Cruz; Albert Picado; María de Los Ángeles Curto; Yutaka Kubota; Diana P Wehrendt; Paula Pavia; Yasuyoshi Mori; Concepción Puerta; Joseph M Ndung'u; Alejandro G Schijman

    2017-01-01

    This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65?C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA ...

  1. Rapid detection assay for the invasive vase tunicate, Ciona intestinalis, using loop-mediated isothermal amplification combined with lateral flow dipstick

    Directory of Open Access Journals (Sweden)

    Ahmed Siah

    2013-01-01

    Full Text Available Invasive tunicate species threaten the shellfish aquaculture industry not only in Prince Edward Island (PEI but also nationally andworldwide. Rapid screening tools for water samples are crucial for the efficient management of aquatic invasive species. This project aims todevelop a rapid detection assay capable of identifying larvae of the vase tunicate, Ciona intestinalis, in seawater. In this study, a loopmediatedisothermal amplification (LAMP method has been developed to detect the 18S ribosomal DNA extracted from C. intestinalis. Thisassay was performed in three steps: 1 a DNA extraction step using a direct lysis buffer, 2 an amplification step using a heating block, and 3a detection step using a lateral flow dipstick. The sensitivity of the assay was estimated at one larva spiked in 100 L of seawater and theturnaround time of the assay was assessed at 80 min including the lysis step. Given the advantages of this assay such as rapid amplification,ease of use and detection, it could be implemented for monitoring bays and estuaries. In the future, rapid diagnostic assays will constitute anew generation of diagnostic platforms enabling the early detection of non-indigenous invasive species.

  2. Competitive autocatalytic reactions in chaotic flows with diffusion: Prediction using finite-time Lyapunov exponents

    Science.gov (United States)

    Schlick, Conor P.; Umbanhowar, Paul B.; Ottino, Julio M.; Lueptow, Richard M.

    2014-03-01

    We investigate chaotic advection and diffusion in autocatalytic reactions for time-periodic sine flow computationally using a mapping method with operator splitting. We specifically consider three different autocatalytic reaction schemes: a single autocatalytic reaction, competitive autocatalytic reactions, which can provide insight into problems of chiral symmetry breaking and homochirality, and competitive autocatalytic reactions with recycling. In competitive autocatalytic reactions, species B and C both undergo an autocatalytic reaction with species A such that A +B→2B and A +C→2C. Small amounts of initially spatially localized B and C and a large amount of spatially homogeneous A are advected by the velocity field, diffuse, and react until A is completely consumed and only B and C remain. We find that local finite-time Lyapunov exponents (FTLEs) can accurately predict the final average concentrations of B and C after the reaction completes. The species that starts in the region with the larger FTLE has, with high probability, the larger average concentration at the end of the reaction. If B and C start in regions with similar FTLEs, their average concentrations at the end of the reaction will also be similar. When a recycling reaction is added, the system evolves towards a single species state, with the FTLE often being useful in predicting which species fills the entire domain and which is depleted. The FTLE approach is also demonstrated for competitive autocatalytic reactions in journal bearing flow, an experimentally realizable flow that generates chaotic dynamics.

  3. Competitive autocatalytic reactions in chaotic flows with diffusion: Prediction using finite-time Lyapunov exponents

    Energy Technology Data Exchange (ETDEWEB)

    Schlick, Conor P., E-mail: conorschlick2015@u.northwestern.edu [Department of Engineering Sciences and Applied Mathematics, Northwestern University, Evanston, Illinois 60208 (United States); Umbanhowar, Paul B. [Department of Mechanical Engineering, Northwestern University, Evanston, Illinois 60208 (United States); Ottino, Julio M. [Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208 (United States); Department of Mechanical Engineering, Northwestern University, Evanston, Illinois 60208 (United States); The Northwestern Institute on Complex Systems (NICO), Northwestern University, Evanston, Illinois 60208 (United States); Lueptow, Richard M., E-mail: r-lueptow@northwestern.edu [Department of Mechanical Engineering, Northwestern University, Evanston, Illinois 60208 (United States); The Northwestern Institute on Complex Systems (NICO), Northwestern University, Evanston, Illinois 60208 (United States)

    2014-03-15

    We investigate chaotic advection and diffusion in autocatalytic reactions for time-periodic sine flow computationally using a mapping method with operator splitting. We specifically consider three different autocatalytic reaction schemes: a single autocatalytic reaction, competitive autocatalytic reactions, which can provide insight into problems of chiral symmetry breaking and homochirality, and competitive autocatalytic reactions with recycling. In competitive autocatalytic reactions, species B and C both undergo an autocatalytic reaction with species A such that A+B→2B and A+C→2C. Small amounts of initially spatially localized B and C and a large amount of spatially homogeneous A are advected by the velocity field, diffuse, and react until A is completely consumed and only B and C remain. We find that local finite-time Lyapunov exponents (FTLEs) can accurately predict the final average concentrations of B and C after the reaction completes. The species that starts in the region with the larger FTLE has, with high probability, the larger average concentration at the end of the reaction. If B and C start in regions with similar FTLEs, their average concentrations at the end of the reaction will also be similar. When a recycling reaction is added, the system evolves towards a single species state, with the FTLE often being useful in predicting which species fills the entire domain and which is depleted. The FTLE approach is also demonstrated for competitive autocatalytic reactions in journal bearing flow, an experimentally realizable flow that generates chaotic dynamics.

  4. Detection of restriction enzyme-digested target DNA by PCR amplification using a stem-loop primer: application to the detection of hypomethylated fetal DNA in maternal plasma.

    Science.gov (United States)

    Tong, Yu K; Chiu, Rossa W K; Leung, Tak Y; Ding, Chunming; Lau, Tze K; Leung, Tse N; Lo, Y M Dennis

    2007-11-01

    The discovery of cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis and monitoring. Among the fetal markers that have been described, methylation markers are sex and polymorphism independent. Methylation-sensitive restriction endonucleases are commonly used to digest hypomethylated DNA molecules, and the hypermethylated molecules remain intact for detection. The positive detection of the cleaved hypomethylated molecules would be useful for certain targets but has not been reported. The use of a stem-loop primer in microRNA detection has previously been described. In this study, DNA assays were designed and performed on maternal plasma, which contained the hypomethylated placental serpin peptidase inhibitor, clade B (ovalbumin), member 5 (SERPINB5; maspin) gene in an excess background of hypermethylated maternal SERPINB5. Detection of the enzyme-digested placenta-derived hypomethylated SERPINB5 molecules was achieved by performing stem-loop extension followed by real-time PCR on maternal plasma. The placental origin of the stem-loop-extended SERPINB5 molecules was confirmed by genotyping. From the real-time PCR results on maternal plasma, stem-loop-extended SERPINB5 promoter sequences were detectable in all 11 enzyme-digested predelivery maternal plasma samples. Postpartum clearance was demonstrated. In 9 cases in which the fetal and maternal SERPINB5 genotypes were distinguishable, the placental-specific genotypes were detected in all predelivery maternal plasma samples. Detection of restriction enzyme-digested hypomethylated placental DNA molecules in maternal plasma by the use of a stem-loop primer represents a novel approach in fetal epigenetic marker detection. The analytical approach may also be generally applicable to the detection of restriction enzyme-digested nucleic acid fragments.

  5. Development of a loop-mediated isothermal amplification method for detecting Streptococcus equi subsp. zooepidemicus and analysis of its use with three simple methods of extracting DNA from equine respiratory tract specimens.

    Science.gov (United States)

    Kinoshita, Yuta; Niwa, Hidekazu; Katayama, Yoshinari

    2014-09-01

    Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a dominant pathogenic bacterium in equine pneumonia. We developed a specific loop-mediated isothermal amplification (LAMP) method, which targets the gene encoding sorbitol-6-phosphate 2-dehydrogenase (sorD), for detecting S. zooepidemicus and examined the clinical efficacies of its use in combination with each of 3 DNA extraction methods easily used by veterinary practitioners, namely the Loopamp PURE DNA Extraction Kit, InstaGene Matrix and a conventional boiling method. The LAMP method plus the Loopamp PURE DNA Extraction Kit gave higher rates of positivity than the other combinations in both clinical and spiked samples containing clinically significant concentrations (>1 × 10(4) CFU/ml) of S. zooepidemicus.

  6. Homogenous, real-time duplex loop-mediated isothermal amplification using a single fluorophore-labeled primer and an intercalator dye: Its application to the simultaneous detection of Shiga toxin genes 1 and 2 in Shiga toxigenic Escherichia coli isolates.

    Science.gov (United States)

    Kouguchi, Yoshihiro; Fujiwara, Takako; Teramoto, Miki; Kuramoto, Mika

    2010-08-01

    We developed a completely homogeneous duplex loop-mediated isothermal amplification (LAMP) method. The present LAMP method employed a combination of a 6-carboxyfluorescein (FAM)-labeled primer (donor) for one target gene, a non-labeled primer for the other, and an intercalator ethidium bromide (EtBr) dye (acceptor) on the basis of fluorescence resonance energy transfer (FRET) between the FAM donor and EtBr acceptor. Measuring changes in fluorescence of FAM enabled the LAMP method to detect two different genes simultaneously. This method was used to detect Shiga toxin genes in Shiga toxigenic Escherichia coli isolates, demonstrating simultaneous detection of two different genes with rapidity and accuracy. Copyright 2010 Elsevier Ltd. All rights reserved.

  7. Assessing the performance of a Loop Mediated Isothermal Amplification (LAMP) assay for the detection and subtyping of high-risk suptypes of Human Papilloma Virus (HPV) for Oropharyngeal Squamous Cell Carcinoma (OPSCC) without DNA purification.

    Science.gov (United States)

    Rohatensky, Mitchell G; Livingstone, Devon M; Mintchev, Paul; Barnes, Heather K; Nakoneshny, Steven C; Demetrick, Douglas J; Dort, Joseph C; van Marle, Guido

    2018-02-08

    Oropharyngeal Squamous Cell Carcinoma (OPSCC) is increasing in incidence despite a decline in traditional risk factors. Human Papilloma Virus (HPV), specifically subtypes 16, 18, 31 and 35, has been implicated as the high-risk etiologic agent. HPV positive cancers have a significantly better prognosis than HPV negative cancers of comparable stage, and may benefit from different treatment regimens. Currently, HPV related carcinogenesis is established indirectly through Immunohistochemistry (IHC) staining for p16, a tumour suppressor gene, or polymerase chain reaction (PCR) that directly tests for HPV DNA in biopsied tissue. Loop mediated isothermal amplification (LAMP) is more accurate than IHC, more rapid than PCR and is significantly less costly. In previous work we showed that a subtype specific HPV LAMP assay performed similar to PCR on purified DNA. In this study we examined the performance of this LAMP assay without DNA purification. We used LAMP assays using established primers for HPV 16 and 18, and new primers for HPV 31 and 35. LAMP reaction conditions were tested on serial dilutions of plasmid HPV DNA to confirm minimum viral copy number detection thresholds. LAMP was then performed directly on different human cell line samples without DNA purification. Our LAMP assays could detect 10 5 , 10 3 , 10 4 , and 10 5 copies of plasmid DNA for HPV 16, 18, 31, and 35, respectively. All primer sets were subtype specific, with no cross-amplification. Our LAMP assays also reliably amplified subtype specific HPV DNA from samples without requiring DNA isolation and purification. The high risk OPSCC HPV subtype specific LAMP primer sets demonstrated, excellent clinically relevant, minimum copy number detection thresholds with an easy readout system. Amplification directly from samples without purification illustrated the robust nature of the assay, and the primers used. This lends further support HPV type specific LAMP assays, and these specific primer sets and assays

  8. Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

    Directory of Open Access Journals (Sweden)

    Xin Zhang

    Full Text Available Fusarium oxysporum f. sp. cubense (Foc, the causal agent of Fusarium wilt (Panama disease, is one of the most devastating diseases of banana (Musa spp.. The Foc tropical race 4 (TR4 is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3 spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05. Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.

  9. Comparison of loop-mediated isothermal amplification (LAMP) and nested-PCR assay targeting the RE and B1 gene for detection of Toxoplasma gondii in blood samples of children with leukaemia.

    Science.gov (United States)

    Fallahi, Shirzad; Seyyed Tabaei, Seyyed Javad; Pournia, Yadollah; Zebardast, Nozhat; Kazemi, Bahram

    2014-07-01

    Toxoplasmosis diagnosis constitutes an important measure for disease prevention and control. In this paper, a newly described DNA amplification technique, loop-mediated isothermal amplification (LAMP), and nested-PCR targeting the repeated element (RE) and B1 gene, were compared to each other for the detection of Toxoplasma gondii DNA in blood samples of children with leukaemia. One hundred ten blood samples from these patients were analyzed by LAMP and nested-PCR. Out of 50 seropositive samples (IgM+, IgG+), positive results were obtained with 92% and 86% on RE, B1-LAMP and 82% and 68% on RE, B1-nested PCR analyses, respectively. Of the 50 seronegative samples, three, two and one samples were detected positive by RE-LAMP, B1-LAMP and RE-nested PCR assays, respectively, while none were detected positive by B1-nested PCR. None of the 10 IgM-, IgG+ samples was detected positive after testing LAMP and nested-PCR assays in duplicate. This is the first report of a study in which the LAMP method was applied with high sensitivity and efficacy for the diagnosis of T. gonii in blood samples of children with leukaemia. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Loop-Mediated Isothermal Amplification Test for Trypanosoma gambiense Group 1 with Stem Primers: A Molecular Xenomonitoring Test for Sleeping Sickness

    Directory of Open Access Journals (Sweden)

    Zablon K. Njiru

    2017-01-01

    Full Text Available The World Health Organization has targeted Human African Trypanosomiasis (HAT for elimination by 2020 with zero incidence by 2030. To achieve and sustain this goal, accurate and easy-to-deploy diagnostic tests for Gambian trypanosomiasis which accounts for over 98% of reported cases will play a crucial role. Most needed will be tools for surveillance of pathogen in vectors (xenomonitoring since population screening tests are readily available. The development of new tests is expensive and takes a long time while incremental improvement of existing technologies that have potential for xenomonitoring may offer a shorter pathway to tools for HAT surveillance. We have investigated the effect of including a second set of reaction accelerating primers (stem primers to the standard T. brucei gambiense LAMP test format. The new test format was analyzed with and without outer primers. Amplification was carried out using Rotorgene 6000 and the portable ESE Quant amplification unit capable of real-time data output. The stem LAMP formats indicated shorter time to results (~8 min, were 10–100-fold more sensitive, and indicated higher diagnostic sensitivity and accuracy compared to the standard LAMP test. It was possible to confirm the predicted product using ESE melt curves demonstrating the potential of combining LAMP and real-time technologies as possible tool for HAT molecular xenomonitoring.

  11. Design, optimisation and preliminary validation of a human specific loop-mediated amplification assay for the rapid detection of human DNA at forensic crime scenes.

    Science.gov (United States)

    Hird, H J; Brown, M K

    2017-11-01

    The identification of samples at a crime scene which require forensic DNA typing has been the focus of recent research interest. We propose a simple, but sensitive analysis system which can be deployed at a crime scene to identify crime scene stains as human or non-human. The proposed system uses the isothermal amplification of DNA in a rapid assay format, which returns results in as little as 30min from sampling. The assay system runs on the Genie II device, a proven in-field detection system which could be deployed at a crime scene. The results presented here demonstrate that the system was sufficiently specific and sensitive and was able to detect the presence of human blood, semen and saliva on mock forensic samples. Copyright © 2017. Published by Elsevier B.V.

  12. Evolution of autocatalytic sets in a competitive percolation model

    International Nuclear Information System (INIS)

    Zhang, Renquan; Pei, Sen; Wei, Wei; Zheng, Zhiming

    2014-01-01

    The evolution of autocatalytic sets (ACSs) is a widespread process in biological, chemical, and ecological systems and is of great significance in many applications such as the evolution of new species or complex chemical organization. In this paper, we propose a competitive model with an m-selection rule in which an abrupt emergence of a macroscopic independent ACS is observed. By numerical simulations, we find that the maximal increase of the size grows linearly with the system size. We analytically derive the threshold t α where the explosive transition occurs and verify it by simulations. Moreover, our analysis explains how this giant independent ACS grows and reveals that, as the selection rule becomes stricter, the phase transition is dramatically postponed, and the number of the largest independent ACSs coexisting in the system increases accordingly. Our result indicates that suppression during evolution could lead to the abrupt appearance of giant ACSs. (paper)

  13. A cubic autocatalytic reaction in a continuous stirred tank reactor

    Energy Technology Data Exchange (ETDEWEB)

    Yakubu, Aisha Aliyu; Yatim, Yazariah Mohd [School of Mathematical Sciences, Universiti Sains Malaysia, 11800 USM, Penang Malaysia (Malaysia)

    2015-10-22

    In the present study, the dynamics of the cubic autocatalytic reaction model in a continuous stirred tank reactor with linear autocatalyst decay is studied. This model describes the behavior of two chemicals (reactant and autocatalyst) flowing into the tank reactor. The behavior of the model is studied analytically and numerically. The steady state solutions are obtained for two cases, i.e. with the presence of an autocatalyst and its absence in the inflow. In the case with an autocatalyst, the model has a stable steady state. While in the case without an autocatalyst, the model exhibits three steady states, where one of the steady state is stable, the second is a saddle point while the last is spiral node. The last steady state losses stability through Hopf bifurcation and the location is determined. The physical interpretations of the results are also presented.

  14. Preliminary validation of direct detection of foot-and-mouth disease virus within clinical samples using reverse transcription loop-mediated isothermal amplification coupled with a simple lateral flow device for detection.

    Directory of Open Access Journals (Sweden)

    Ryan A Waters

    Full Text Available Rapid, field-based diagnostic assays are desirable tools for the control of foot-and-mouth disease (FMD. Current approaches involve either; 1 Detection of FMD virus (FMDV with immuochromatographic antigen lateral flow devices (LFD, which have relatively low analytical sensitivity, or 2 portable RT-qPCR that has high analytical sensitivity but is expensive. Loop-mediated isothermal amplification (LAMP may provide a platform upon which to develop field based assays without these drawbacks. The objective of this study was to modify an FMDV-specific reverse transcription-LAMP (RT-LAMP assay to enable detection of dual-labelled LAMP products with an LFD, and to evaluate simple sample processing protocols without nucleic acid extraction. The limit of detection of this assay was demonstrated to be equivalent to that of a laboratory based real-time RT-qPCR assay and to have a 10,000 fold higher analytical sensitivity than the FMDV-specific antigen LFD currently used in the field. Importantly, this study demonstrated that FMDV RNA could be detected from epithelial suspensions without the need for prior RNA extraction, utilising a rudimentary heat source for amplification. Once optimised, this RT-LAMP-LFD protocol was able to detect multiple serotypes from field epithelial samples, in addition to detecting FMDV in the air surrounding infected cattle, pigs and sheep, including pre-clinical detection. This study describes the development and evaluation of an assay format, which may be used as a future basis for rapid and low cost detection of FMDV. In addition it provides providing "proof of concept" for the future use of LAMP assays to tackle other challenging diagnostic scenarios encompassing veterinary and human health.

  15. Preliminary Validation of Direct Detection of Foot-And-Mouth Disease Virus within Clinical Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Simple Lateral Flow Device for Detection

    Science.gov (United States)

    Waters, Ryan A.; Fowler, Veronica L.; Armson, Bryony; Nelson, Noel; Gloster, John; Paton, David J.; King, Donald P.

    2014-01-01

    Rapid, field-based diagnostic assays are desirable tools for the control of foot-and-mouth disease (FMD). Current approaches involve either; 1) Detection of FMD virus (FMDV) with immuochromatographic antigen lateral flow devices (LFD), which have relatively low analytical sensitivity, or 2) portable RT-qPCR that has high analytical sensitivity but is expensive. Loop-mediated isothermal amplification (LAMP) may provide a platform upon which to develop field based assays without these drawbacks. The objective of this study was to modify an FMDV-specific reverse transcription–LAMP (RT-LAMP) assay to enable detection of dual-labelled LAMP products with an LFD, and to evaluate simple sample processing protocols without nucleic acid extraction. The limit of detection of this assay was demonstrated to be equivalent to that of a laboratory based real-time RT-qPCR assay and to have a 10,000 fold higher analytical sensitivity than the FMDV-specific antigen LFD currently used in the field. Importantly, this study demonstrated that FMDV RNA could be detected from epithelial suspensions without the need for prior RNA extraction, utilising a rudimentary heat source for amplification. Once optimised, this RT-LAMP-LFD protocol was able to detect multiple serotypes from field epithelial samples, in addition to detecting FMDV in the air surrounding infected cattle, pigs and sheep, including pre-clinical detection. This study describes the development and evaluation of an assay format, which may be used as a future basis for rapid and low cost detection of FMDV. In addition it provides providing “proof of concept” for the future use of LAMP assays to tackle other challenging diagnostic scenarios encompassing veterinary and human health. PMID:25165973

  16. Simultaneous detection and differentiation of dengue virus serotypes 1-4, Japanese encephalitis virus, and West Nile virus by a combined reverse-transcription loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Yin Jianhua

    2011-07-01

    Full Text Available Abstract Background Rapid identification and differentiation of mosquito-transmitted flaviviruses in acute-phase sera of patients and field-caught vector mosquitoes are important for the prediction and prevention of large-scale epidemics. Results We developed a flexible reverse-transcription loop-mediated isothermal amplification (RT-LAMP unit for the detection and differentiation of dengue virus serotypes 1-4 (DENV1-4, Japanese encephalitis virus (JEV, and West Nile virus (WNV. The unit efficiently amplified the viral genomes specifically at wide ranges of viral template concentrations, and exhibited similar amplification curves as monitored by a real-time PCR engine. The detection limits of the RT-LAMP unit were 100-fold higher than that of RT-PCR in 5 of the six flaviviruses. The results on specificity indicated that the six viruses in the assay had no cross-reactions with each other. By examining 66 viral strains of DENV1-4 and JEV, the unit identified the viruses with 100% accuracy and did not cross-react with influenza viruses and hantaviruses. By screening a panel of specimens containing sera of 168 patients and 279 pools of field-caught blood sucked mosquitoes, results showed that this unit is high feasible in clinical settings and epidemiologic field, and it obtained results 100% correlated with real-time RT-PCR. Conclusions The RT-LAMP unit developed in this study is able to quickly detect and accurately differentiate the six kinds of flaviviruses, which makes it extremely feasible for screening these viruses in acute-phase sera of the patients and in vector mosquitoes without the need of high-precision instruments.

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

    Science.gov (United States)

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

    2017-08-01

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

  18. Rapid detection of Pseudomonas aeruginosa and Acinetobacter baumannii Harboring bla(VIM-2), bla(IMP-1) and bla(OXA-23) genes by using loop-mediated isothermal amplification methods.

    Science.gov (United States)

    Kim, Hye Jin; Kim, Hyung Sun; Lee, Jae Myun; Yoon, Sang Sun; Yong, Dongeun

    2016-01-01

    Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of bla(VIM-2), bla(IMP-1), and bla(OXA-23), which are critical components for carbapenem resistance. Two sets of primers, inner and outer primers, were manually designed as previously described. The LAMP buffer was optimized (at 2mM MgSO₄) by testing different concentrations of MgSO₄. The optimal reaction temperature and incubation time were determined by using a gradient thermocycler. Then, the optimized bla(VIM-2), bla(IMP-1), and bla(OXA-23) LAMP reactions were evaluated by using 120 P. aeruginosa and 99 A. baumannii clinical isolates. Only one strain of the 100 CRPA isolates harbored bla(IMP-1), whereas none of them harbored bla(VIM-2). These results indicate that the acquisition of bla(VIM-2) or bla(IMP-1) may not play a major role in carbapenem resistance in Korea. Fifty two strains of the 75 CRAB isolates contained bla(OXA-23), but none contained bla(VIM-2) and bla(IMP-1) alleles. Our results demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB.

  19. Hemoculture and Direct Sputum Detection of mecA-Mediated Methicillin-Resistant Staphylococcus aureus by Loop-Mediated Isothermal Amplification in Combination With a Lateral-Flow Dipstick.

    Science.gov (United States)

    Nawattanapaiboon, Kawin; Prombun, Photchanathorn; Santanirand, Pitak; Vongsakulyanon, Apirom; Srikhirin, Toemsak; Sutapun, Boonsong; Kiatpathomchai, Wansika

    2016-09-01

    This study reports loop-mediated isothermal amplification (LAMP) for rapid detection of methicillin-resistant Staphylococcus aureus from direct clinical specimens. Four primers including outer and inner primers were specifically designed on the two target sequences-femB to identify S. aureus and mecA to identify antibiotic-resistant gene. Reference strains including various species of gram-positive/gram-negative isolates were used to evaluate and optimize LAMP assays. The optimum LAMP condition was found at 63°C within 70 min assay time (include hybridization with FITC probe for 5 min and further 5 min for reading the results on the lateral flow dipstick). The detection limits of LAMP for mecA was 10 pg of total DNA or 100 CFU/ml. The LAMP assays were applied to a total of 155 samples of direct DNA extraction from sputum and hemoculture bottles. The sensitivity of LAMP for mecA detection in sputum and hemoculture bottles was 93.3% (28/30) and 100% (52/52), respectively. In conclusion, LAMP assay is an alternative technique for rapid detection of MRSA infection with a technical simplicity and cost-effective method in a routine diagnostic laboratory. © 2016 Wiley Periodicals, Inc.

  20. One-tube loop-mediated isothermal amplification combined with restriction endonuclease digestion and ELISA for colorimetric detection of resistance to isoniazid, ethambutol and streptomycin in Mycobacterium tuberculosis isolates.

    Science.gov (United States)

    Lee, Mei-Feng; Chen, Yen-Hsu; Hsu, Hui-Jine; Peng, Chien-Fang

    2010-10-01

    In this study, we designed a simple and rapid colorimetric detection method, a one-tube loop-mediated isothermal amplification (LAMP)-PCR-hybridization-restriction endonuclease-ELISA [one-tube LAMP-PCR-HY-RE-ELISA] system, to detect resistance to isoniazid, ethambutol and streptomycin in strains of Mycobacterium tuberculosis isolated from clinical specimens. The clinical performance of this method for detecting isoniazid-resistant, ethambutol-resistant and streptomycin-resistant isolates of M. tuberculosis showed 98.9%, 94.3% and 93.8%, respectively. This assay is rapid and convenient that can be performed within one working day. One-tube LAMP-PCR-HY-RE-ELISA system was designed based on hot spot point mutations in target drug-resistant genes, using LAMP-PCR, hybridization, digestion with restriction endonuclease and colorimetric method of ELISA. In this study, LAMP assay was used to amplify DNA from drug-resistant M. tuberculosis, and ELISA was used for colorimetrical determination. This assay will be a useful tool for rapid diagnosis of mutant codons in strains of M. tuberculosis for isoniazid at katG 315 and katG 463, ethambutol at embB 306 and embB 497, and streptomycin at rpsL 43. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

  1. Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP kit prototype for detection of Trypanosoma cruzi DNA in human blood samples.

    Directory of Open Access Journals (Sweden)

    Susana A Besuschio

    2017-07-01

    Full Text Available This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs, in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I and CL Brener (Tc VI stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener. LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after "Boil & Spin" rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23, as well as seronegative controls (N = 10 were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2 fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10-1 fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2 parasite equivalents/mL in spiked EDTA blood and 1x10-1 par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2 par

  2. Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples.

    Science.gov (United States)

    Besuschio, Susana A; Llano Murcia, Mónica; Benatar, Alejandro F; Monnerat, Severine; Cruz, Israel; Picado, Albert; Curto, María de Los Ángeles; Kubota, Yutaka; Wehrendt, Diana P; Pavia, Paula; Mori, Yasuyoshi; Puerta, Concepción; Ndung'u, Joseph M; Schijman, Alejandro G

    2017-07-01

    This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after "Boil & Spin" rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2 fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10-1 fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2 parasite equivalents/mL in spiked EDTA blood and 1x10-1 par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2 par.eq./mL in EDTA blood

  3. A Mechanistic Model of a Passive Autocatalytic Hydrogen Recombiner

    Directory of Open Access Journals (Sweden)

    Rożeń Antoni

    2015-03-01

    Full Text Available : A passive autocatalytic hydrogen recombiner (PAR is a self-starting device, without operator action or external power input, installed in nuclear power plants to remove hydrogen from the containment building of a nuclear reactor. A new mechanistic model of PAR has been presented and validated by experimental data and results of Computational Fluid Dynamics (CFD simulations. The model allows to quickly and accurately predict gas temperature and composition, catalyst temperature and hydrogen recombination rate. It is assumed in the model that an exothermic recombination reaction of hydrogen and oxygen proceeds at the catalyst surface only, while processes of heat and mass transport occur by assisted natural and forced convection in non-isothermal and laminar gas flow conditions in vertical channels between catalyst plates. The model accounts for heat radiation from a hot catalyst surface and has no adjustable parameters. It can be combined with an equation of chimney draft and become a useful engineering tool for selection and optimisation of catalytic recombiner geometry.

  4. First experience with the installation of passive autocatalytic recombiners

    International Nuclear Information System (INIS)

    Snoeck, J.; Solaro, C.; Moeyaert, P.

    1997-01-01

    From the principle decision of installing Passive Autocatalytic Recombiners (PARs) in all Belgian NPPs to the installation on site, several problems needed to be identified and solved; it is the goal of this paper to address these issues and to present the solutions which were adopted. The first section deals with the sizing and the distribution of the catalytic surface inside the containment to be equipped. Rather than to rely on numerous multicompartment calculations, the procedure is based on a single volume approach combined with engineering judgement. The criteria applied in the successive design steps are explained and illustrated for the Doel1 unit. The second section is devoted to additional requirements concerning the resistance to poisoning agents and the maintenance constraints, and to some recommendations for the design of the supports. Because the effectiveness of the PARs entirely relies on the catalyst material, a periodic control of the active parts is of primary importance. Therefore, the Transportable In-Service Inspection Equipment (TIIE) and the test procedures are presented in the third section. Finally, recommendations are made for further work, derived from this first experience. (author)

  5. Analysis of passive autocatalytic recombiners installation in Cofrentes NPP

    International Nuclear Information System (INIS)

    Del Carmen Molina, Cesar Serrano; Jimenez, Gonzalo

    2014-01-01

    The analysis of Passive Autocatalytic Recombiners (PAR) installation in Cofrentes NPP is a regulatory requirement from the stress tests carried out due to the Fukushima accident. The analysis has been developed by IIC with the support of UPM-ETSII, and defines the PARs configuration to be installed into the containment/drywell to minimize the risks derived from hydrogen generation during a severe accident. The analysis has been developed in three stages: - Generation and release of hydrogen in containment/drywell: The mass and energy sources releases are analysed with MAAP4.0.7 code and covers all phases of the severe accident in-vessel and ex-vessel. Probabilistic and deterministic criteria were used to select the accident sequences. Model sensitivities analyses were included maximizing the global hydrogen releases as well as the peak values. - Distribution of hydrogen in containment/drywell: Hydrogen and other gases distribution is developed with GOTHIC 8.0 code using a 3D detailed model. Long term simulation necessities were taken into account during model adjustment. Model verification was carried out by comparing the results with several experimental data and simulations with other thermohydraulic codes, both covering the range of simulation. The model considers mass and energy discharges in the suppression pool as well as and in the containment atmosphere and takes into account heat transfer and condensation process. In addition, the influence of containment systems activation has been evaluated. - PAR sizing and locating: The location and size analysis of PAR considers the hydrogen depletion rates and operating conditions of different equipment. Furthermore, a post processing treatment has been developed to deeply analyse all parameters involved in the combustion phenomena and thus identify the risks zones. The study takes into account different distribution options regarding cost and operational criteria. Walk-downs were carried out for a realistic

  6. Autocatalytic surface reduction and its role in controlling seed-mediated growth of colloidal metal nanocrystals.

    Science.gov (United States)

    Yang, Tung-Han; Zhou, Shan; Gilroy, Kyle D; Figueroa-Cosme, Legna; Lee, Yi-Hsien; Wu, Jenn-Ming; Xia, Younan

    2017-12-26

    The growth of colloidal metal nanocrystals typically involves an autocatalytic process, in which the salt precursor adsorbs onto the surface of a growing nanocrystal, followed by chemical reduction to atoms for their incorporation into the nanocrystal. Despite its universal role in the synthesis of colloidal nanocrystals, it is still poorly understood and controlled in terms of kinetics. Through the use of well-defined nanocrystals as seeds, including those with different types of facets, sizes, and internal twin structure, here we quantitatively analyze the kinetics of autocatalytic surface reduction in an effort to control the evolution of nanocrystals into predictable shapes. Our kinetic measurements demonstrate that the activation energy barrier to autocatalytic surface reduction is highly dependent on both the type of facet and the presence of twin boundary, corresponding to distinctive growth patterns and products. Interestingly, the autocatalytic process is effective not only in eliminating homogeneous nucleation but also in activating and sustaining the growth of octahedral nanocrystals. This work represents a major step forward toward achieving a quantitative understanding and control of the autocatalytic process involved in the synthesis of colloidal metal nanocrystals.

  7. Diagnostic Accuracy of Loop-mediated Isothermal Amplification Assay as a Field Molecular Tool for Rapid Mass Screening of Old WorldLeishmaniaInfections in Sand Flies and In Vitro Culture.

    Science.gov (United States)

    Ghodrati, Mehdi; Spotin, Adel; Hazratian, Teimour; Mahami-Oskouei, Mahmoud; Bordbar, Ali; Ebrahimi, Sahar; Fallahi, Shirzad; Parvizi, Parviz

    2017-01-01

    We employed a highly sensitive loop-mediated isothermal amplification (LAMP) by targeting 18S rRNA gene to identify the rapid mass screening of Leishmania infections in captured sand flies of southwest Iran and In vitro culture. One hundred fifty sand flies were collected from 11 sites adjacent to Iraqi's borders in southern parts of Khuzestan Province by using sticky sheets of paper and CDC miniature light traps during late May 2014 to Nov 2015. Following morphological identification of sand flies species, the DNA of infected samples was extracted and amplified by PCR and LAMP assays by targeting ITS-rDNA and 18S rRNA genes. The PCR amplicons were directly sequenced to conduct the phylogenetic analysis. Ten (6.6%) Leishmania infections were identified by LAMP assay (detection limit 0.01 parasites DNA) among infected Sergentomyia baghdadis , S. sintoni and Phlebotomus papatasi sand flies that was more sensitive than PCR (n=6.4%; (detection limit 10 1 parasites DNA). LAMP can identify 10 1 -10 6 promastigotes/100 μl RPMI 1640 while PCR recognized 10 4 -10 6 promastigotes. The majority infection rate of sand flies was confirmed to L. major inferred by phylogenetic analysis. This is the first exploration characterized the Old World Leishmania infections by LAMP technique in both infected sand flies and In vitro conditions. The LAMP method because of its shorter reaction time, robustness, more sensitivity, lack of requirement of complicated equipment and visual discriminatory of positivity can be appeared a promising tool instead of PCR to identify low Leishmania loads and entomological monitoring of leishmaniasis in resource-limited endemic of the world.

  8. Rapid and sensitive detection of novel avian-origin influenza A (H7N9 virus by reverse transcription loop-mediated isothermal amplification combined with a lateral-flow device.

    Directory of Open Access Journals (Sweden)

    Yiyue Ge

    Full Text Available A severe disease in humans caused by a novel avian-origin influenza A (H7N9 virus emerged in China recently, which has caused at least 128 cases and 26 deaths. Rapid detection of the novel H7N9 virus is urgently needed to differentiate the disease from other infections, and to facilitate infection control as well as epidemiologic investigations. In this study, a reverse transcription loop-mediated isothermal amplification combined with a lateral flow device (RT-LAMP-LFD assay to rapidly detect H7N9 virus was developed and evaluated. The RT-LAMP primers were designed to target the haemagglutinin (HA and neuraminidase (NA genes of H7N9 virus. Results of 10-fold dilution series assays showed that analysis of RT-LAMP products by the LFD method was as sensitive as real-time turbidity detection, and that the analytic sensitivities of the HA and NA RT-LAMP assays were both 10 copies of synthetic RNA. Furthermore, both the assays showed 100% clinical specificity for identification of H7N9 virus. The performance characteristics of the RT-LAMP-LFD assay were evaluated with 80 clinical specimens collected from suspected H7N9 patients. The NA RT-LAMP-LFD assay was more sensitive than real time RT-PCR assay. Compared with a combination of virus culture and real-time RT-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the RT-LAMP-LFD assay were all 100%. Overall, The RT-LAMP-LFD assay established in this study can be used as a reliable method for early diagnosis of the avian-origin influenza A (H7N9 virus infection.

  9. Evaluation of commercial kit based on loop-mediated isothermal amplification for rapid detection of low levels of uninjured and injured Salmonella on duck meat, bean sprouts, and fishballs in Singapore.

    Science.gov (United States)

    Lim, Hazel Sin Yue; Zheng, Qianwang; Miks-Krajnik, Marta; Turner, Matthew; Yuk, Hyun-Gyun

    2015-06-01

    The objective of this study was to evaluate performance of the commercial kit based on loop-mediated isothermal amplification (LAMP) in comparison with the International Organization for Standardization method for detecting uninjured and sublethally injured Salmonella cells artificially inoculated at levels of 10(0) and 10(1) CFU/25 g on raw duck wing, raw mung bean sprouts, and processed fishballs. Injured cells were prepared by a heat treatment for duck wings and fishball samples and a chlorine treatment for bean sprout samples. Additionally, a validation study was performed on naturally contaminated food samples sold in Singapore. A total of 110 samples of each commodity were analyzed in this study. Regardless of inoculum levels, the detection by the commercial LAMP kit showed 100% sensitivity and specificity for both inoculated and uninoculated samples compared with the International Organization for Standardization method, with the exception of bean sprout samples. Only 20% of bean sprout samples inoculated with 10(0) CFU/25 g injured Salmonella cells were positive by using the commercial LAMP-based kit. However, all negative samples became positive following a secondary enrichment in Rappaport-Vassiliadis medium with soy broth or after concentration by centrifugation. These results suggest that secondary enrichment or centrifugation should be considered as an additional step to increase the sensitivity of the commercial LAMP-based kit with low numbers of injured target cells in samples with high background microflora (such as mung bean sprouts). The validation study also showed that the commercial LAMP-based kit provided 91% sensitivity and 95% specificity for naturally contaminated samples. Thus, this study demonstrates that the commercial LAMP-based kit might be a cost-effective method, as this system could provide rapid, accurate detection of both uninjured and injured Salmonella cells on raw duck wings, raw mung bean sprouts, and processed fishballs in

  10. 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.

    Science.gov (United States)

    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 10⁵ conidia per reaction with the primer set ID9 for A. nomius and 10⁴ 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

  11. Input to Resin Column Structural Analysis if Autocatalytic Resin Reaction Occurs in HB-Line Phase II

    Energy Technology Data Exchange (ETDEWEB)

    Hallman, D.F.

    2001-07-10

    Solutions of plutonium in nitric acid are purified and concentrated using anion resin prior to precipitation. There have been instances of resin column explosions caused by autocatalytic reactions of anion resins in nitric acid within the DOE complex

  12. Autocatalytic microtubule nucleation determines the size and mass of Xenopus laevis egg extract spindles.

    Science.gov (United States)

    Decker, Franziska; Oriola, David; Dalton, Benjamin; Brugués, Jan

    2018-01-11

    Regulation of size and growth is a fundamental problem in biology. A prominent example is the formation of the mitotic spindle, where protein concentration gradients around chromosomes are thought to regulate spindle growth by controlling microtubule nucleation. Previous evidence suggests that microtubules nucleate throughout the spindle structure. However, the mechanisms underlying microtubule nucleation and its spatial regulation are still unclear. Here, we developed an assay based on laser ablation to directly probe microtubule nucleation events in Xenopus laevis egg extracts. Combining this method with theory and quantitative microscopy, we show that the size of a spindle is controlled by autocatalytic growth of microtubules, driven by microtubule-stimulated microtubule nucleation. The autocatalytic activity of this nucleation system is spatially regulated by the limiting amounts of active microtubule nucleators, which decrease with distance from the chromosomes. This mechanism provides an upper limit to spindle size even when resources are not limiting. © 2018, Decker et al.

  13. State of the art on hydrogen passive auto-catalytic recombiner (european union Parsoar project)

    International Nuclear Information System (INIS)

    Arnould, F.; Bachellerie, E.; Auglaire, M.; Boeck, B. de; Braillard, O.; Eckardt, B.; Ferroni, F.; Moffett, R.; Van Goethem, G.

    2001-01-01

    This paper presents an overview of the European Union PARSOAR project, which consists in carrying out a state of the art on hydrogen passive auto-catalytic recombiner (PAR) and a handbook guide for implementing these devices in nuclear power plants. This work is performed in the area ''Operational Safety of Existing Installations'' of the key action ''Nuclear Fission'' of the fifth Euratom Framework Programme (1998-2002). (author)

  14. State of the art on hydrogen passive auto-catalytic recombiner (european union Parsoar project)

    Energy Technology Data Exchange (ETDEWEB)

    Arnould, F.; Bachellerie, E. [Technicatome, 13 - Aix en Provence (France); Auglaire, M. [Tractebel Energy Engineering, Brussels (Belgium); Boeck, B. de [Association Vincotte Nuclear, Brussels (Belgium); Braillard, O. [CEA Cadarache, 13 - Saint Paul lez Durance (France); Eckardt, B. [Siemens AG, Offenbach am Main (Germany); Ferroni, F. [Electrowatt Engineering Limited, Zurich (Switzerland); Moffett, R. [Atomic Energy Canada Limited, Pinawa (Canada); Van Goethem, G. [European Commission, Brussels (Belgium)

    2001-07-01

    This paper presents an overview of the European Union PARSOAR project, which consists in carrying out a state of the art on hydrogen passive auto-catalytic recombiner (PAR) and a handbook guide for implementing these devices in nuclear power plants. This work is performed in the area ''Operational Safety of Existing Installations'' of the key action ''Nuclear Fission'' of the fifth Euratom Framework Programme (1998-2002). (author)

  15. Amplification factor variable amplifier

    NARCIS (Netherlands)

    Akitsugu, Oshita; Nauta, Bram

    2007-01-01

    PROBLEM TO BE SOLVED: To provide an amplification factor variable amplifier capable of achieving temperature compensation of an amplification factor over a wide variable amplification factor range. ; SOLUTION: A Gilbert type amplification factor variable amplifier 11 amplifies an input signal and

  16. Amplification factor variable amplifier

    NARCIS (Netherlands)

    Akitsugu, Oshita; Nauta, Bram

    2010-01-01

    PROBLEM TO BE SOLVED: To provide an amplification factor variable amplifier capable of achieving temperature compensation of an amplification factor over a wide variable amplification factor range. ;SOLUTION: A Gilbert type amplification factor variable amplifier 11 amplifies an input signal and can

  17. Underground autocatalytic-criticality potential and its implications to weapons fissile- material disposition

    International Nuclear Information System (INIS)

    Choi, J.-S.

    1998-01-01

    Several options for weapons fissile-material disposition, such as once-through mixed- oxide (MOX) fuel in reactors or immobilisation in waste glass, would result in end products requiring geologic disposal. The criticality potential of the fissile end products containing U-235 and Pu-239 and the associated consequences in a geologic setting are important considerations for the final disposal of these materials. The possibility of underground criticality, and especially autocatalytic criticality, is affected by (1) groundwater leaking into a failed waste container, (2) preferential leaching of neutron absorbers or of fissile material from a failed container, and (3) preferential deposition of fissile material in the surrounding rock. Bowman and Venneri have pointed out that fissile material mixed with varying compositions of water and silica can undergo a nuclear chain reaction. Some configurations can become autocatalytically supercritical resulting in considerable energy release, terminated finally by disassembly. Some reviews rejected the Bowman and Venneri warning as implausible because of low probabilities of scenarios that could lead to such configurations. Sanchez et al. reported possible supercritical conditions in systems of Pu-SiO 2 -H 2 O and Pu-tuff-H 2 O but concluded that the probability of forming such combinations is extremely low. Kastenberg et al. studied the potential for autocatalytic criticality of plutonium or highly enriched uranium in the proposed Yucca Mountain geologic repository. They concluded that plutonium or uranium could, theoretically, become supercritical, but that such criticality is unlikely given the hydrology, geology and geochemistry of the Yucca Mountain site. These studies are not definitive. The possibility of criticality exists. Detailed mechanisms have not been sufficiently studied for clear conclusions on the probabilities of occurrence. More technical analysis is needed to understand the potential for underground

  18. Design and Analysis of Compact DNA Strand Displacement Circuits for Analog Computation Using Autocatalytic Amplifiers.

    Science.gov (United States)

    Song, Tianqi; Garg, Sudhanshu; Mokhtar, Reem; Bui, Hieu; Reif, John

    2018-01-19

    A main goal in DNA computing is to build DNA circuits to compute designated functions using a minimal number of DNA strands. Here, we propose a novel architecture to build compact DNA strand displacement circuits to compute a broad scope of functions in an analog fashion. A circuit by this architecture is composed of three autocatalytic amplifiers, and the amplifiers interact to perform computation. We show DNA circuits to compute functions sqrt(x), ln(x) and exp(x) for x in tunable ranges with simulation results. A key innovation in our architecture, inspired by Napier's use of logarithm transforms to compute square roots on a slide rule, is to make use of autocatalytic amplifiers to do logarithmic and exponential transforms in concentration and time. In particular, we convert from the input that is encoded by the initial concentration of the input DNA strand, to time, and then back again to the output encoded by the concentration of the output DNA strand at equilibrium. This combined use of strand-concentration and time encoding of computational values may have impact on other forms of molecular computation.

  19. The CP-based electrochemical biosensors with autocatalytic stage in their function and the mathematical description of their work

    Directory of Open Access Journals (Sweden)

    Volodymyr Valentynovych Tkach

    2012-07-01

    Full Text Available The electroanalytic process of the detection of biosubstances, realized by the biosensor, based in conducting polyheterocyclic compounds, the function of which contained autocatalytic stage, was mathematically described. The correspondent mathematical model was analyzed by linear stability theory and bifurcational analysis. The electrochemical instabilities, capable to succeed in this process, were explained in the terms of this model.

  20. Amplification of Chirality through Self-Replication of Micellar Aggregates in Water

    KAUST Repository

    Bukhriakov, Konstantin

    2015-03-17

    We describe a system in which the self-replication of micellar aggregates results in a spontaneous amplification of chirality in the reaction products. In this system, amphiphiles are synthesized from two "clickable" fragments: a water-soluble "head" and a hydrophobic "tail". Under biphasic conditions, the reaction is autocatalytic, as aggregates facilitate the transfer of hydrophobic molecules to the aqueous phase. When chiral, partially enantioenriched surfactant heads are used, a strong nonlinear induction of chirality in the reaction products is observed. Preseeding the reaction mixture with an amphiphile of one chirality results in the amplification of this product and therefore information transfer between generations of self-replicating aggregates. Because our amphiphiles are capable of catalysis, information transfer, and self-assembly into bounded structures, they present a plausible model for prenucleic acid "lipid world" entities. © 2015 American Chemical Society.

  1. A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers.

    Directory of Open Access Journals (Sweden)

    Geoffrey P Noble

    2015-06-01

    Full Text Available Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.

  2. A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers

    Science.gov (United States)

    Noble, Geoffrey P.; Wang, Daphne W.; Walsh, Daniel J.; Barone, Justin R.; Miller, Michael B.; Nishina, Koren A.; Li, Sheng; Supattapone, Surachai

    2015-01-01

    Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure. PMID:26125623

  3. An advanced uracil DNA glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP) technique used in the sensitive and specific detection of Cryptosporidium parvum, Cryptosporidium hominis, and Cryptosporidium meleagridis in AIDS patients.

    Science.gov (United States)

    Fallahi, Shirzad; Moosavi, Seyedeh Fatemeh; Karimi, Azadeh; Chegeni, Ali Sharafi; Saki, Mohammad; Namdari, Parsa; Rashno, Mohammad Menati; Varzi, Ali Mohamad; Tarrahi, Mohammad Javad; Almasian, Mohammad

    2017-12-24

    The rapid and accurate detection of Cryptosporidium spp. is critically important for the prevention and timely treatment of cryptosporidiosis in AIDS patients (APs). This study was conducted to examine a UDG-LAMP technique for the first time to diagnose cryptosporidiosis in APs. After collecting demographic and clinical data, three stool samples were collected from the participants (120 volunteering APs). The microscopic examination of stained smears using the acid-fast method and the UDG-LAMP assay were performed for each sample. 10% of APs were infected with Cryptosporidium spp. The number of detected cryptosporidiosis cases using the acid-fast staining and UDG-LAMP methods were significantly different (P < 0.001). Diarrhea and weight loss were found to be significantly associated with cryptosporidiosis in patients (P < 0.05). The pretreatment of LAMP reagents with UDG successfully eliminated the likelihood of product re-amplification remaining from previous reactions. The UDG-LAMP technique could detect cryptosporidiosis in APs with high sensitivity and rapidity without carryover contamination. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Quantitative detection of DNA by autocatalytic enlargement of hybridized gold nanoprobes

    DEFF Research Database (Denmark)

    Zhan, Zongrui; Cao, Cuong; Sim, Sang Jun

    2010-01-01

    Quantitative detection of specific viral DNA has become a pressing issue for the earlier clinical diagnosis of viral infectious diseases. Therefore, in this paper, we report a simple, sensitive, and inexpensive quantitative approach for DNA detection based on the autocatalytic Au deposition of gold...... nanoprobes via the surface reduction of AuCl4− to Au0 on their surface in the presence of ascorbic acid (AA) and cetyltrimethylammonium bromide (CTAB). On this basis, signal enhancements in the absorbance intensity and kinetic behavior of gold enlargement in the aqueous phase have been well investigated...... and explained for the selection of analytical parameters. To achieve high sensitivity, magnetic particles conjugated with capture probes (PMPs) were employed for the collection of gold nanoprobes. After denaturated by ion a pH 11 solution, the amplified signals of gold nanoprobes, which is proportional...

  5. Evaluation of Passive Autocatalytic Recombiner (PAR) Implementation in a Konvoi NPP Containment Type

    Energy Technology Data Exchange (ETDEWEB)

    Lopez-Alonso, E.; Papini, D.; Jimenez, G.

    2015-07-01

    The evaluation of Passive Autocatalytic Recombiner (PAR) implementation has been developed under the methodology extracted from the IAEA document, analysing the size, location and number of the PARs capable to minimize the combustion risk, which arises from a hydrogen release generated during a severe accident and its distribution in containment building. A detailed three-dimensional model of Konvoi (PWR) containment with GOTHIC 8.1 code was used for the simulations. The hydrogen preferential pathways and the accumulation points were studied and identified on the basis of a base case scenario without any mitigation measure. The PAR configuration offers an improvement in the chosen accidental scenario; decreasing the possibility of hydrogen combustion and leading to concentration values below the flammability limit (hydrogen concentration below 7%), in all the containment compartments at the end of the transient. Furthermore, from the analysis, it is concluded that the time required to reach hydrogen concentrations below the combustion limit is considerably reduced. (Author)

  6. CFD simulation of hydrogen mixing and mitigation by means of passive auto-catalytic recombiners

    International Nuclear Information System (INIS)

    Kelm, S.; Reinecke, E-A.; Jahn, W.; Allelein, H-J.

    2011-01-01

    Modeling of passive auto-catalytic recombiners (PARs) operation in containment geometries involves a large variety of scales; thus, a CFD calculation resolving all these scales would be much too expensive. Therefore, the mechanistic PAR model REKO-DIREKT, developed at Forschungszentrum Juelich, has been coupled with the commercial CFD code ANSYS CFX in order to simulate PAR operation as well as the induced flow and transport phenomena. Based on a short introduction of REKO-DIREKT, its interface to CFX and the explicit coupling scheme is discussed. The paper is finalized by a first demonstration of simulation capabilities on the basis of the ThAI PAR-4 experiment (Becker Technologies GmbH, Eschborn, Germany). (author)

  7. LASER INDUCED SELECTIVE ACTIVATION UTILIZING AUTO-CATALYTIC ELECTROLESS PLATING ON POLYMER SURFACE

    DEFF Research Database (Denmark)

    Zhang, Yang; Nielsen, Jakob Skov; Tang, Peter Torben

    2009-01-01

    . Characterization of the deposited copper layer was used to select and improve laser parameters. Several types of polymers with different melting points were used as substrate. Using the above mentioned laser treatment, standard grades of thermoplastic materials such as ABS, SAN, PE, PC and others have been......This paper presents a new method for selective micro metallization of polymers induced by laser. An Nd: YAG laser was employed to draw patterns on polymer surfaces using a special set-up. After subsequent activation and auto-catalytic electroless plating, copper only deposited on the laser tracks....... Induced by the laser, porous and rough structures are formed on the surface, which favours the palladium attachment during the activation step prior to the metallization. Laser focus detection, scanning electron microscopy (SEM) and other instruments were used to analyze the topography of the laser track...

  8. Associated technologies ensures complete loop mediated ...

    African Journals Online (AJOL)

    Loop Mediated Isothermal Amplification (LAMP) assay could be a useful adjunct diagnostic assay along with the conventional methods that would preclude the requirement of continuous maintenance of pure cultures. Moreover, LAMP assay is simple, rapid, specific and sensitive for the detection of pathogens. Having ...

  9. Loop-to-loop coupling.

    Energy Technology Data Exchange (ETDEWEB)

    Warne, Larry Kevin; Lucero, Larry Martin; Langston, William L.; Salazar, Robert Austin; Coleman, Phillip Dale; Basilio, Lorena I.; Bacon, Larry Donald

    2012-05-01

    This report estimates inductively-coupled energy to a low-impedance load in a loop-to-loop arrangement. Both analytical models and full-wave numerical simulations are used and the resulting fields, coupled powers and energies are compared. The energies are simply estimated from the coupled powers through approximations to the energy theorem. The transmitter loop is taken to be either a circular geometry or a rectangular-loop (stripline-type) geometry that was used in an experimental setup. Simple magnetic field models are constructed and used to estimate the mutual inductance to the receiving loop, which is taken to be circular with one or several turns. Circuit elements are estimated and used to determine the coupled current and power (an equivalent antenna picture is also given). These results are compared to an electromagnetic simulation of the transmitter geometry. Simple approximate relations are also given to estimate coupled energy from the power. The effect of additional loads in the form of attached leads, forming transmission lines, are considered. The results are summarized in a set of susceptibility-type curves. Finally, we also consider drives to the cables themselves and the resulting common-to-differential mode currents in the load.

  10. Modelling of a passive autocatalytic hydrogen recombiner – a parametric study

    Directory of Open Access Journals (Sweden)

    Rożeń Antoni

    2015-03-01

    Full Text Available Operation of a passive autocatalytic hydrogen recombiner (PAR has been investigated by means of computational fluid dynamics methods (CFD. The recombiner is a self-active and self-adaptive device used to remove hydrogen from safety containments of light water nuclear reactors (LWR by means of a highly exothermic reaction with oxygen at the surface of a platinum or palladium catalyst. Different turbulence models (k-ω, k-ɛ, intermittency, RSM were applied in numerical simulations of: gas flow, heat and mass transport and chemical surface reactions occurring in PAR. Turbulence was found to improve mixing and mass transfer and increase hydrogen recombination rate for high gas flow rates. At low gas flow rates, simulation results converged to those obtained for the limiting case of laminar flow. The large eddy simulation technique (LES was used to select the best RANS (Reynolds average stress model. Comparison of simulation results obtained for two- and three-dimensional computational grids showed that heat and mass transfer occurring in PAR were virtually two-dimensional processes. The effect of hydrogen thermal diffusion was also discussed in the context of possible hydrogen ignition inside the recombiner.

  11. Evaluation of passive autocatalytic recombiners operation efficiency by means of the lumped parameter approach*

    Directory of Open Access Journals (Sweden)

    Bury Tomasz

    2015-06-01

    Full Text Available The problem of hydrogen behavior in containment buildings of nuclear reactors belongs to thermal-hydraulic area. Taking into account the size of systems under consideration and, first of all, safety issues, such type of analyses cannot be done by means of full-scale experiments. Therefore, mathematical modeling and numerical simulations are widely used for these purposes. A lumped parameter approach based code HEPCAL has been elaborated in the Institute of Thermal Technology of the Silesian University of Technology for simulations of pressurized water reactor containment transient response. The VVER-440/213 and European pressurised water reactor (EPR reactors containments are the subjects of analysis within the framework of this paper. Simulations have been realized for the loss-of-coolant accident scenarios with emergency core cooling system failure. These scenarios include core overheating and hydrogen generation. Passive autocatalytic recombiners installed for removal of hydrogen has been taken into account. The operational efficiency of the hydrogen removal system has been evaluated by comparing with an actual hydrogen concentration and flammability limit. This limit has been determined for the three-component mixture of air, steam and hydrogen. Some problems related to the lumped parameter approach application have been also identified.

  12. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro.

    Science.gov (United States)

    Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas; Choi, Kyung H

    2014-03-01

    Pestivirus N(pro) is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N(pro) blocks the host׳s interferon response by inducing degradation of interferon regulatory factor-3. N(pro׳)s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N(pro)-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N(pro) proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N(pro׳)s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N(pro) does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro

    International Nuclear Information System (INIS)

    Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas; Choi, Kyung H.

    2014-01-01

    Pestivirus N pro is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N pro blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N pro' s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N pro -GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N pro proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N pro' s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N pro does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N pro' s autoproteolysis is studied using N pro -GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N pro prefers small amino acids with non-branched beta carbons at the P1 position

  14. Simultaneous fingering, double-diffusive convection, and thermal plumes derived from autocatalytic exothermic reaction fronts

    Science.gov (United States)

    Eskew, Matthew W.; Harrison, Jason; Simoyi, Reuben H.

    2016-11-01

    Oxidation reactions of thiourea by chlorite in a Hele-Shaw cell are excitable, autocatalytic, exothermic, and generate a lateral instability upon being triggered by the autocatalyst. Reagent concentrations used to develop convective instabilities delivered a temperature jump at the wave front of 2.1 K. The reaction zone was 2 mm and due to normal cooling after the wave front, this generated a spike rather than the standard well-studied front propagation. The reaction front has solutal and thermal contributions to density changes that act in opposite directions due to the existence of a positive isothermal density change in the reaction. The competition between these effects generates thermal plumes. The fascinating feature of this system is the coexistence of plumes and fingering in the same solution which alternate in frequency as the front propagates, generating hot and cold spots within the Hele-Shaw cell, and subsequently spatiotemporal inhomogeneities. The small ΔT at the wave front generated thermocapillary convection which competed effectively with thermogravitational forces at low Eötvös Numbers. A simplified reaction-diffusion-convection model was derived for the system. Plume formation is heavily dependent on boundary effects from the cell dimensions. This work was supported by Grant No. CHE-1056366 from the NSF and a Research Professor Grant from the University of KwaZulu-Natal.

  15. Gene amplification in carcinogenesis

    Directory of Open Access Journals (Sweden)

    Lucimari Bizari

    2006-01-01

    Full Text Available Gene amplification increases the number of genes in a genome and can give rise to karyotype abnormalities called double minutes (DM and homogeneously staining regions (HSR, both of which have been widely observed in human tumors but are also known to play a major role during embryonic development due to the fact that they are responsible for the programmed increase of gene expression. The etiology of gene amplification during carcinogenesis is not yet completely understood but can be considered a result of genetic instability. Gene amplification leads to an increase in protein expression and provides a selective advantage during cell growth. Oncogenes such as CCND1, c-MET, c-MYC, ERBB2, EGFR and MDM2 are amplified in human tumors and can be associated with increased expression of their respective proteins or not. In general, gene amplification is associated with more aggressive tumors, metastases, resistance to chemotherapy and a decrease in the period during which the patient stays free of the disease. This review discusses the major role of gene amplification in the progression of carcinomas, formation of genetic markers and as possible therapeutic targets for the development of drugs for the treatment of some types of tumors.

  16. Rapid and simple identification of carbapenemase genes, blaNDM, blaOXA-48, blaVIM, blaIMP-14and blaKPCgroups, in Gram-negative bacilli by in-house loop-mediated isothermal amplification with hydroxynaphthol blue dye.

    Science.gov (United States)

    Srisrattakarn, Arpasiri; Lulitanond, Aroonlug; Wilailuckana, Chotechana; Charoensri, Nicha; Wonglakorn, Lumyai; Saenjamla, Pimjai; Chaimanee, Prajuab; Daduang, Jureerut; Chanawong, Aroonwadee

    2017-07-01

    Carbapenem-resistant Enterobacteriaceae isolates by carbapenemase production are being reported globally with increasing frequency, leading to limited therapeutic options. We therefore developed a loop-mediated isothermal amplification method with hydroxynaphthol blue dye (LAMP-HNB) for rapid confirmation of bla NDM , bla OXA-48 , bla VIM , bla IMP-14 and bla KPC groups. Sixty-two Enterobacteriaceae and Pseudomonas spp. isolates carrying various carbapenemase genes (28 bla NDM-1 , 9 bla IMP-14a , 2 bla IMP-48 , 1 bla IMP-1 , 1 bla IMP-4 , 1 bla IMP-9 , 1 bla IMP-15 , 4 bla VIM-2 , 1 bla VIM-1 , 1 bla IMP-14a & bla VIM-2 , 7 bla KPC-2 , 3 bla OXA-48 and 3 bla OXA-181 ) and 37 non-carbapenemase-producing Enterobacteriaceae isolates as confirmed by the PCR methods were included. Bacterial DNA was extracted by a simple boiling method. The LAMP-HNB method for each target gene was carried out using a set of six primers under isothermal condition at 65 °C in an ordinary water bath within 60 min and visual measurement of reaction by the change from violet to sky blue. This method had high efficiency (100% sensitivity and specificity) for identifying the bla NDM , bla OXA-48 , bla VIM , bla IMP-14 and bla KPC groups compared with the PCR method. The HNB is easy to prepare, inexpensive and provides reliable results. Therefore, this method could be used as a confirmatory carbapenemase test in routine laboratory or for epidemiological purposes.

  17. Social amplification of risk

    International Nuclear Information System (INIS)

    Kasperson, R.E.; Renn, O.; Slovic, P.; Kasperson, J.X.; Emani, S.

    1989-01-01

    The risks associated with radioactive and other hazardous waste disposal may be expected to interact with societal processes to enlarge or attenuate the consequences of risks and risk events. This article summarizes a conceptual framework that depicts the social amplification of risk. Using a data base of 128 hazard events that have occurred largely over the past ten years, the authors examine the role of physical consequences, media coverage, and public perceptions of risk in generating social and economic impacts. The analysis concludes that social amplification processes substantially shape the nature and magnitude of those impacts but also that such social amplification appears to be systematically related to characteristics of the risks and risk events

  18. Nucleic acid amplification: Alternative methods of polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Md Fakruddin

    2013-01-01

    Full Text Available Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically.

  19. Development and testing of passive autocatalytic recombiners cooled by heat pipes

    International Nuclear Information System (INIS)

    Granzow, Christoph

    2012-01-01

    A severe accident in a nuclear power plant (NPP) can lead to core damage in conjunction with the release of large amounts of hydrogen. As hydrogen mitigation measure, passive autocatalytic recombiners (PARs) are used in today's pressurized water reactors. PARs recombine hydrogen and oxygen contained in the air to steam. The heat from this exothermic reaction causes the catalyst and its surroundings to heat up. If parts of the PAR heat up above the ignition temperature of the gas mixture, a spontaneous deflagration or detonation can occur. The aim of this work is the prevention of such high temperatures by means of passive cooling of the catalyst with heat pipes. Heat pipes are completely passive heat exchanger with a very high effective thermal conductivity. For a deeper understanding of the reaction kinetics at lower temperatures, single catalytic coated heat pipes are studied in a flow reactor. The development of a modular small-scale PAR model is then based on a test series with cooled catalyst sheets. Finally, the PAR model is tested inside a pressure vessel under boundary conditions similar to a real NPP. The experiments show, that the temperatures of the cooled catalytic sheets stay significantly below the temperature of the uncooled sheets and below the ignition temperature of the gas mixture under any set boundary conditions, although no significant reduction of the conversion efficiency can be observed. As a last point, a mathematical model of the reaction kinetics of the recombination process as well as a model of the fluid dynamic and thermohydraulic processes in a heat pipe are developed with the data obtained from the experiments.

  20. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

    Energy Technology Data Exchange (ETDEWEB)

    Gottipati, Keerthi; Acholi, Sudheer [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States); Ruggli, Nicolas [Institute of Virology and Immunology, CH-3147 Mittelhäusern (Switzerland); Choi, Kyung H., E-mail: kychoi@utmb.edu [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States)

    2014-03-15

    Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.

  1. On soliton amplification

    Science.gov (United States)

    Leibovich, S.; Randall, J. D.

    1979-01-01

    The paper considers a modified Korteweg-de Vries equation that permits wave amplification or damping. A 'terminal similarity' solution is identified for large times in amplified systems. Numerical results are given which confirm that the terminal similarity solution is a valid local approximation for mu t sufficiently large and positive, even though the approximation is not uniformly valid in space.

  2. Biomaterials in light amplification

    Science.gov (United States)

    Mysliwiec, Jaroslaw; Cyprych, Konrad; Sznitko, Lech; Miniewicz, Andrzej

    2017-03-01

    Biologically produced or inspired materials can serve as optical gain media, i.e. they can exhibit the phenomenon of light amplification. Some of these materials, under suitable dye-doping and optical pumping conditions, show lasing phenomena. The emerging branch of research focused on obtaining lasing action in highly disordered and highly light scattering materials, i.e. research on random lasing, is perfectly suited for biological materials. The use of biomaterials in light amplification has been extensively reported in the literature. In this review we attempt to report on progress in the development of biologically derived systems able to show the phenomena of light amplification and random lasing together with the contribution of our group to this field. The rich world of biopolymers modified with molecular aggregates and nanocrystals, and self-organized at the nanoscale, offers a multitude of possibilities for tailoring luminescent and light scattering properties that are not easily replicated in conventional organic or inorganic materials. Of particular importance and interest are light amplification and lasing, or random lasing studies in biological cells and tissues. In this review we will describe nucleic acids and their complexes employed as gain media due to their favorable optical properties and ease of manipulation. We will report on research conducted on various biomaterials showing structural analogy to nucleic acids such as fluorescent proteins, gelatins in which the first distributed feedback laser was realized, and also amyloids or silks, which, due to their dye-doped fiber-like structure, allow for light amplification. Other materials that were investigated in that respect include polysaccharides, like starch exhibiting favorable photostability in comparison to other biomaterials, and chitosan, which forms photonic crystals or cellulose. Light amplification and random lasing was not only observed in processed biomaterials but also in living

  3. Development of loop-mediated isothermal amplification method for ...

    African Journals Online (AJOL)

    DR. NJ TONUKARI

    2011-10-10

    mail: wuwenxue@cau.edu.cn. Tel: 86-10-62733048. Fax: 86-10-62733048. was identified in Asia (Murakami et al., 1994; Tian et al.,. 2007). PRRSV was first confirmed in China in 1996 and later became widely spread. Most of ...

  4. Loop-mediated isothermal amplification (LAMP) based detection of ...

    African Journals Online (AJOL)

    Various diseases are caused by pathogenic bacteria and their diagnosis depends on accurate detection of pathogen from clinical samples. Several molecular methods have been developed including PCR, Real Time PCR or multiplex PCR which detects the pathogen accurately. However, every method has some ...

  5. Loop-mediated isothermal amplification (LAMP) based detection of ...

    African Journals Online (AJOL)

    SAM

    2014-05-07

    May 7, 2014 ... used to extract template for the LAMP process. These methods vary depending on the source material and whether RNA or DNA is required for the procedure. Commercial column based kits are most frequently used and have been used successfully for extraction from microbial cell cultures (En et al., 2008; ...

  6. LOOP mediated isothermal AMPlification (LAMP) in diagnosis of ...

    African Journals Online (AJOL)

    We collected a quantity of 35 serum samples of HIVpositive patients and a number of 107 cerebrospinal fluid (CSF) samples of patients who had shown symptoms of meningitis. We designed target specific primers for PCR and LAMP techniques to trace C. neoformans and C. gattii. From the total 142 clinical specimens, five ...

  7. Feedback amplification loop drives malignant growth in epithelial tissues.

    Science.gov (United States)

    Muzzopappa, Mariana; Murcia, Lada; Milán, Marco

    2017-08-29

    Interactions between cells bearing oncogenic mutations and the surrounding microenvironment, and cooperation between clonally distinct cell populations, can contribute to the growth and malignancy of epithelial tumors. The genetic techniques available in Drosophila have contributed to identify important roles of the TNF-α ligand Eiger and mitogenic molecules in mediating these interactions during the early steps of tumor formation. Here we unravel the existence of a tumor-intrinsic-and microenvironment-independent-self-reinforcement mechanism that drives tumor initiation and growth in an Eiger-independent manner. This mechanism relies on cell interactions between two functionally distinct cell populations, and we present evidence that these cell populations are not necessarily genetically different. Tumor-specific and cell-autonomous activation of the tumorigenic JNK stress-activated pathway drives the expression of secreted signaling molecules and growth factors to delaminating cells, which nonautonomously promote proliferative growth of the partially transformed epithelial tissue. We present evidence that cross-feeding interactions between delaminating and nondelaminating cells increase each other's sizes and that these interactions can explain the unlimited growth potential of these tumors. Our results will open avenues toward our molecular understanding of those social cell interactions with a relevant function in tumor initiation in humans.

  8. Application of loop-mediated isothermal amplification (LAMP) of the ...

    African Journals Online (AJOL)

    Administrator

    2011-05-23

    LAMP) of the random insertion mobile element (RIME - LAMP) to diagnose camel Trypanosomiasis in Sudan. Extracted DNA from camels was used to detect Trypanosoma evansi infection in camels using RIME-LAMP technique.

  9. Validation of modified directed graph drawing clustering method for fuzzy autocatalytic set using fuzzy C-means

    Science.gov (United States)

    Nurfatiha, Che Wan; Bakar, Sumarni Abu

    2017-04-01

    Fuzzy Autocatalytic Sets (FACS) is a concept that has been formed from a combination of autocatalytic theory in fuzzy graph theory, particularly on fuzzy graph type-3. This concept has been applied to model a complex system whereby the model is in a form of non-coordinated weighted directed graph. A method to transform the non-coordinated FACS to coordinated FACS known as Modified Directed Graph Drawing (MDGD) had been developed and implemented to the FACS of clinical waste incineration process and circulating fluidized bed boiler. The obvious observation made from the coordinated graphs of FACS show some group of nodes appeared in the graphs, thus making MDGD possible to be used as a clustering technique to cluster the graphs. However, no validation of the technique has been made. Therefore, in this paper, the number of group obtained from MDGD technique for both graphs are validated using Fuzzy C-Means clustering technique. Several clustering validity indices are calculated for several number of clusters set for Fuzzy C-Means and is compared to the group of nodes in the coordinated FACS. The result shows that appropriate number of clusters obtained by using Fuzzy C-Means for clustered FACS with the help of PBM-index is in accordance with the number of groups obtained from MDGD technique. Thus MDGD can be used as a clustering technique to cluster any non-coordinated weighted directed graph.

  10. Derivation of an Analytical Solution to a Reaction-Diffusion Model for Autocatalytic Degradation and Erosion in Polymer Microspheres.

    Science.gov (United States)

    Ford Versypt, Ashlee N; Arendt, Paul D; Pack, Daniel W; Braatz, Richard D

    2015-01-01

    A mathematical reaction-diffusion model is defined to describe the gradual decomposition of polymer microspheres composed of poly(D,L-lactic-co-glycolic acid) (PLGA) that are used for pharmaceutical drug delivery over extended periods of time. The partial differential equation (PDE) model treats simultaneous first-order generation due to chemical reaction and diffusion of reaction products in spherical geometry to capture the microsphere-size-dependent effects of autocatalysis on PLGA erosion that occurs when the microspheres are exposed to aqueous media such as biological fluids. The model is solved analytically for the concentration of the autocatalytic carboxylic acid end groups of the polymer chains that comprise the microspheres as a function of radial position and time. The analytical solution for the reaction and transport of the autocatalytic chemical species is useful for predicting the conditions under which drug release from PLGA microspheres transitions from diffusion-controlled to erosion-controlled release, for understanding the dynamic coupling between the PLGA degradation and erosion mechanisms, and for designing drug release particles. The model is the first to provide an analytical prediction for the dynamics and spatial heterogeneities of PLGA degradation and erosion within a spherical particle. The analytical solution is applicable to other spherical systems with simultaneous diffusive transport and first-order generation by reaction.

  11. Alternative loop rings

    CERN Document Server

    Goodaire, EG; Polcino Milies, C

    1996-01-01

    For the past ten years, alternative loop rings have intrigued mathematicians from a wide cross-section of modern algebra. As a consequence, the theory of alternative loop rings has grown tremendously. One of the main developments is the complete characterization of loops which have an alternative but not associative, loop ring. Furthermore, there is a very close relationship between the algebraic structures of loop rings and of group rings over 2-groups. Another major topic of research is the study of the unit loop of the integral loop ring. Here the interaction between loop rings and group ri

  12. Hardness amplification in nondeterministic logspace

    OpenAIRE

    Gupta, Sushmita

    2007-01-01

    A hard problem is one which cannot be easily computed by efficient algorithms. Hardness amplification is a procedure which takes as input a problem of mild hardness and returns a problem of higher hardness. This is closely related to the task of decoding certain error-correcting codes. We show amplification from mild average case hardness to higher average case hardness for nondeterministic logspace and worst-to-average amplification for nondeterministic linspace. Finally we explore possible ...

  13. Evidence of high-elevation amplification versus Arctic amplification.

    Science.gov (United States)

    Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

    2016-01-12

    Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961-2010 period, we find that the warming for the world's high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification but also a latitudinal amplification. The warming for the high-elevation stations is linearly proportional to the temperature lapse rates along altitudinal and latitudinal gradients, as a result of the functional shape of Stefan-Boltzmann law in both vertical and latitudinal directions. In contrast, neither altitudinal amplification nor latitudinal amplification is found within the Arctic region despite its greater warming than lower latitudes. Further analysis shows that the Arctic amplification is an integrated part of the latitudinal amplification trend for the low-elevation stations (≤500 m above sea level) across the entire low- to high-latitude Northern Hemisphere, also a result of the mathematical shape of Stefan-Boltzmann law but only in latitudinal direction.

  14. Evidence of high-elevation amplification versus Arctic amplification

    Science.gov (United States)

    Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

    2016-01-01

    Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961-2010 period, we find that the warming for the world’s high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification but also a latitudinal amplification. The warming for the high-elevation stations is linearly proportional to the temperature lapse rates along altitudinal and latitudinal gradients, as a result of the functional shape of Stefan-Boltzmann law in both vertical and latitudinal directions. In contrast, neither altitudinal amplification nor latitudinal amplification is found within the Arctic region despite its greater warming than lower latitudes. Further analysis shows that the Arctic amplification is an integrated part of the latitudinal amplification trend for the low-elevation stations (≤500 m above sea level) across the entire low- to high-latitude Northern Hemisphere, also a result of the mathematical shape of Stefan-Boltzmann law but only in latitudinal direction.

  15. Efficient audio power amplification - challenges

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Michael A.E.

    2005-07-01

    For more than a decade efficient audio power amplification has evolved and today switch-mode audio power amplification in various forms are the state-of-the-art. The technical steps that lead to this evolution are described and in addition many of the challenges still to be faced and where extensive research and development are needed is covered. (au)

  16. Efficient Audio Power Amplification - Challenges

    DEFF Research Database (Denmark)

    Andersen, Michael Andreas E.

    2005-01-01

    For more than a decade efficient audio power amplification has evolved and today switch-mode audio power amplification in various forms are the state-of-the-art. The technical steps that lead to this evolution are described and in addition many of the challenges still to be faced and where...... extensive research and development are needed is covered....

  17. Comparison of polymerase chain reaction (PCR) and loop-mediated ...

    African Journals Online (AJOL)

    Comparison of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) for diagnosis of Fusarium solani in human immunodeficiency virus (HIV) positive patients. ... The test was carried out in 1 h reaction at 65°C in a heater block. The specificity of the test was 100% and its sensitivity was a ...

  18. Development and application of a loop-mediated isothermal ...

    African Journals Online (AJOL)

    Haemophilus parasuis is the causative agent of Glässer's disease that has received much attention recently, due to the increasing economic losses this disease inflicts upon the pig industry worldwide. In this study, loop-mediated isothermal amplification method (LAMP) methodology was designed for diagnosing H.

  19. Loop Transfer Matrix and Loop Quantum Mechanics

    International Nuclear Information System (INIS)

    Savvidy, George K.

    2000-01-01

    The gonihedric model of random surfaces on a 3d Euclidean lattice has equivalent representation in terms of transfer matrix K(Q i ,Q f ), which describes the propagation of loops Q. We extend the previous construction of the loop transfer matrix to the case of nonzero self-intersection coupling constant κ. We introduce the loop generalization of Fourier transformation which allows to diagonalize transfer matrices, that depend on symmetric difference of loops only and express all eigenvalues of 3d loop transfer matrix through the correlation functions of the corresponding 2d statistical system. The loop Fourier transformation allows to carry out the analogy with quantum mechanics of point particles, to introduce conjugate loop momentum P and to define loop quantum mechanics. We also consider transfer matrix on 4d lattice which describes propagation of memebranes. This transfer matrix can also be diagonalized by using the generalized Fourier transformation, and all its eigenvalues are equal to the correlation functions of the corresponding 3d statistical system. In particular the free energy of the 4d membrane system is equal to the free energy of 3d gonihedric system of loops and is equal to the free energy of 2d Ising model. (author)

  20. DNAzyme Feedback Amplification: Relaying Molecular Recognition to Exponential DNA Amplification.

    Science.gov (United States)

    Liu, Meng; Yin, Qingxin; McConnell, Erin M; Chang, Yangyang; Brennan, John D; Li, Yingfu

    2018-03-26

    Technologies capable of linking DNA amplification to molecular recognition are very desirable for ultrasensitive biosensing applications. We have developed a simple but powerful isothermal DNA amplification method, termed DNAzyme feedback amplification (DFA), that is capable of relaying molecular recognition to exponential DNA amplification. The method incorporates both an RNA-cleaving DNAzyme (RCD) and rolling circle amplification (RCA) carried out by a special DNA polymerase using a circular DNA template. DFA begins with a stimulus-dependent RCA reaction, producing tandemly linked RCDs in long-chain DNA products. These RCDs cleave an RNA-containing DNA sequence to form additional primers that hybridize to the circular DNA molecule, giving rise to DNA assemblies that act as the new inputs for RCA. The RCA reaction and the cleavage event keep on feeding each other autonomously, resulting in exponential growth of repetitive DNA sequences that can be easily detected. This method can be used for the detection of both nucleic acid based targets and non-nucleic acid analytes. In this article, we discuss the conceptual framework of the feedback amplification approach, the essential features of this method as well as remaining challenges and possible solutions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Comparing the Caputo, Caputo-Fabrizio and Atangana-Baleanu derivative with fractional order: Fractional cubic isothermal auto-catalytic chemical system

    Science.gov (United States)

    Saad, K. M.

    2018-03-01

    In this work we extend the standard model for a cubic isothermal auto-catalytic chemical system (CIACS) to a new model of a fractional cubic isothermal auto-catalytic chemical system (FCIACS) based on Caputo (C), Caputo-Fabrizio (CF) and Atangana-Baleanu in the Liouville-Caputo sense (ABC) fractional time derivatives, respectively. We present approximate solutions for these extended models using the q -homotopy analysis transform method ( q -HATM). We solve the FCIACS with the C derivative and compare our results with those obtained using the CF and ABC derivatives. The ranges of convergence of the solutions are found and the optimal values of h , the auxiliary parameter, are derived. Finally, these solutions are compared with numerical solutions of the various models obtained using finite differences and excellent agreement is found.

  2. Next generation Chirped Pulse Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Nees, J.; Biswal, S.; Mourou, G. [Univ. Michigan, Center for Ultrafast Optical Science, Ann Arbor, MI (United States); Nishimura, Akihiko; Takuma, Hiroshi

    1998-03-01

    The limiting factors of Chirped Pulse Amplification (CPA) are discussed and experimental results of CPA in Yb:glass regenerative amplifier are given. Scaling of Yb:glass to the petawatt level is briefly discussed. (author)

  3. Reliability Tests of Aluminium Wedge Wire Bonding on Auto-catalytic Silver Immersion Gold (ASIG) PCB Metallization

    CERN Document Server

    Drozd, A; Kaufmann, S; Manolescu, F; McGill, I

    2011-01-01

    The Auto-catalytic Silver Immersion Gold (ASIG) PCB metallization is a new process that has clear advantages for PCB assembly especially with regard to lead-free soldering. As it may become a popular process in the future for electronics used in physics experiments, the quality of this metallization for aluminium wire bonding has been studied. Aluminium wedge wire bonding continues to be the interconnection method of choice for many physics detector sensors, for high density signal routing and for unpackaged die. Although advertised as having good quality for aluminium wire bonding, this study was performed to verify this claim as well as to test the longer term reliability of the wire bonds taking into consideration the environmental conditions and life-expectancy of devices, in particular for high energy physics detector applications. The tests were performed on PCBs made with the ASIG and ENIG (Electro-less Nickel Immersion Gold) processes at the same time in order to make a comparison with the current ind...

  4. Evaluation of passive autocatalytic recombiners (PARS) performance for a PWR-konvoi containment type with Gothic 8.1 code

    International Nuclear Information System (INIS)

    Lopez-Alonso Conty, E.; Papini, D.; Jimenez Varas, G.

    2016-01-01

    The study presented in this work analyses the evaluation of Passive Autocatalytic Recombiners (PARs) performance for a PWR-Konvoi containment type as a result of an international collaboration between the Paul Scherrer institute (PSI) and the Universidad Politecnica de Madrid (UPM). The implementation study analyzes the size, location and number of the PARs to minimize the risk arising from a hydrogen release and its distribution in the containment building during a hypothetical severe accident. A detailed 3D model of containment was used for the simulations developed for the Gothic 8.1 code. In the first place, the hydrogen preferential pathways and points of hydrogen accumulation were studies and identified starting from the base case scenario without any mitigation measure. The severe accident scenario chosen is a fast release of hydrogen-steam mixture from hot leg creep rupture during SBO (Station Black-Out) accident. Secondly a configuration of PARs was simulated under the same conditions of the unmitigated case. The PAR configuration offered an improvement in the chosen accident scenario, decreasing the hydrogen concentration values below the flammability limit /hydrogen concentration below 7%) in all the containment compartments. (Author)

  5. Renormalization of loop functions for all loops

    International Nuclear Information System (INIS)

    Brandt, R.A.; Neri, F.; Sato, M.

    1981-01-01

    It is shown that the vacuum expectation values W(C 1 ,xxx, C/sub n/) of products of the traces of the path-ordered phase factors P exp[igcontour-integral/sub C/iA/sub μ/(x)dx/sup μ/] are multiplicatively renormalizable in all orders of perturbation theory. Here A/sub μ/(x) are the vector gauge field matrices in the non-Abelian gauge theory with gauge group U(N) or SU(N), and C/sub i/ are loops (closed paths). When the loops are smooth (i.e., differentiable) and simple (i.e., non-self-intersecting), it has been shown that the generally divergent loop functions W become finite functions W when expressed in terms of the renormalized coupling constant and multiplied by the factors e/sup -K/L(C/sub i/), where K is linearly divergent and L(C/sub i/) is the length of C/sub i/. It is proved here that the loop functions remain multiplicatively renormalizable even if the curves have any finite number of cusps (points of nondifferentiability) or cross points (points of self-intersection). If C/sub γ/ is a loop which is smooth and simple except for a single cusp of angle γ, then W/sub R/(C/sub γ/) = Z(γ)W(C/sub γ/) is finite for a suitable renormalization factor Z(γ) which depends on γ but on no other characteristic of C/sub γ/. This statement is made precise by introducing a regularization, or via a loop-integrand subtraction scheme specified by a normalization condition W/sub R/(C-bar/sub γ/) = 1 for an arbitrary but fixed loop C-bar/sub γ/. Next, if C/sub β/ is a loop which is smooth and simple except for a cross point of angles β, then W(C/sub β/) must be renormalized together with the loop functions of associated sets S/sup i//sub β/ = ]C/sup i/ 1 ,xxx, C/sup i//sub p/i] (i = 2,xxx,I) of loops C/sup i//sub q/ which coincide with certain parts of C/sub β/equivalentC 1 1 . Then W/sub R/(S/sup i//sub β/) = Z/sup i/j(β)W(S/sup j//sub β/) is finite for a suitable matrix Z/sup i/j

  6. Does classroom amplification aid comprehension?

    Science.gov (United States)

    Arnold, P; Canning, D

    1999-06-01

    Many classrooms are noisy and this interferes with listening and teaching. FM soundfield (FM) amplification systems have been developed which provide a uniform soundfield throughout the classroom and increase the speech-signal:noise ratio. The effect on comprehension of such a system was investigated. Forty-nine pupils (comprising the two top classes of a mainstream primary school) participated in this study, with a mean age of 9.92 years (range 8.58-11.42 years). The Neale Analysis of Reading Ability (Neale, 1988a, b) was modified and administered as a spoken comprehension test. Tests of nonverbal intelligence, auditory memory and a questionnaire were given. The passages spoken though the FM amplification system were understood better than the comparable unamplified passages. Auditory memory, sex and non-verbal intelligence had no effect on improved comprehension. FM amplification significantly improved comprehension and could be considered for use in other schools.

  7. Study of anti corrosive behaviour on A I 6061 samples covered with Ni-P alloys obtained by autocatalytic method

    International Nuclear Information System (INIS)

    Castro, M. E; Barbero, J. A; Bubach, E

    2006-01-01

    There are many ways to keep safe an industrial material from corrosion attack.One is covering the piece with a layer of another material which corrosion resistance is higher to the one of the element to protect.The anticorrosion protection mechanism is achieved by the formation of a physical pore less barrier without any defects.This avoid the arrival of those agents from environment responsible of electrochemical attack.In this paper, corrosion resistance of metallic coatings over nuclear usage aluminum samples is analyzed.Our interest is aimed on nickel I phosphorous alloy coatings (Ni I P) obtained by electroless method (autocatalytic) over Al 6061 alloy samples.A comparative study is carried on with different phosphorous contents but always under 12 %.This job is completed with other nickel coating, Vitro vac 0080 (with no phosphorous content) in order to compare structures and anti corrosive properties.Besides, the comparison between mentioned materials and aluminum samples is made.The study is carried on using superficial characterization of each sample with or without coating through a series of complementary techniques such as chemical, electrochemical (linear sweep voltammetry, cyclic voltammetry, polarization resistance determination) and physical (scanning electronic microscopy, hardness determination) techniques.Finally, variable correlation is made as a function of the phosphorous content in the samples used in the experiences.The coating structure obtained is amorphous.It presents no pore or failure and its hardness shows important values.The electrochemical analysis allows to check that anti corrosive protection capacity of Ni-P alloy increases with the phosphorous content in the coat. Al 6061 by itself demonstrate an electrochemically bad behaviour.Substrate I coating adherence is very good [es

  8. Genome position and gene amplification

    Czech Academy of Sciences Publication Activity Database

    Jirsová, Pavla; Snijders, A.M.; Kwek, S.; Roydasgupta, R.; Fridlyand, J.; Tokuyasu, T.; Pinkel, D.; Albertson, D. G.

    2007-01-01

    Roč. 8, č. 6 (2007), r120 ISSN 1474-760X Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : gene amplification * array comparative genomic hybridization * oncogene Subject RIV: BO - Biophysics Impact factor: 6.589, year: 2007

  9. On loop extensions and cohomology of loops

    OpenAIRE

    Benítez, Rolando Jiménez; Meléndez, Quitzeh Morales

    2015-01-01

    In this paper are defined cohomology-like groups that classify loop extensions satisfying a given identity in three variables for association identities, and in two variables for the case of commutativity. It is considered a large amount of identities. This groups generalize those defined in works of Nishigori [2] and of Jhonson and Leedham-Green [4]. It is computed the number of metacyclic extensions for trivial action of the quotient on the kernel in one particular case for left Bol loops a...

  10. Neutron transport in irradiation loops (IRENE loop)

    International Nuclear Information System (INIS)

    Sarsam, Maher.

    1980-09-01

    This thesis is composed of two parts with different aspects. Part one is a technical description of the loop and its main ancillary facilities as well as of the safety and operational regulations. The measurement methods on the model of the ISIS reactor and on the loop in the OSIRIS reactor are described. Part two deals with the possibility of calculating the powers dissipated by each rod of the fuel cluster, using appropriate computer codes, not only in the reflector but also in the core and to suggest a method of calculation [fr

  11. MATLAB: For While Loops

    OpenAIRE

    2005-01-01

    sim tut Simulation Tutorial Interactive Media Element This interactive tutorial on MATLAB covers the For Loop and the While Loop functions. Examples are provided with step-by-step animated explanations. The interactions involve entering MATLAB instructions and observing the outcomes. Self-check questions are provided to help learners determine their level of understanding of the content presented. EC1010 Introduction to MATLAB

  12. Water loop for training

    International Nuclear Information System (INIS)

    Moeller, S.V.

    1983-02-01

    The procedures used to operate the water loop of the Institute of Nuclear Enginering (IEN) in Brazil are presented. The aim is to help future operators of the training water loop in the operation technique and in a better comprehension of the phenomena occured during the execution of an experience. (E.G.) [pt

  13. Accelerator for amplification of energy

    International Nuclear Information System (INIS)

    Mori, Yoshiharu

    1998-01-01

    As a forming method of new nuclear energy, an energy amplification system using accelerator driven subcritical reactor is focussed. In order to realize amplification of energy driven by accelerator, development of an accelerator with excellent electric power efficiency is one of the most important problems. The necessary beam power of accelerator is 10 MW, and when reducing used electric power of the accelerator to under 25% of total power generation, more than 30% of electric power efficiency is required for the accelerator. Therefore, an accelerator with excellent electric power efficiency without experiencing before now is required to realize such an aim. A prominent candidate of the accelerator is FFAG (Fixed Field Alternating Gradient) synchrotron (may be called ring synchrotron). In this paper, some simple considerations of electric power efficiency of accelerators and basic parameter of FFAG synchrotron were described. (G.K.)

  14. Laser amplification in excited dielectrics

    Science.gov (United States)

    Winkler, Thomas; Haahr-Lillevang, Lasse; Sarpe, Cristian; Zielinski, Bastian; Götte, Nadine; Senftleben, Arne; Balling, Peter; Baumert, Thomas

    2018-01-01

    Wide-bandgap dielectrics such as glasses or water are transparent at visible and infrared wavelengths. This changes when they are exposed to ultrashort and highly intense laser pulses. Different interaction mechanisms lead to the appearance of various transient nonlinear optical phenomena. Using these, the optical properties of dielectrics can be controlled from the transparent to the metal-like state. Here we expand this range by a yet unexplored mechanism in excited dielectrics: amplification. In a two-colour pump-probe experiment, we show that a 400 nm femtosecond laser pulse is coherently amplified inside an excited sapphire sample on a scale of a few micrometres. Simulations strongly support the proposed two-photon stimulated emission process, which is temporally and spatially controllable. Consequently, we expect applications in all fields that demand strongly localized amplification.

  15. Frequency domain optical parametric amplification.

    Science.gov (United States)

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

    2014-05-07

    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 μm wavelength.

  16. Laser amplification in excited dielectrics

    DEFF Research Database (Denmark)

    Winkler, Thomas; Haahr-Lillevang, Lasse; Sarpe, Cristian

    2018-01-01

    Wide-bandgap dielectrics such as glasses or water are transparent at visible and infrared wavelengths. This changes when they are exposed to ultrashort and highly intense laser pulses. Different interaction mechanisms lead to the appearance of various transient nonlinear optical phenomena. Using...... these, the optical properties of dielectrics can be controlled from the transparent to the metal-like state. Here we expand this range by a yet unexplored mechanism in excited dielectrics: amplification. In a two-colour pump-probe experiment, we show that a 400nm femtosecond laser pulse is coherently...... amplified inside an excited sapphire sample on a scale of a few micrometres. Simulations strongly support the proposed two-photon stimulated emission process, which is temporally and spatially controllable. Consequently, we expect applications in all fields that demand strongly localized amplification....

  17. Differential voltage amplification from ferroelectric negative capacitance

    Science.gov (United States)

    Khan, Asif I.; Hoffmann, Michael; Chatterjee, Korok; Lu, Zhongyuan; Xu, Ruijuan; Serrao, Claudy; Smith, Samuel; Martin, Lane W.; Hu, Chenming; Ramesh, Ramamoorthy; Salahuddin, Sayeef

    2017-12-01

    We demonstrate that a ferroelectric can cause a differential voltage amplification without needing an external energy source. As the ferroelectric switches from one polarization state to the other, a transfer of energy takes place from the ferroelectric to the dielectric, determined by the ratio of their capacitances, which, in turn, leads to the differential amplification. This amplification is very different in nature from conventional inductor-capacitor based circuits where an oscillatory amplification can be observed. The demonstration of differential voltage amplification from completely passive capacitor elements only has fundamental ramifications for next generation electronics.

  18. Study of the behavior to corrosion of samples of nuclear grade aluminium sheathed in nickel-phosphorous alloys obtained autocatalytically

    International Nuclear Information System (INIS)

    Castro, Maria Eugenia; Barbero, Jose Alfredo; Bubach, Ernesto

    2006-01-01

    One of the ways to protect an industrially important metallic material against corrosion is by covering the piece with an approximately 1 μm layer of a material whose resistance to corrosion is greater than the element being protected. The mechanism by which the anticorrosive protection is obtained is with the formation of a pore free physical barrier without defects that impedes the arrival of the agents responsible for the electrochemical attack. Other sacrifice anodes such as aluminum or zinc have protective forms based on their dissolution as a consequence of their less electrochemically noble behavior to preserve the material. This work studies the resistance to the corrosion of metallic coatings on nuclear grade aluminum substrates. The focus is on coating nickel-phosphorous (ni-P) alloys obtained autocatalytically from aluminum 6061. A comparative study is carried out of a series of electroless nickel coatings containing different amounts of the latter element, but without surpassing the threshold of 12%. The work includes the study of another nickel coating, Vitrovac 0080 (without phosphorous content) in order to compare structures and anticorrosive properties. These materials are also compared with the Al6061 substrate without any kind of coating. The study is carried out with surface characterization of each one of the samples with or without coating using a series of complementary techniques, such as chemical and electrochemical techniques (linear-sweep voltammetry, cyclic voltammetry, determination of the polarization resistance) and physical techniques (SEM microscopy, determination of micro-hardness). The correlation of variables is carried out later as a function of the phosphorous content of the test samples. The structures obtained from the coatings are amorphous. They have no pores or faults and have high hardness values. The electrochemical study proves that the anticorrosive protection capacity of the Ni-P alloy increases along with the

  19. Spheromak Impedance and Current Amplification

    International Nuclear Information System (INIS)

    Fowler, T K; Hua, D D; Stallard, B W

    2002-01-01

    It is shown that high current amplification can be achieved only by injecting helicity on the timescale for reconnection, τ REC , which determines the effective impedance of the spheromak. An approximate equation for current amplification is: dI TOR 2 /dt ∼ I 2 /τ REC - I TOR 2 /τ closed where I is the gun current, I TOR is the spheromak toroidal current and τ CLOSED is the ohmic decay time of the spheromak. Achieving high current amplification, I TOR >> I, requires τ REC CLOSED . For resistive reconnection, this requires reconnection in a cold zone feeding helicity into a hot zone. Here we propose an impedance model based on these ideas in a form that can be implemented in the Corsica-based helicity transport code. The most important feature of the model is the possibility that τ REC actually increases as the spheromak temperature increases, perhaps accounting for the ''voltage sag'' observed in some experiments, and a tendency toward a constant ratio of field to current, B ∝ I, or I TOR ∼ I. Program implications are discussed

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

    Directory of Open Access Journals (Sweden)

    Kristina Roskos

    Full Text Available 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.

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

    Science.gov (United States)

    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

  2. Blind Loop Syndrome

    Science.gov (United States)

    ... of tissue that protrude through the intestinal wall (diverticulosis) Certain medical conditions, including Crohn's disease, radiation enteritis, ... History of radiation therapy to the abdomen Diabetes Diverticulosis of the small intestine Complications A blind loop ...

  3. Diffusion of Wilson loops

    International Nuclear Information System (INIS)

    Brzoska, A.M.; Lenz, F.; Thies, M.; Negele, J.W.

    2005-01-01

    A phenomenological analysis of the distribution of Wilson loops in SU(2) Yang-Mills theory is presented in which Wilson loop distributions are described as the result of a diffusion process on the group manifold. It is shown that, in the absence of forces, diffusion implies Casimir scaling and, conversely, exact Casimir scaling implies free diffusion. Screening processes occur if diffusion takes place in a potential. The crucial distinction between screening of fundamental and adjoint loops is formulated as a symmetry property related to the center symmetry of the underlying gauge theory. The results are expressed in terms of an effective Wilson loop action and compared with various limits of SU(2) Yang-Mills theory

  4. Mashup the OODA Loop

    National Research Council Canada - National Science Library

    Heier, Jeffrey E

    2008-01-01

    ...) processes via the Observe, Orient, Decide, and Act (OODA) Loop concept. As defined by Wikipedia, a mashup is a Website or application that combines the content from more than one source into an integrated presentation...

  5. Dechanneling by dislocation loops

    International Nuclear Information System (INIS)

    Chalant, Gerard.

    1976-09-01

    Ion implantation always induces the creation of dislocation loops. When the damage profile is determined by a backscattering technique, the dechanneling by these loops is implicitely at the origin of these measurements. The dechanneling of alpha particles by dislocation loops produced by the coalescence of quenched-in vacancies in aluminium is studied. The dechanneling and the concentration of loops were determined simultaneously. The dechanneling width around dislocation was found equal to lambda=6A, both for perfect and imperfect loops having a mean diameter d=250A. In the latter case, a dechanneling probability chi=0.34 was determined for the stacking fault, in good agreement with previous determination in gold. A general formula is proposed which takes into account the variation of lambda with the curvature (or the diameter d) of the loops. Finally, by a series of isothermal anneals, the self-diffusion energy ΔH of aluminium was measured. The value obtained ΔH=1.32+-0.10eV is in good agreement with the values obtained by other methods [fr

  6. Looping in OLSRv2 in Mobile Ad-Hoc Networks, Loop Suppression and Loop Correction

    Science.gov (United States)

    Speakman, Lee; Owada, Yasunori; Mase, Kenichi

    Transient routing loops have been observed to form in Mobile Ad-hoc Networks running the OLSRv2 proactive link-state routing protocol. The packets falling into loops impact the surrounding network thus degrading throughput even though only a small proportion of the traffic may enter these loops and only for a short time. This becomes significantly more evident when Link Layer Notification is used to catch broken links, inadvertently leading to an increase in the number of loops. Two methods of Loop Detection are introduced and are used to trigger either Loop Suppression by selectively and preemptively discarding the looping packets that are unlikely to reach their destination, or Loop Correction by the notification of the routing protocol to cut the link over which the packet is looping. The newly introduced Loop Suppression and Loop Correction techniques used with Link Layer Notification are shown to significantly increase network performance over plain OLSRv2 and OLSRv2 with Link Layer Notification.

  7. Resonant primordial gravitational waves amplification

    Directory of Open Access Journals (Sweden)

    Chunshan Lin

    2016-01-01

    Full Text Available 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.

  8. The importance of carry out studies about the use of passive autocatalytic recombiners for hydrogen control in reactors type ESBWR; La importancia de realizar estudios sobre el uso de recombinadores autocataliticos pasivos para control de hidrogeno en reactores tipo ESBWR

    Energy Technology Data Exchange (ETDEWEB)

    Sanchez J, J.; Morales S, J. B. [UNAM, Facultad de Ingenieria, Departamento de Sistemas Energeticos, Ciudad Universitaria, 04510 Mexico D. F. (Mexico)], e-mail: jersonsanchez@gmail.com

    2009-10-15

    A way to satisfy and to guarantee the energy necessities in the future is increasing in a gradual way the creation of nuclear power plants, introducing advanced designs in its systems that contribute in way substantial in the security of the same nuclear plants. The tendency of new designs of these nuclear plants is the incorporation of systems more reliable and sure, and that the operation does not depend on external factors as the electric power, motors diesel or the action of the operator of nuclear plant, what is known as security passive systems. In this sense, the passive autocatalytic recombiners are a contribution toward the use of this type of systems. At the present time it is had studies of the incorporation of passive autocatalytic recombiners in nuclear plants in operation and that they have contributed to minimize the danger associated to hydrogen. The present work contains a first approach to the study of hydrogen recombiners incorporation in advanced nuclear plants, for this case in a nuclear power plant of ESBWR type. To achieve our objective it seeks to use specialized codes as RELAP/SCDAP to obtain simulations of passive autocatalytic recombiners behaviour and we can to estimate their operation inside the reactor contention, contemplating the possibility to use other codes like SCILAB and/or MATLAB for the simulation of a passive autocatalytic recombiner. (Author)

  9. Chirped pulse Raman amplification in plasma

    Energy Technology Data Exchange (ETDEWEB)

    Vieux, G; Lyachev, A; Yang, X; Ersfeld, B; Farmer, J P; Brunetti, E; Issac, R C; Raj, G; Welsh, G H; Wiggins, S M; Jaroszynski, D A, E-mail: d.a.jaroszynski@strath.ac.uk [Department of Physics, Scottish Universities Physics Alliance, University of Strathclyde, Glasgow G4 0NG (United Kingdom)

    2011-06-15

    Raman amplification in plasma has been proposed to be a promising method of amplifying short radiation pulses. Here, we investigate chirped pulse Raman amplification (CPRA) where the pump pulse is chirped and leads to spatiotemporal distributed gain, which exhibits superradiant scaling in the linear regime, usually associated with the nonlinear pump depletion and Compton amplification regimes. CPRA has the potential to serve as a high-efficiency high-fidelity amplifier/compressor stage.

  10. Risk Perception and Social Amplification

    International Nuclear Information System (INIS)

    Smith, R.E.

    2001-01-01

    This paper seeks to consider social amplification as it applies to risk perception. Perceptions of the magnitude of a risk are conditioned by issues such as the degree of uncertainty in probability and consequences, the nature of the consequences and the relative weightings placed on probability and consequences. Risk perceptions are also influenced by factors such as confidence in the operator of an industrial process, trust in the regulator and the perceived fairness of regulatory decision-making. Different people may hold different views about these issues and there may also be difficulties in communication. The paper identifies and discusses self-reinforcing mechanisms, which will be labelled 'lock-in' here. They appear to apply in many situations where social amplification is observed. Historically, the term 'lock-in' has been applied mainly in the technological context but, in this paper, four types of lock-in are identified, namely scientific/technological, economic, social and institutional lock-in. One type of lock-in tends to lead to the next and all are buttressed by people's general acceptance of the familiar, fear of the unknown and resistance to change. The regulator seeks to make decisions which achieve the common good rather than supporting or perpetuating any set of vested interests. In this regard the locked-in positions of stakeholders, whether organisations, interest groups, or individual members of the public, are obstacles and challenges. Existing methods of consultation are unsatisfactory in terms of achieving a proper and productive level of dialogue with stakeholders

  11. Closing global material loops

    DEFF Research Database (Denmark)

    Prosman, Ernst-Jan; Wæhrens, Brian Vejrum; Liotta, Giacomo

    2017-01-01

    Replacing virgin materials with waste materials, a practice known as Industrial Symbiosis (IS), has been identified as a key strategy for closing material loops. This article adopts a critical view on geographic proximity and external coordinators – two key enablers of IS. By ‘uncovering’ a case ...... for geographic proximity and external coordinators. In doing so, our insights into firm-level challenges of long-distance IS exchanges contribute to closing global material loops by increasing the number of potential circular pathways....

  12. Closed Loop Subspace Identification

    Directory of Open Access Journals (Sweden)

    Geir W. Nilsen

    2005-07-01

    Full Text Available A new three step closed loop subspace identifications algorithm based on an already existing algorithm and the Kalman filter properties is presented. The Kalman filter contains noise free states which implies that the states and innovation are uneorre lated. The idea is that a Kalman filter found by a good subspace identification algorithm will give an output which is sufficiently uncorrelated with the noise on the output of the actual process. Using feedback from the output of the estimated Kalman filter in the closed loop system a subspace identification algorithm can be used to estimate an unbiased model.

  13. Electricity-free amplification and detection for molecular point-of-care diagnosis of HIV-1.

    Science.gov (United States)

    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 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.

  14. Multiscale image contrast amplification (MUSICA)

    Science.gov (United States)

    Vuylsteke, Pieter; Schoeters, Emile P.

    1994-05-01

    This article presents a novel approach to the problem of detail contrast enhancement, based on multiresolution representation of the original image. The image is decomposed into a weighted sum of smooth, localized, 2D basis functions at multiple scales. Each transform coefficient represents the amount of local detail at some specific scale and at a specific position in the image. Detail contrast is enhanced by non-linear amplification of the transform coefficients. An inverse transform is then applied to the modified coefficients. This yields a uniformly contrast- enhanced image without artefacts. The MUSICA-algorithm is being applied routinely to computed radiography images of chest, skull, spine, shoulder, pelvis, extremities, and abdomen examinations, with excellent acceptance. It is useful for a wide range of applications in the medical, graphical, and industrial area.

  15. Loop Quantum Gravity

    Directory of Open Access Journals (Sweden)

    Rovelli Carlo

    1998-01-01

    Full Text Available The problem of finding the quantum theory of the gravitational field, and thus understanding what is quantum spacetime, is still open. One of the most active of the current approaches is loop quantum gravity. Loop quantum gravity is a mathematically well-defined, non-perturbative and background independent quantization of general relativity, with its conventional matter couplings. Research in loop quantum gravity today forms a vast area, ranging from mathematical foundations to physical applications. Among the most significant results obtained are: (i The computation of the physical spectra of geometrical quantities such as area and volume, which yields quantitative predictions on Planck-scale physics. (ii A derivation of the Bekenstein-Hawking black hole entropy formula. (iii An intriguing physical picture of the microstructure of quantum physical space, characterized by a polymer-like Planck scale discreteness. This discreteness emerges naturally from the quantum theory and provides a mathematically well-defined realization of Wheeler's intuition of a spacetime ``foam''. Long standing open problems within the approach (lack of a scalar product, over-completeness of the loop basis, implementation of reality conditions have been fully solved. The weak part of the approach is the treatment of the dynamics: at present there exist several proposals, which are intensely debated. Here, I provide a general overview of ideas, techniques, results and open problems of this candidate theory of quantum gravity, and a guide to the relevant literature.

  16. Justification by Infinite Loops

    NARCIS (Netherlands)

    Peijnenburg, A.J.M.; Atkinson, David

    2010-01-01

    In an earlier paper we have shown that a proposition can have a well-defined probability value, even if its justification consists of an infinite linear chain. In the present paper we demonstrate that the same holds if the justification takes the form of a closed loop. Moreover, in the limit that

  17. Improving Loop Dependence Analysis

    DEFF Research Database (Denmark)

    Jensen, Nicklas Bo; Karlsson, Sven

    2017-01-01

    Programmers can no longer depend on new processors to have significantly improved single-thread performance. Instead, gains have to come from other sources such as the compiler and its optimization passes. Advanced passes make use of information on the dependencies related to loops. We improve th...

  18. Risk Perception and Social Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Smith, R.E. [Environment Agency (United Kingdom)

    2001-07-01

    This paper seeks to consider social amplification as it applies to risk perception. Perceptions of the magnitude of a risk are conditioned by issues such as the degree of uncertainty in probability and consequences, the nature of the consequences and the relative weightings placed on probability and consequences. Risk perceptions are also influenced by factors such as confidence in the operator of an industrial process, trust in the regulator and the perceived fairness of regulatory decision-making. Different people may hold different views about these issues and there may also be difficulties in communication. The paper identifies and discusses self-reinforcing mechanisms, which will be labelled 'lock-in' here. They appear to apply in many situations where social amplification is observed. Historically, the term 'lock-in' has been applied mainly in the technological context but, in this paper, four types of lock-in are identified, namely scientific/technological, economic, social and institutional lock-in. One type of lock-in tends to lead to the next and all are buttressed by people's general acceptance of the familiar, fear of the unknown and resistance to change. The regulator seeks to make decisions which achieve the common good rather than supporting or perpetuating any set of vested interests. In this regard the locked-in positions of stakeholders, whether organisations, interest groups, or individual members of the public, are obstacles and challenges. Existing methods of consultation are unsatisfactory in terms of achieving a proper and productive level of dialogue with stakeholders.

  19. Ototoxicity of loop diuretics.

    Science.gov (United States)

    Rybak, L P

    1993-10-01

    The loop diuretics are drugs that increase the excretion of water and electrolytes in the urine by their action on the cells in the loop of Henle. Clinical reports of ototoxicity of these agents are reviewed, and the results of a number of studies in experimental animals are discussed. These drugs can cause either a temporary, or in some cases, a permanent loss of hearing in patients. Animal experiments show that these drugs act on the stria vascularis, producing edema of these tissues and a temporary loss of function, resulting in a decrease of the endocochlear potential. This can result in secondary effects on sound-evoked measures of hearing. As new information unfolds about protective agents, it may be possible to preserve hearing and maintain the desired therapeutic effect.

  20. Loop Quantum Cosmology

    Directory of Open Access Journals (Sweden)

    Bojowald Martin

    2008-07-01

    Full Text Available Quantum gravity is expected to be necessary in order to understand situations in which classical general relativity breaks down. In particular in cosmology one has to deal with initial singularities, i.e., the fact that the backward evolution of a classical spacetime inevitably comes to an end after a finite amount of proper time. This presents a breakdown of the classical picture and requires an extended theory for a meaningful description. Since small length scales and high curvatures are involved, quantum effects must play a role. Not only the singularity itself but also the surrounding spacetime is then modified. One particular theory is loop quantum cosmology, an application of loop quantum gravity to homogeneous systems, which removes classical singularities. Its implications can be studied at different levels. The main effects are introduced into effective classical equations, which allow one to avoid the interpretational problems of quantum theory. They give rise to new kinds of early-universe phenomenology with applications to inflation and cyclic models. To resolve classical singularities and to understand the structure of geometry around them, the quantum description is necessary. Classical evolution is then replaced by a difference equation for a wave function, which allows an extension of quantum spacetime beyond classical singularities. One main question is how these homogeneous scenarios are related to full loop quantum gravity, which can be dealt with at the level of distributional symmetric states. Finally, the new structure of spacetime arising in loop quantum gravity and its application to cosmology sheds light on more general issues, such as the nature of time.

  1. Loop Quantum Cosmology.

    Science.gov (United States)

    Bojowald, Martin

    2008-01-01

    Quantum gravity is expected to be necessary in order to understand situations in which classical general relativity breaks down. In particular in cosmology one has to deal with initial singularities, i.e., the fact that the backward evolution of a classical spacetime inevitably comes to an end after a finite amount of proper time. This presents a breakdown of the classical picture and requires an extended theory for a meaningful description. Since small length scales and high curvatures are involved, quantum effects must play a role. Not only the singularity itself but also the surrounding spacetime is then modified. One particular theory is loop quantum cosmology, an application of loop quantum gravity to homogeneous systems, which removes classical singularities. Its implications can be studied at different levels. The main effects are introduced into effective classical equations, which allow one to avoid the interpretational problems of quantum theory. They give rise to new kinds of early-universe phenomenology with applications to inflation and cyclic models. To resolve classical singularities and to understand the structure of geometry around them, the quantum description is necessary. Classical evolution is then replaced by a difference equation for a wave function, which allows an extension of quantum spacetime beyond classical singularities. One main question is how these homogeneous scenarios are related to full loop quantum gravity, which can be dealt with at the level of distributional symmetric states. Finally, the new structure of spacetime arising in loop quantum gravity and its application to cosmology sheds light on more general issues, such as the nature of time. Supplementary material is available for this article at 10.12942/lrr-2008-4.

  2. Closing the loop.

    Science.gov (United States)

    Dassau, E; Atlas, E; Phillip, M

    2010-02-01

    The dream of closing the loop is actually the dream of creating an artificial pancreas and freeing the patients from being involved with the care of their own diabetes. Insulin-dependent diabetes (type 1) is a chronic incurable disease which requires constant therapy without the possibility of any 'holidays' or insulin-free days. It means that patients have to inject insulin every day of their life, several times per day, and in order to do it safely they also have to measure their blood glucose levels several times per day. Patients need to plan their meals, their physical activities and their insulin regime - there is only very small room for spontaneous activities. This is why the desire for an artificial pancreas is so strong despite the fact that it will not cure the diabetic patients. Attempts to develop a closed-loop system started in the 1960s but never got to a clinical practical stage of development. In recent years the availability of continuous glucose sensors revived those efforts and stimulated the clinician and researchers to believe that closing the loop might be possible nowadays. Many papers have been published over the years describing several different ideas on how to close the loop. Most of the suggested systems have a sensing arm that measures the blood glucose repeatedly or continuously, an insulin delivery arm that injects insulin upon command and a computer that makes the decisions of when and how much insulin to deliver. The differences between the various published systems in the literature are mainly in their control algorithms. However, there are also differences related to the method and site of glucose measurement and insulin delivery. SC glucose measurements and insulin delivery are the most studied option but other combinations of insulin measurements and glucose delivery including intravascular and intraperitoneal (IP) are explored. We tried to select recent publications that we believe had influenced and inspired people interested

  3. Loop Quantum Cosmology

    Directory of Open Access Journals (Sweden)

    Bojowald Martin

    2005-12-01

    Full Text Available Quantum gravity is expected to be necessary in order to understand situations where classical general relativity breaks down. In particular in cosmology one has to deal with initial singularities, i.e., the fact that the backward evolution of a classical space-time inevitably comes to an end after a finite amount of proper time. This presents a breakdown of the classical picture and requires an extended theory for a meaningful description. Since small length scales and high curvatures are involved, quantum effects must play a role. Not only the singularity itself but also the surrounding space-time is then modified. One particular realization is loop quantum cosmology, an application of loop quantum gravity to homogeneous systems, which removes classical singularities. Its implications can be studied at different levels. Main effects are introduced into effective classical equations which allow to avoid interpretational problems of quantum theory. They give rise to new kinds of early universe phenomenology with applications to inflation and cyclic models. To resolve classical singularities and to understand the structure of geometry around them, the quantum description is necessary. Classical evolution is then replaced by a difference equation for a wave function which allows to extend space-time beyond classical singularities. One main question is how these homogeneous scenarios are related to full loop quantum gravity, which can be dealt with at the level of distributional symmetric states. Finally, the new structure of space-time arising in loop quantum gravity and its application to cosmology sheds new light on more general issues such as time.

  4. LoopIng: a template-based tool for predicting the structure of protein loops.

    KAUST Repository

    Messih, Mario Abdel

    2015-08-06

    Predicting the structure of protein loops is very challenging, mainly because they are not necessarily subject to strong evolutionary pressure. This implies that, unlike the rest of the protein, standard homology modeling techniques are not very effective in modeling their structure. However, loops are often involved in protein function, hence inferring their structure is important for predicting protein structure as well as function.We describe a method, LoopIng, based on the Random Forest automated learning technique, which, given a target loop, selects a structural template for it from a database of loop candidates. Compared to the most recently available methods, LoopIng is able to achieve similar accuracy for short loops (4-10 residues) and significant enhancements for long loops (11-20 residues). The quality of the predictions is robust to errors that unavoidably affect the stem regions when these are modeled. The method returns a confidence score for the predicted template loops and has the advantage of being very fast (on average: 1 min/loop).www.biocomputing.it/loopinganna.tramontano@uniroma1.itSupplementary data are available at Bioinformatics online.

  5. Amplification of chirality in liquid crystals

    NARCIS (Netherlands)

    Eelkema, Rienk; Feringa, Ben L.

    2006-01-01

    The amplification of molecular chirality by liquid crystalline systems is widely applied in investigations towards enantioselective solvent - solute interactions, chiral supramolecular assemblies, smart materials, and the development of liquid crystal displays. Here we present an overview of recent

  6. Privacy amplification for quantum key distribution

    International Nuclear Information System (INIS)

    Watanabe, Yodai

    2007-01-01

    This paper examines classical privacy amplification using a universal family of hash functions. In quantum key distribution, the adversary's measurement can wait until the choice of hash functions is announced, and so the adversary's information may depend on the choice. Therefore the existing result on classical privacy amplification, which assumes the independence of the choice from the other random variables, is not applicable to this case. This paper provides a security proof of privacy amplification which is valid even when the adversary's information may depend on the choice of hash functions. The compression rate of the proposed privacy amplification can be taken to be the same as that of the existing one with an exponentially small loss in secrecy of a final key. (fast track communication)

  7. Rolling circle amplification of metazoan mitochondrialgenomes

    Energy Technology Data Exchange (ETDEWEB)

    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.

  8. Four-photon parametric amplification in semiconductors

    International Nuclear Information System (INIS)

    Jain, M.; Gersten, J.; Tzoar, N.

    1975-01-01

    A theoretical study of four-photon parametric amplification in narrow-band-gap semiconductors is made. It is shown that phase matching is achievable in a linear geometry if a magnetic field is employed. Furthermore, a substantial cyclotron resonance enhancement occurs in the presence of a magnetic field. We calculate the growth rates and threshold fields associated with the parametric amplification and conclude that an efficient laser may be designed based on this process

  9. Sm doped mesoporous CeO2 nanocrystals: aqueous solution-based surfactant assisted low temperature synthesis, characterization and their improved autocatalytic activity.

    Science.gov (United States)

    Mandal, Bappaditya; Mondal, Aparna; Ray, Sirsendu Sekhar; Kundu, Amar

    2016-01-28

    Mesoporous Sm(3+) doped CeO2 (Ce-Sm) with a nanocrystalline framework, a high content of Ce(3+) and surface area (184 m(2) g(-1)), have been synthesized through a facile aqueous solution-based surfactant assisted route by using inorganic precursors and sodium dodecyl sulphate as a template. The XRD results indicate that the calcined Ce-Sm and even the as-prepared material have a cubic fluorite structure of CeO2 with no crystalline impurity phase. XRD studies along with HRTEM results confirmed the formation of mesoporous nanocrystalline CeO2 at a lower temperature as low as 100 °C. A detailed analysis revealed that Sm(3+) doping in CeO2 has increased the lattice volume, surface area, mesopore volume and engineered the surface defects. Higher concentrations of Ce(3+) and oxygen vacancies of Ce-Sm resulted in lowering of the band gap. It is evident from the H2-TPR results that Sm(3+) doping in CeO2 strongly modified the reduction behavior of CeO2 by shifting the bulk reduction at a much lower temperature, indicating increased oxygen mobility in the sample which enables enhanced oxygen diffusion at lower temperatures, thus promoting reducibility, i.e., the process of Ce(4+)→ Ce(3+). UV-visible transmission studies revealed improved autocatalytic performance due to easier Ce(4+)/Ce(3+) recycling in the Sm(3+) doped CeO2 nanoparticles. From the in vitro cytotoxicity of both pure CeO2 and Sm(3+) doped CeO2 calcined at 500 °C in a concentration as high as 100 μg mL(-1) (even after 120 h) on MG-63 cells, no obvious decrease in cell viability is observed, confirming their excellent biocompatibility. The presence of an increased amount of surface hydroxyl groups, mesoporosity, and surface defects have contributed towards an improved autocatalytic activity of mesoporous Ce-Sm, which appear to be a potential candidate for biomedical (antioxidant) applications.

  10. On some properties of conjugacy closed loops

    International Nuclear Information System (INIS)

    Adeniran, John Olusola

    2002-07-01

    It is shown that central loops are not conjugacy closed loops but instead are loops of units in their loop algebras that are conjugacy closed. It is also shown that certain inner mappings of a conjugacy closed loop are nuclear. Some invariants of left conjugacy closed loops are obtained. (author)

  11. Development of Temperature Control Solutions for Non-Instrumented Nucleic Acid Amplification Tests (NINAAT

    Directory of Open Access Journals (Sweden)

    Tamás Pardy

    2017-06-01

    Full Text Available Non-instrumented nucleic acid amplification tests (NINAAT are a novel paradigm in portable molecular diagnostics. They offer the high detection accuracy characteristic of nucleic acid amplification tests (NAAT in a self-contained device, without the need for any external instrumentation. These Point-of-Care tests typically employ a Lab-on-a-Chip for liquid handling functionality, and perform isothermal nucleic acid amplification protocols that require low power but high accuracy temperature control in a single well-defined temperature range. We propose temperature control solutions based on commercially available heating elements capable of meeting these challenges, as well as demonstrate the process by which such elements can be fitted to a NINAAT system. Self-regulated and thermostat-controlled resistive heating elements were evaluated through experimental characterization as well as thermal analysis using the finite element method (FEM. We demonstrate that the proposed solutions can support various NAAT protocols, as well as demonstrate an optimal solution for the loop-mediated isothermal amplification (LAMP protocol. Furthermore, we present an Arduino-compatible open-source thermostat developed for NINAAT applications.

  12. Heat induces gene amplification in cancer cells

    International Nuclear Information System (INIS)

    Yan, Bin; Ouyang, Ruoyun; Huang, Chenghui; Liu, Franklin; Neill, Daniel; Li, Chuanyuan; Dewhirst, Mark

    2012-01-01

    Highlights: ► This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. ► Hyperthermia induces DNA double strand breaks. ► DNA double strand breaks are considered to be required for the initiation of gene amplification. ► 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 °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) γH2AX immunostaining to detect γH2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 °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 °C for 30 min induces DNA double strand breaks in HCT116 cells as shown by γH2AX 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.

  13. High temperature storage loop :

    Energy Technology Data Exchange (ETDEWEB)

    Gill, David Dennis; Kolb, William J.

    2013-07-01

    A three year plan for thermal energy storage (TES) research was created at Sandia National Laboratories in the spring of 2012. This plan included a strategic goal of providing test capability for Sandia and for the nation in which to evaluate high temperature storage (>650ÀC) technology. The plan was to scope, design, and build a flow loop that would be compatible with a multitude of high temperature heat transfer/storage fluids. The High Temperature Storage Loop (HTSL) would be reconfigurable so that it was useful for not only storage testing, but also for high temperature receiver testing and high efficiency power cycle testing as well. In that way, HTSL was part of a much larger strategy for Sandia to provide a research and testing platform that would be integral for the evaluation of individual technologies funded under the SunShot program. DOEs SunShot program seeks to reduce the price of solar technologies to 6/kWhr to be cost competitive with carbon-based fuels. The HTSL project sought to provide evaluation capability for these SunShot supported technologies. This report includes the scoping, design, and budgetary costing aspects of this effort

  14. Loop‑mediated Isothermal Amplification assay (LAMP) based detection of Pasteurella multocida in cases of haemorrhagic septicaemia and fowl cholera.

    Science.gov (United States)

    Bhimani, Mayurkumar; Bhanderi, Bharat; Roy, Ashish

    2015-01-01

    Twenty two isolates of Pasteurella multocida were obtained from different tissues of dead birds and animals (cattle, buffalo, sheep, and goat) suspected of fowl cholera and haemorrhagic septicaemia. The isolates were confirmed as P. multocida by various biochemical tests and PM PCR. An attempt was made to standardize Loop mediated isothermal amplification (LAMP) using newly designed primer sequences of KMT1 gene. Loop mediated isothermal amplification was conducted using 6 sets of primers at 65°C for 30 minutes and the result was confirmed by visual observation using SYBR green fluorescence dye as marker of positive reaction under UV transilluminator. On electrophoretic analysis of the products on 2% agarose gel, a ladder like pattern was observed, which suggested a positive amplification, whereas no amplification was observed in negative controls. Additionally, product of positive reaction yielded a green fluorescence following addition of SYBR green under UV transilluminator. It was observed that LAMP is a more sensitive test than polymerase chain reaction (PCR), as the former could detect DNA to lower limit of 22.8 pg/µl, while the latter could detect DNA to lower limit of 2.28 ng/ µl, thus LAMP could detect 100 times lesser concentration of DNA in comparison to PCR. Loop mediated isothermal amplification is a rather newer molecular technique, which can be used for rapid detection of infectious agent at field level and which does not require sophisticated instrument, i.e. thermal cycler. Furthermore, unlike the conventional PCR technique, LAMP requires lesser time to perform and result can be read visually.

  15. A method for phenomenological and chemical kinetics study of autocatalytic reactive dissolution by optical microscopy. The case of uranium dioxide dissolution in nitric acid media

    Science.gov (United States)

    Marc, Philippe; Magnaldo, Alastair; Godard, Jérémy; Schaer, Éric

    2018-03-01

    Dissolution is a milestone of the head-end of hydrometallurgical processes, as the stabilization rates of the chemical elements determine the process performance and hold-up. This study aims at better understanding the chemical and physico-chemical phenomena of uranium dioxide dissolution reactions in nitric acid media in the Purex process, which separates the reusable materials and the final wastes of the spent nuclear fuels. It has been documented that the attack of sintering-manufactured uranium dioxide solids occurs through preferential attack sites, which leads to the development of cracks in the solids. Optical microscopy observations show that in some cases, the development of these cracks leads to the solid cleavage. It is shown here that the dissolution of the detached fragments is much slower than the process of the complete cleavage of the solid, and occurs with no disturbing phenomena, like gas bubbling. This fact has motivated the measurement of dissolution kinetics using optical microscopy and image processing. By further discriminating between external resistance and chemical reaction, the "true" chemical kinetics of the reaction have been measured, and the highly autocatalytic nature of the reaction confirmed. Based on these results, the constants of the chemical reactions kinetic laws have also been evaluated.

  16. A method for phenomenological and chemical kinetics study of autocatalytic reactive dissolution by optical microscopy. The case of uranium dioxide dissolution in nitric acid media

    Directory of Open Access Journals (Sweden)

    Marc Philippe

    2018-01-01

    Full Text Available Dissolution is a milestone of the head-end of hydrometallurgical processes, as the stabilization rates of the chemical elements determine the process performance and hold-up. This study aims at better understanding the chemical and physico-chemical phenomena of uranium dioxide dissolution reactions in nitric acid media in the Purex process, which separates the reusable materials and the final wastes of the spent nuclear fuels. It has been documented that the attack of sintering-manufactured uranium dioxide solids occurs through preferential attack sites, which leads to the development of cracks in the solids. Optical microscopy observations show that in some cases, the development of these cracks leads to the solid cleavage. It is shown here that the dissolution of the detached fragments is much slower than the process of the complete cleavage of the solid, and occurs with no disturbing phenomena, like gas bubbling. This fact has motivated the measurement of dissolution kinetics using optical microscopy and image processing. By further discriminating between external resistance and chemical reaction, the “true” chemical kinetics of the reaction have been measured, and the highly autocatalytic nature of the reaction confirmed. Based on these results, the constants of the chemical reactions kinetic laws have also been evaluated.

  17. Modeling of compact loop antennas

    International Nuclear Information System (INIS)

    Baity, F.W.

    1987-01-01

    A general compact loop antenna model which treats all elements of the antenna as lossy transmission lines has been developed. In addition to capacitively-tuned resonant double loop (RDL) antennas the model treats stub-tuned resonant double loop antennas. Calculations using the model have been compared with measurements on full-scale mockups of resonant double loop antennas for ATF and TFTR in order to refine the transmission line parameters. Results from the model are presented for RDL antenna designs for ATF, TFTR, Tore Supra, and for the Compact Ignition Tokamak

  18. Noise propagation in gene regulation networks involving interlinked positive and negative feedback loops.

    Directory of Open Access Journals (Sweden)

    Hui Zhang

    Full Text Available It is well known that noise is inevitable in gene regulatory networks due to the low-copy numbers of molecules and local environmental fluctuations. The prediction of noise effects is a key issue in ensuring reliable transmission of information. Interlinked positive and negative feedback loops are essential signal transduction motifs in biological networks. Positive feedback loops are generally believed to induce a switch-like behavior, whereas negative feedback loops are thought to suppress noise effects. Here, by using the signal sensitivity (susceptibility and noise amplification to quantify noise propagation, we analyze an abstract model of the Myc/E2F/MiR-17-92 network that is composed of a coupling between the E2F/Myc positive feedback loop and the E2F/Myc/miR-17-92 negative feedback loop. The role of the feedback loop on noise effects is found to depend on the dynamic properties of the system. When the system is in monostability or bistability with high protein concentrations, noise is consistently suppressed. However, the negative feedback loop reduces this suppression ability (or improves the noise propagation and enhances signal sensitivity. In the case of excitability, bistability, or monostability, noise is enhanced at low protein concentrations. The negative feedback loop reduces this noise enhancement as well as the signal sensitivity. In all cases, the positive feedback loop acts contrary to the negative feedback loop. We also found that increasing the time scale of the protein module or decreasing the noise autocorrelation time can enhance noise suppression; however, the systems sensitivity remains unchanged. Taken together, our results suggest that the negative/positive feedback mechanisms in coupled feedback loop dynamically buffer noise effects rather than only suppressing or amplifying the noise.

  19. Vortex loops and Majoranas

    International Nuclear Information System (INIS)

    Chesi, Stefano; Jaffe, Arthur; Loss, Daniel; Pedrocchi, Fabio L.

    2013-01-01

    We investigate the role that vortex loops play in characterizing eigenstates of interacting Majoranas. We give some general results and then focus on ladder Hamiltonian examples as a test of further ideas. Two methods yield exact results: (i) A mapping of certain spin Hamiltonians to quartic interactions of Majoranas shows that the spectra of these two examples coincide. (ii) In cases with reflection-symmetric Hamiltonians, we use reflection positivity for Majoranas to characterize vortices in the ground states. Two additional methods suggest wider applicability of these results: (iii) Numerical evidence suggests similar behavior for certain systems without reflection symmetry. (iv) A perturbative analysis also suggests similar behavior without the assumption of reflection symmetry

  20. Dynamic PID loop control

    International Nuclear Information System (INIS)

    Pei, L.; Klebaner, A.; Theilacker, J.; Soyars, W.; Martinez, A.; Bossert, R.; DeGraff, B.; Darve, C.

    2011-01-01

    The Horizontal Test Stand (HTS) SRF Cavity and Cryomodule 1 (CM1) of eight 9-cell, 1.3GHz SRF cavities are operating at Fermilab. For the cryogenic control system, how to hold liquid level constant in the cryostat by regulation of its Joule-Thompson JT-valve is very important after cryostat cool down to 2.0 K. The 72-cell cryostat liquid level response generally takes a long time delay after regulating its JT-valve; therefore, typical PID control loop should result in some cryostat parameter oscillations. This paper presents a type of PID parameter self-optimal and Time-Delay control method used to reduce cryogenic system parameters oscillation.

  1. Closed loop reflux system

    International Nuclear Information System (INIS)

    De Witt, R.; Jepson, B.E.; Schwind, R.A.

    1975-01-01

    Sulfur isotopes are continuously separated and enriched using a closed loop reflux system wherein sulfur dioxide (SO 2 ) is reacted with sodium hydroxide (NaOH) or the like to form sodium hydrogen sulfite (NaHSO 3 ). Heavier sulfur isotopes are preferentially attracted to the NaHSO 3 , and subsequently reacted with sulfuric acid (H 2 SO 4 ) forming sodium hydrogen sulfate (NaHSO 4 ) and SO 2 gas, which contains increased concentrations of the heavier sulfur isotopes. This heavy isotope enriched SO 2 gas is subsequently separated and the NaHSO 4 is reacted with NaOH to form sodium sulfate (Na 2 SO 4 ), which is subsequently decomposed in an electrodialysis unit to form the NaOH and H 2 SO 4 components, which are used in the aforesaid reactions thereby effecting sulfur isotope separation and enrichment without objectionable loss of feed materials. (U.S.)

  2. Loop Quantum Gravity

    Directory of Open Access Journals (Sweden)

    Rovelli Carlo

    2008-07-01

    Full Text Available The problem of describing the quantum behavior of gravity, and thus understanding quantum spacetime, is still open. Loop quantum gravity is a well-developed approach to this problem. It is a mathematically well-defined background-independent quantization of general relativity, with its conventional matter couplings. Today research in loop quantum gravity forms a vast area, ranging from mathematical foundations to physical applications. Among the most significant results obtained so far are: (i The computation of the spectra of geometrical quantities such as area and volume, which yield tentative quantitative predictions for Planck-scale physics. (ii A physical picture of the microstructure of quantum spacetime, characterized by Planck-scale discreteness. Discreteness emerges as a standard quantum effect from the discrete spectra, and provides a mathematical realization of Wheeler’s “spacetime foam” intuition. (iii Control of spacetime singularities, such as those in the interior of black holes and the cosmological one. This, in particular, has opened up the possibility of a theoretical investigation into the very early universe and the spacetime regions beyond the Big Bang. (iv A derivation of the Bekenstein–Hawking black-hole entropy. (v Low-energy calculations, yielding n-point functions well defined in a background-independent context. The theory is at the roots of, or strictly related to, a number of formalisms that have been developed for describing background-independent quantum field theory, such as spin foams, group field theory, causal spin networks, and others. I give here a general overview of ideas, techniques, results and open problems of this candidate theory of quantum gravity, and a guide to the relevant literature.

  3. Loop Quantum Gravity.

    Science.gov (United States)

    Rovelli, Carlo

    2008-01-01

    The problem of describing the quantum behavior of gravity, and thus understanding quantum spacetime , is still open. Loop quantum gravity is a well-developed approach to this problem. It is a mathematically well-defined background-independent quantization of general relativity, with its conventional matter couplings. Today research in loop quantum gravity forms a vast area, ranging from mathematical foundations to physical applications. Among the most significant results obtained so far are: (i) The computation of the spectra of geometrical quantities such as area and volume, which yield tentative quantitative predictions for Planck-scale physics. (ii) A physical picture of the microstructure of quantum spacetime, characterized by Planck-scale discreteness. Discreteness emerges as a standard quantum effect from the discrete spectra, and provides a mathematical realization of Wheeler's "spacetime foam" intuition. (iii) Control of spacetime singularities, such as those in the interior of black holes and the cosmological one. This, in particular, has opened up the possibility of a theoretical investigation into the very early universe and the spacetime regions beyond the Big Bang. (iv) A derivation of the Bekenstein-Hawking black-hole entropy. (v) Low-energy calculations, yielding n -point functions well defined in a background-independent context. The theory is at the roots of, or strictly related to, a number of formalisms that have been developed for describing background-independent quantum field theory, such as spin foams, group field theory, causal spin networks, and others. I give here a general overview of ideas, techniques, results and open problems of this candidate theory of quantum gravity, and a guide to the relevant literature.

  4. Uranyl Nitrate Flow Loop

    International Nuclear Information System (INIS)

    Ladd-Lively, Jennifer L

    2008-01-01

    The objectives of the work discussed in this report were to: (1) develop a flow loop that would simulate the purified uranium-bearing aqueous stream exiting the solvent extraction process in a natural uranium conversion plant (NUCP); (2) develop a test plan that would simulate normal operation and disturbances that could be anticipated in an NUCP; (3) use the flow loop to test commercially available flowmeters for use as safeguards monitors; and (4) recommend a flowmeter for production-scale testing at an NUCP. There has been interest in safeguarding conversion plants because the intermediate products [uranium dioxide (UO 2 ), uranium tetrafluoride (UF 4 ), and uranium hexafluoride (UF 6 )] are all suitable uranium feedstocks for producing special nuclear materials. Furthermore, if safeguards are not applied virtually any nuclear weapons program can obtain these feedstocks without detection by the International Atomic Energy Agency (IAEA). Historically, IAEA had not implemented safeguards until the purified UF 6 product was declared as feedstock for enrichment plants. H. A. Elayat et al. provide a basic definition of a safeguards system: 'The function of a safeguards system on a chemical conversion plant is in general terms to verify that no useful nuclear material is being diverted to use in a nuclear weapons program'. The IAEA now considers all highly purified uranium compounds as candidates for safeguarding. DOE is currently interested in 'developing instruments, tools, strategies, and methods that could be of use to the IAEA in the application of safeguards' for materials found in the front end of the nuclear fuel cycle-prior to the production of the uranium hexafluoride or oxides that have been the traditional starting point for IAEA safeguards. Several national laboratories, including Oak Ridge, Los Alamos, Lawrence Livermore, and Brookhaven, have been involved in developing tools or techniques for safeguarding conversion plants. This study was sponsored by

  5. Development and clinical performance of high throughput loop-mediated isothermal amplification for detection of malaria.

    Directory of Open Access Journals (Sweden)

    Rushini S Perera

    Full Text Available Accurate and efficient detection of sub-microscopic malaria infections is crucial for enabling rapid treatment and interruption of transmission. Commercially available malaria LAMP kits have excellent diagnostic performance, though throughput is limited by the need to prepare samples individually. Here, we evaluate the clinical performance of a newly developed high throughput (HTP sample processing system for use in conjunction with the Eiken malaria LAMP kit.The HTP system utilised dried blood spots (DBS and liquid whole blood (WB, with parallel sample processing of 94 samples per run. The system was evaluated using 699 samples of known infection status pre-determined by gold standard nested PCR.The sensitivity and specificity of WB-HTP-LAMP was 98.6% (95% CI, 95.7-100, and 99.7% (95% CI, 99.2-100; sensitivity of DBS-HTP-LAMP was 97.1% (95% CI, 93.1-100, and specificity 100% against PCR. At parasite densities greater or equal to 2 parasites/μL, WB and DBS HTP-LAMP showed 100% sensitivity and specificity against PCR. At densities less than 2 p/μL, WB-HTP-LAMP sensitivity was 88.9% (95% CI, 77.1-100 and specificity was 99.7% (95% CI, 99.2-100; sensitivity and specificity of DBS-HTP-LAMP was 77.8% (95% CI, 54.3-99.5 and 100% respectively.The HTP-LAMP system is a highly sensitive diagnostic test, with the potential to allow large scale population screening in malaria elimination campaigns.

  6. Sulfur Mustard Induces Apoptosis in Lung Epithelial Cells via a Caspase Amplification Loop

    Science.gov (United States)

    2010-01-01

    SM inhalation by normal breathing. Twenty-four hours after SM exposure, bronchoalveolar lavage (BAL) samples were collected to analyze cellular com...grafted onto nude mice . ol et al. (2009) recently showed that peptide inhibitors specific or caspase-8 or -9 could prevent SM-induced microvesication...ntibodies and reprobed with other antibodies to compare different proteins from he same blot as described in Rosenthal et al. (2003). .5. Bronchoalveolar

  7. Rapid and specific identification of Brucella abortus using the loop-mediated isothermal amplification (LAMP) assay.

    Science.gov (United States)

    Kang, Sung-Il; Her, Moon; Kim, Ji-Yeon; Lee, Jin Ju; Lee, Kichan; Sung, So-Ra; Jung, Suk Chan

    2015-06-01

    A rapid and accurate diagnosis of brucellosis is required to reduce and prevent the spread of disease among animals and the risk of transfer to humans. In this study, a Brucella abortus-specific (Ba) LAMP assay was developed, that had six primers designed from the BruAb2_0168 region of chromosome I. The specificity of this LAMP assay was confirmed with Brucella reference strains, B. abortus vaccine strains, B. abortus isolates and phylogenetically or serologically related strains. The detection limit of target DNA was up to 20 fg/μl within 60 min. The sensitivity of the new LAMP assay was equal to or slightly higher than other PCR based assays. Moreover, this Ba-LAMP assay could specifically amplify all B. abortus biovars compared to previous PCR assays. To our knowledge, this is the first report of specific detection of B. abortus using a LAMP assay. The Ba-LAMP assay can offer a rapid, sensitive and accurate diagnosis of bovine brucellosis in the field. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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.

  9. Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Common Strains of Escherichia coli▿

    Science.gov (United States)

    Hill, Joshua; Beriwal, Shilpa; Chandra, Ishwad; Paul, Vinod K.; Kapil, Aarti; Singh, Tripti; Wadowsky, Robert M.; Singh, Vinita; Goyal, Ankur; Jahnukainen, Timo; Johnson, James R.; Tarr, Phillip I.; Vats, Abhay

    2008-01-01

    We developed a highly sensitive and specific LAMP assay for Escherichia coli. It does not require DNA extraction and can detect as few as 10 copies. It detected all 36 of 36 E. coli isolates and all 22 urine samples (out of 89 samples tested) that had E. coli. This assay is rapid, low in cost, and simple to perform. PMID:18550738

  10. Loop-mediated isothermal amplification assay for rapid detection of common strains of Escherichia coli.

    Science.gov (United States)

    Hill, Joshua; Beriwal, Shilpa; Chandra, Ishwad; Paul, Vinod K; Kapil, Aarti; Singh, Tripti; Wadowsky, Robert M; Singh, Vinita; Goyal, Ankur; Jahnukainen, Timo; Johnson, James R; Tarr, Phillip I; Vats, Abhay

    2008-08-01

    We developed a highly sensitive and specific LAMP assay for Escherichia coli. It does not require DNA extraction and can detect as few as 10 copies. It detected all 36 of 36 E. coli isolates and all 22 urine samples (out of 89 samples tested) that had E. coli. This assay is rapid, low in cost, and simple to perform.

  11. ESTIMATION OF AMPLIFICATION FACTOR IN EARTHQUAKE ENGINEERING

    Directory of Open Access Journals (Sweden)

    Nazarov Yuriy Pavlovich

    2015-03-01

    Full Text Available The authors are the developers of Odyssey Software (Eurosoft Co. for the analysis of seismological data and computing of seismic loads and their parameters. While communicating with the users of the software, the authors have revealed some uncertainty about both understanding of the term "amplification factor (AF" and calculation of the amplification factor using various methods. In this article, a simple example shows that the determination of the amplification factor as the ratio of the acceleration’s spectrum to the maximal acceleration is derived from the classical definition of AF in the form of the ratio of maximal dynamic displacement to the displacement by the action of static load. Deterministic and probabilistic ap-proaches for the calculating of the AF were discussed. There was an example of AFs calculation and their envelopes for translational and rotational components of seismic impact by using Odyssey Software.

  12. Time varying arctic climate change amplification

    Energy Technology Data Exchange (ETDEWEB)

    Chylek, Petr [Los Alamos National Laboratory; Dubey, Manvendra K [Los Alamos National Laboratory; Lesins, Glen [DALLHOUSIE U; Wang, Muyin [NOAA/JISAO

    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.

  13. Amplification, Redundancy, and Quantum Chernoff Information

    Science.gov (United States)

    Zwolak, Michael; Riedel, C. Jess; Zurek, Wojciech H.

    2014-04-01

    Amplification was regarded, since the early days of quantum theory, as a mysterious ingredient that endows quantum microstates with macroscopic consequences, key to the "collapse of the wave packet," and a way to avoid embarrassing problems exemplified by Schrödinger's cat. Such a bridge between the quantum microworld and the classical world of our experience was postulated ad hoc in the Copenhagen interpretation. Quantum Darwinism views amplification as replication, in many copies, of the information about quantum states. We show that such amplification is a natural consequence of a broad class of models of decoherence, including the photon environment we use to obtain most of our information. This leads to objective reality via the presence of robust and widely accessible records of selected quantum states. The resulting redundancy (the number of copies deposited in the environment) follows from the quantum Chernoff information that quantifies the information transmitted by a typical elementary subsystem of the environment.

  14. Loop quantum cosmology: Recent progress

    Indian Academy of Sciences (India)

    Aspects of the full theory of loop quantum gravity can be studied in a simpler context by reducing to symmetric models like cosmological ones. This leads to several applications where loop effects play a significant role when one is sensitive to the quantum regime. As a consequence, the structure of and the approach to ...

  15. RCD+: Fast loop modeling server.

    Science.gov (United States)

    López-Blanco, José Ramón; Canosa-Valls, Alejandro Jesús; Li, Yaohang; Chacón, Pablo

    2016-07-08

    Modeling loops is a critical and challenging step in protein modeling and prediction. We have developed a quick online service (http://rcd.chaconlab.org) for ab initio loop modeling combining a coarse-grained conformational search with a full-atom refinement. Our original Random Coordinate Descent (RCD) loop closure algorithm has been greatly improved to enrich the sampling distribution towards near-native conformations. These improvements include a new workflow optimization, MPI-parallelization and fast backbone angle sampling based on neighbor-dependent Ramachandran probability distributions. The server starts by efficiently searching the vast conformational space from only the loop sequence information and the environment atomic coordinates. The generated closed loop models are subsequently ranked using a fast distance-orientation dependent energy filter. Top ranked loops are refined with the Rosetta energy function to obtain accurate all-atom predictions that can be interactively inspected in an user-friendly web interface. Using standard benchmarks, the average root mean squared deviation (RMSD) is 0.8 and 1.4 Å for 8 and 12 residues loops, respectively, in the challenging modeling scenario in where the side chains of the loop environment are fully remodeled. These results are not only very competitive compared to those obtained with public state of the art methods, but also they are obtained ∼10-fold faster. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Higher dimensional loop quantum cosmology

    International Nuclear Information System (INIS)

    Zhang, Xiangdong

    2016-01-01

    Loop quantum cosmology (LQC) is the symmetric sector of loop quantum gravity. In this paper, we generalize the structure of loop quantum cosmology to the theories with arbitrary spacetime dimensions. The isotropic and homogeneous cosmological model in n + 1 dimensions is quantized by the loop quantization method. Interestingly, we find that the underlying quantum theories are divided into two qualitatively different sectors according to spacetime dimensions. The effective Hamiltonian and modified dynamical equations of n + 1 dimensional LQC are obtained. Moreover, our results indicate that the classical big bang singularity is resolved in arbitrary spacetime dimensions by a quantum bounce. We also briefly discuss the similarities and differences between the n + 1 dimensional model and the 3 + 1 dimensional one. Our model serves as a first example of higher dimensional loop quantum cosmology and offers the possibility to investigate quantum gravity effects in higher dimensional cosmology. (orig.)

  17. Gravitational Wave Detection via Weak Measurements Amplification

    OpenAIRE

    Hu, Meng-Jun; Zhang, Yong-Sheng

    2017-01-01

    A universal amplification scheme of ultra-small phase based on weak measurements is given and a weak measurements amplification based laser interferometer gravitational-wave observatory (WMA-LIGO) is suggested. The WMA-LIGO has potential to amplify the ultra-small phase signal to at least $10^{3}$ order of magnitude such that the sensitivity and bandwidth of gravitational-wave detector can be further improved. Our results not only shed a new light on the quantum measurement but also open a ne...

  18. Optical Pattern Recognition With Self-Amplification

    Science.gov (United States)

    Liu, Hua-Kuang

    1994-01-01

    In optical pattern recognition system with self-amplification, no reference beam used in addressing mode. Polarization of laser beam and orientation of photorefractive crystal chosen to maximize photorefractive effect. Intensity of recognition signal is orders of magnitude greater than other optical correlators. Apparatus regarded as real-time or quasi-real-time optical pattern recognizer with memory and reprogrammability.

  19. Social amplification of risk: a conceptual framework

    International Nuclear Information System (INIS)

    Kasperson, R.E.; Renn, O.; Slovic, P.; Brown, H.S.; Emel, J.; Goble, R.; Kasperson, J.X.; Ratick, S.

    1988-01-01

    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

  20. Desert Amplification in a Warming Climate

    Science.gov (United States)

    Zhou, Liming

    2016-01-01

    Here I analyze the observed and projected surface temperature anomalies over land between 50°S-50°N for the period 1950–2099 by large-scale ecoregion and find strongest warming consistently and persistently seen over driest ecoregions such as the Sahara desert and the Arabian Peninsula during various 30-year periods, pointing to desert amplification in a warming climate. This amplification enhances linearly with the global mean greenhouse gases(GHGs) radiative forcing and is attributable primarily to a stronger GHGs-enhanced downward longwave radiation forcing reaching the surface over drier ecoregions as a consequence of a warmer and thus moister atmosphere in response to increasing GHGs. These results indicate that desert amplification may represent a fundamental pattern of global warming associated with water vapor feedbacks over land in low- and mid- latitudes where surface warming rates depend inversely on ecosystem dryness. It is likely that desert amplification might involve two types of water vapor feedbacks that maximize respectively in the tropical upper troposphere and near the surface over deserts, with both being very dry and thus extremely sensitive to changes of water vapor. PMID:27538725

  1. Intelligence amplification framework for enhancing scheduling processes

    NARCIS (Netherlands)

    Dobrkovic, Andrej; Liu, Luyao; Iacob, Maria Eugenia; van Hillegersberg, Jos

    2016-01-01

    The scheduling process in a typical business environment consists of predominantly repetitive tasks that have to be completed in limited time and often containing some form of uncertainty. The intelligence amplification is a symbiotic relationship between a human and an intelligent agent. This

  2. Optical parametric amplification beyond the slowly varying ...

    Indian Academy of Sciences (India)

    The coupled-wave equations describing optical parametric amplification (OPA) are usually solved in the slowly varying amplitude (SVA) approximation regime, in which the second-order derivatives of the signal and idler amplitudes are ignored and in fact the electromagnetic effects due to exit face of the medium is not ...

  3. Desert Amplification in a Warming Climate.

    Science.gov (United States)

    Zhou, Liming

    2016-08-19

    Here I analyze the observed and projected surface temperature anomalies over land between 50°S-50°N for the period 1950-2099 by large-scale ecoregion and find strongest warming consistently and persistently seen over driest ecoregions such as the Sahara desert and the Arabian Peninsula during various 30-year periods, pointing to desert amplification in a warming climate. This amplification enhances linearly with the global mean greenhouse gases(GHGs) radiative forcing and is attributable primarily to a stronger GHGs-enhanced downward longwave radiation forcing reaching the surface over drier ecoregions as a consequence of a warmer and thus moister atmosphere in response to increasing GHGs. These results indicate that desert amplification may represent a fundamental pattern of global warming associated with water vapor feedbacks over land in low- and mid- latitudes where surface warming rates depend inversely on ecosystem dryness. It is likely that desert amplification might involve two types of water vapor feedbacks that maximize respectively in the tropical upper troposphere and near the surface over deserts, with both being very dry and thus extremely sensitive to changes of water vapor.

  4. BMN correlators by loop equations

    International Nuclear Information System (INIS)

    Eynard, Bertrand; Kristjansen, Charlotte

    2002-01-01

    In the BMN approach to N=4 SYM a large class of correlators of interest are expressible in terms of expectation values of traces of words in a zero-dimensional gaussian complex matrix model. We develop a loop-equation based, analytic strategy for evaluating such expectation values to any order in the genus expansion. We reproduce the expectation values which were needed for the calculation of the one-loop, genus one correction to the anomalous dimension of BMN-operators and which were earlier obtained by combinatorial means. Furthermore, we present the expectation values needed for the calculation of the one-loop, genus two correction. (author)

  5. Isothermal amplification of environmental DNA (eDNA for direct field-based monitoring and laboratory confirmation of Dreissena sp.

    Directory of Open Access Journals (Sweden)

    Maggie R Williams

    Full Text Available Loop-mediated isothermal amplification (LAMP of aquatic invasive species environmental DNA (AIS eDNA was used for rapid, sensitive, and specific detection of Dreissena sp. relevant to the Great Lakes (USA basin. The method was validated for two uses including i direct amplification of eDNA using a hand filtration system and ii confirmation of the results after DNA extraction using a conventional thermal cycler run at isothermal temperatures. Direct amplification eliminated the need for DNA extraction and purification and allowed detection of target invasive species in grab or concentrated surface water samples, containing both free DNA as well as larger cells and particulates, such as veligers, eggs, or seeds. The direct amplification method validation was conducted using Dreissena polymorpha and Dreissena bugensis and uses up to 1 L grab water samples for high target abundance (e.g., greater than 10 veligers (larval mussels per L for Dreissena sp. or 20 L samples concentrated through 35 μm nylon screens for low target abundance, at less than 10 veligers per liter water. Surface water concentrate samples were collected over a period of three years, mostly from inland lakes in Michigan with the help of a network of volunteers. Field samples collected from 318 surface water locations included i filtered concentrate for direct amplification validation and ii 1 L grab water sample for eDNA extraction and confirmation. Though the extraction-based protocol was more sensitive (resulting in more positive detections than direct amplification, direct amplification could be used for rapid screening, allowing for quicker action times. For samples collected between May and August, results of eDNA direct amplification were consistent with known presence/absence of selected invasive species. A cross-platform smartphone application was also developed to disseminate the analyzed results to volunteers. Field tests of the direct amplification protocol using a

  6. Optical loop framing

    International Nuclear Information System (INIS)

    Kalibjian, R.; Chong, Y.P.; Prono, D.S.; Cavagnolo, H.R.

    1984-06-01

    The ATA provides an electron beam pulse of 70-ns duration at a 1-Hz rate. Our present optical diagnostics technique involve the imaging of the visible light generated by the beam incident onto the plant of a thin sheet of material. It has already been demonstrated that the light generated has a sufficiently fast temporal reponse in performing beam diagnostics. Notwithstanding possible beam emittance degradation due to scattering in the thin sheet, the observation of beam spatial profiles with relatively high efficiencies has provided data complementary to that obtained from beam wall current monitors and from various x-ray probes and other electrical probes. The optical image sensor consists of a gated, intensified television system. The gate pulse of the image intensifier can be appropriately delayed to give frames that are time-positioned from the head to the tail of the beam with a minimum gate time of 5-ns. The spatial correlation of the time frames from pulse to pulse is very good for a stable electron beam; however, when instabilities do occur, it is difficult to properly assess the spatial composition of the head and the tail of the beam on a pulse-to-pulse basis. Multiple gating within a pulse duration becomes desirable but cannot be performed because the recycle time (20-ms) of the TV system is much longer than the beam pulse. For this reason we have developed an optical-loop framing technique that will allow the recording of two frames within one pulse duration with our present gated/intensified TV system

  7. Loop quantum cosmology: Recent progress

    Indian Academy of Sciences (India)

    . These techniques and their implications can be illustrated and tested in simple sit- uations by introducing symmetries, which is the origin of loop quantum cosmology. The symmetry reduction can be done in such a way that the characteristic ...

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

    Science.gov (United States)

    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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

    Directory of Open Access Journals (Sweden)

    La Mura Maurizio

    2009-02-01

    Full Text Available Abstract 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.

  10. Dynamical behaviour in coronal loops

    Science.gov (United States)

    Haisch, Bernhard M.

    1986-01-01

    Rapid variability has been found in two active region coronal loops observed by the X-ray Polychromator (XRP) and the Hard X-ray Imaging Spectrometer (HXIS) onboard the Solar Maximum Mission (SMM). There appear to be surprisingly few observations of the short-time scale behavior of hot loops, and the evidence presented herein lends support to the hypothesis that coronal heating may be impulsive and driven by flaring.

  11. Closed Loop Fluid Delivery System

    Science.gov (United States)

    2014-02-28

    loop fluid delivery system (CLFDS) will integrate a vital signs monitor ( VSM ) and high speed infusion pump (Pump) to respond quickly to drops in...Interface (GUI) shows VSM data, allows the user to select from several injury types (head, uncontrolled hemorrhage, controlled hemorrhage, and three total...the bedrock for future Closed Loop Fluid System Pre-Market Approval application(s) to FDA. 6. Major Issues Clinical study testing revealed a

  12. Plasmonic Terahertz Amplification in Graphene-Based Asymmetric Hyperbolic Metamaterial

    Directory of Open Access Journals (Sweden)

    Igor Nefedov

    2015-05-01

    Full Text Available We propose and theoretically explore terahertz amplification, based on stimulated generation of plasmons in graphene asymmetric hyperbolic metamaterials (AHMM, strongly coupled to terahertz radiation. In contrast to the terahertz amplification in resonant nanocavities, AHMM provides a wide-band THz amplification without any reflection in optically thin graphene multilayers.

  13. RNA amplification for successful gene profiling analysis

    Directory of Open Access Journals (Sweden)

    Wang Ena

    2005-07-01

    Full Text Available Abstract The study of clinical samples is often limited by the amount of material available to study. While proteins cannot be multiplied in their natural form, DNA and RNA can be amplified from small specimens and used for high-throughput analyses. Therefore, genetic studies offer the best opportunity to screen for novel insights of human pathology when little material is available. Precise estimates of DNA copy numbers in a given specimen are necessary. However, most studies investigate static variables such as the genetic background of patients or mutations within pathological specimens without a need to assess proportionality of expression among different genes throughout the genome. Comparative genomic hybridization of DNA samples represents a crude exception to this rule since genomic amplification or deletion is compared among different specimens directly. For gene expression analysis, however, it is critical to accurately estimate the proportional expression of distinct RNA transcripts since such proportions directly govern cell function by modulating protein expression. Furthermore, comparative estimates of relative RNA expression at different time points portray the response of cells to environmental stimuli, indirectly informing about broader biological events affecting a particular tissue in physiological or pathological conditions. This cognitive reaction of cells is similar to the detection of electroencephalographic patterns which inform about the status of the brain in response to external stimuli. As our need to understand human pathophysiology at the global level increases, the development and refinement of technologies for high fidelity messenger RNA amplification have become the focus of increasing interest during the past decade. The need to increase the abundance of RNA has been met not only for gene specific amplification, but, most importantly for global transcriptome wide, unbiased amplification. Now gene

  14. Comparison of the Soil Dynamic Amplification Factor and Soil Amplification by Using Microtremor and MASW Methods Respectively

    Science.gov (United States)

    Tuncel, Aykut; Cevdet Özdag, Özkan; Pamuk, Eren; Akgün, Mustafa

    2017-12-01

    Single Station Microtremor method, which is widely used nowadays, is an effective and easy applicable method. In this study, dynamic amplification factor distributions of the study area were obtained using scenario earthquake parameters with single station microtremor data gathered at 112 points. In addition, a surface wave active method, which is known as MASW (Multichannel Analysis of Surface Waves), was applied at 43 profiles to calculate the soil amplification values. Dynamic amplification factor (DAF), soil amplification, the predominant soil period (PSP), geology and topography data of the study area were analysed together. Dynamic amplification factor and soil amplification values were obtained 2 or higher at about sea level parts of the study area which are generally composed of alluvial units. Additionally, in high altitude regions that are composed of volcanic rocks, relatively lower dynamic amplification factor and soil amplification values were obtained. The minimum amplification value in the study area was 1.15, while the maximum amplification value was 3.05 according to the dynamic amplification results and the soil amplification values were between 1.16 and 3.85 in harmony. It is seen that the obtained DAF values and the soil amplification values calculated from the seismic velocities are very similar to each other numerically and regionally. Because of this, it is concluded that the values of the soil amplification obtained by the MASW method and the calculated DAF values in this study are in harmony with each other. Although the depths of research in these two calculation methods are different from each other, the similarity of the results allows us to arrive at the result of how effective the ground layer is on the amplification. It has a great importance to calculate the amplification values and other dynamic parameters by in situ measurements for a planned plot because geological units can vary even at very short distances in heterogeneously

  15. Parametric nanomechanical amplification at very high frequency.

    Science.gov (United States)

    Karabalin, R B; Feng, X L; Roukes, M L

    2009-09-01

    Parametric resonance and amplification are important in both fundamental physics and technological applications. Here we report very high frequency (VHF) parametric resonators and mechanical-domain amplifiers based on nanoelectromechanical systems (NEMS). Compound mechanical nanostructures patterned by multilayer, top-down nanofabrication are read out by a novel scheme that parametrically modulates longitudinal stress in doubly clamped beam NEMS resonators. Parametric pumping and signal amplification are demonstrated for VHF resonators up to approximately 130 MHz and provide useful enhancement of both resonance signal amplitude and quality factor. We find that Joule heating and reduced thermal conductance in these nanostructures ultimately impose an upper limit to device performance. We develop a theoretical model to account for both the parametric response and nonequilibrium thermal transport in these composite nanostructures. The results closely conform to our experimental observations, elucidate the frequency and threshold-voltage scaling in parametric VHF NEMS resonators and sensors, and establish the ultimate sensitivity limits of this approach.

  16. Digital Microfluidics for Nucleic Acid Amplification

    Directory of Open Access Journals (Sweden)

    Beatriz Coelho

    2017-06-01

    Full Text Available Digital Microfluidics (DMF has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

  17. Digital Microfluidics for Nucleic Acid Amplification.

    Science.gov (United States)

    Coelho, Beatriz; Veigas, Bruno; Fortunato, Elvira; Martins, Rodrigo; Águas, Hugo; Igreja, Rui; Baptista, Pedro V

    2017-06-25

    Digital Microfluidics (DMF) has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

  18. Preventing PCR amplification carryover contamination in a clinical laboratory.

    Science.gov (United States)

    Aslanzadeh, Jaber

    2004-01-01

    During the past two decades PCR and several other DNA/RNA amplification techniques have become important diagnostic tools in clinical laboratories. Amplification products contamination has been the main impediment to using these techniques routinely in diagnostic laboratories. Over the years, several creative pre- and post-amplification methods have been developed that prevent amplicon carryover contamination. These procedures, coupled with automated systems that employ real-time amplification and simultaneous detection in a closed system, have substantially reduced the possibility of false positive results due to amplification products carryover contamination.

  19. Amplification of trace amounts of nucleic acids

    Science.gov (United States)

    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).

  20. Temperature measurement of accelerator cell solenoid loop

    International Nuclear Information System (INIS)

    Mu Fan; Dong Pan; Dai Zhiyong

    2010-01-01

    This paper presents the research on temperature measurement of solenoid loop. The measuring temperature fiber is layered in solenoid loop for the accelerator cell. When the solenoid loop is supplied with high current form a constant current source, its temperature increases rapidly. The temperature fiber can measure the temperature of the solenoid loop and get temperature measurement rule. Experiment and simulation show temperature of interior solenoid loop the highest and it decreases from the interior to the exterior of solenoid loop. To control temperature of solenoid loop under 60 degree C, simulation displays load interval of constant current source with 80 A current should be at least is 17.5 minutes. (authors)

  1. Tritium Management Loop Design Status

    Energy Technology Data Exchange (ETDEWEB)

    Rader, Jordan D. [ORNL; Felde, David K. [ORNL; McFarlane, Joanna [ORNL; Greenwood, Michael Scott [ORNL; Qualls, A L. [ORNL; Calderoni, Pattrick [Idaho National Laboratory (INL)

    2017-12-01

    This report summarizes physical, chemical, and engineering analyses that have been done to support the development of a test loop to study tritium migration in 2LiF-BeF2 salts. The loop will operate under turbulent flow and a schematic of the apparatus has been used to develop a model in Mathcad to suggest flow parameters that should be targeted in loop operation. The introduction of tritium into the loop has been discussed as well as various means to capture or divert the tritium from egress through a test assembly. Permeation was calculated starting with a Modelica model for a transport through a nickel window into a vacuum, and modifying it for a FLiBe system with an argon sweep gas on the downstream side of the permeation interface. Results suggest that tritium removal with a simple tubular permeation device will occur readily. Although this system is idealized, it suggests that rapid measurement capability in the loop may be necessary to study and understand tritium removal from the system.

  2. A large ungated TPC with GEM amplification

    Science.gov (United States)

    Berger, M.; Ball, M.; Fabbietti, L.; Ketzer, B.; Arora, R.; Beck, R.; Böhmer, F. V.; Chen, J.-C.; Cusanno, F.; Dørheim, S.; García, F.; Hehner, J.; Herrmann, N.; Höppner, C.; Kaiser, D.; Kis̆, M.; Kleipa, V.; Konorov, I.; Kunkel, J.; Kurz, N.; Leifels, Y.; Müllner, P.; Münzer, R.; Neubert, S.; Rauch, J.; Schmidt, C. J.; Schmitz, R.; Soyk, D.; Vandenbroucke, M.; Voss, B.; Walther, D.; Zmeskal, J.

    2017-10-01

    A Time Projection Chamber (TPC) is an ideal device for the detection of charged particle tracks in a large volume covering a solid angle of almost 4 π. The high density of hits on a given particle track facilitates the task of pattern recognition in a high-occupancy environment and in addition provides particle identification by measuring the specific energy loss for each track. For these reasons, TPCs with Multiwire Proportional Chamber (MWPC) amplification have been and are widely used in experiments recording heavy-ion collisions. A significant drawback, however, is the large dead time of the order of 1 ms per event generated by the use of a gating grid, which is mandatory to prevent ions created in the amplification region from drifting back into the drift volume, where they would severely distort the drift path of subsequent tracks. For experiments with higher event rates this concept of a conventional TPC operating with a triggered gating grid can therefore not be applied without a significant loss of data. A continuous readout of the signals is the more appropriate way of operation. This, however, constitutes a change of paradigm with considerable challenges to be met concerning the amplification region, the design and bandwidth of the readout electronics, and the data handling. A mandatory prerequisite for such an operation is a sufficiently good suppression of the ion backflow from the avalanche region, which otherwise limits the tracking and particle identification capabilities of such a detector. Gas Electron Multipliers (GEM) are a promising candidate to combine excellent spatial resolution with an intrinsic suppression of ions. In this paper we describe the design, construction and the commissioning of a large TPC with GEM amplification and without gating grid (GEM-TPC). The design requirements have driven innovations in the construction of a light-weight field-cage, a supporting media flange, the GEM amplification and the readout system, which are

  3. Implementation of antimicrobial peptides for sample preparation prior to nucleic acid amplification in point-of-care settings.

    Science.gov (United States)

    Krõlov, Katrin; Uusna, Julia; Grellier, Tiia; Andresen, Liis; Jevtuševskaja, Jekaterina; Tulp, Indrek; Langel, Ülo

    2017-12-01

    A variety of sample preparation techniques are used prior to nucleic acid amplification. However, their efficiency is not always sufficient and nucleic acid purification remains the preferred method for template preparation. Purification is difficult and costly to apply in point-of-care (POC) settings and there is a strong need for more robust, rapid, and efficient biological sample preparation techniques in molecular diagnostics. Here, the authors applied antimicrobial peptides (AMPs) for urine sample preparation prior to isothermal loop-mediated amplification (LAMP). AMPs bind to many microorganisms such as bacteria, fungi, protozoa and viruses causing disruption of their membrane integrity and facilitate nucleic acid release. The authors show that incubation of E. coli with antimicrobial peptide cecropin P1 for 5 min had a significant effect on the availability of template DNA compared with untreated or even heat treated samples resulting in up to six times increase of the amplification efficiency. These results show that AMPs treatment is a very efficient sample preparation technique that is suitable for application prior to nucleic acid amplification directly within biological samples. Furthermore, the entire process of AMPs treatment was performed at room temperature for 5 min thereby making it a good candidate for use in POC applications.

  4. Thermodynamics in Loop Quantum Cosmology

    International Nuclear Information System (INIS)

    Li, L.F.; Zhu, J.Y.

    2009-01-01

    Loop quantum cosmology (LQC) is very powerful to deal with the behavior of early universe. Moreover, the effective loop quantum cosmology gives a successful description of the universe in the semiclassical region. We consider the apparent horizon of the Friedmann-Robertson-Walker universe as a thermodynamical system and investigate the thermodynamics of LQC in the semiclassical region. The effective density and effective pressure in the modified Friedmann equation from LQC not only determine the evolution of the universe in LQC scenario but also are actually found to be the thermodynamic quantities. This result comes from the energy definition in cosmology (the Misner-Sharp gravitational energy) and is consistent with thermodynamic laws. We prove that within the framework of loop quantum cosmology, the elementary equation of equilibrium thermodynamics is still valid.

  5. High pressure experimental water loop

    International Nuclear Information System (INIS)

    Grenon, M.

    1958-01-01

    A high pressure experimental water loop has been made for studying the detection and evolution of cladding failure in a pressurized reactor. The loop has been designed for a maximum temperature of 360 deg. C, a maximum of 160 kg/cm 2 and flow rates up to 5 m 3 /h. The entire loop consists of several parts: a main circuit with a canned rotor circulation pump, steam pressurizer, heating tubes, two hydro-cyclones (one de-gasser and one decanter) and one tubular heat exchanger; a continuous purification loop, connected in parallel, comprising pressure reducing valves and resin pots which also allow studies of the stability of resins under pressure, temperature and radiation; following the gas separator is a gas loop for studying the recombination of the radiolytic gases in the steam phase. The preceding circuits, as well as others, return to a low pressure storage circuit. The cold water of the low pressure storage flask is continuously reintroduced into the high pressure main circuit by means of a return pump at a maximum head of 160 kg /cm 2 , and adjusted to the pressurizer level. This loop is also a testing bench for the tight high pressure apparatus. The circulating pump and the connecting flanges (Oak Ridge type) are water-tight. The feed pump and the pressure reducing valves are not; the un-tight ones have a system of leak recovery. To permanently check the tightness the circuit has been fitted with a leak detection system (similar to the HRT one). (author) [fr

  6. Lack of correlation between reaction speed and analytical sensitivity in isothermal amplification reveals the value of digital methods for optimization: validation using digital real-time RT-LAMP.

    Science.gov (United States)

    Khorosheva, Eugenia M; Karymov, Mikhail A; Selck, David A; Ismagilov, Rustem F

    2016-01-29

    In this paper, we asked if it is possible to identify the best primers and reaction conditions based on improvements in reaction speed when optimizing isothermal reactions. We used digital single-molecule, real-time analyses of both speed and efficiency of isothermal amplification reactions, which revealed that improvements in the speed of isothermal amplification reactions did not always correlate with improvements in digital efficiency (the fraction of molecules that amplify) or with analytical sensitivity. However, we observed that the speeds of amplification for single-molecule (in a digital device) and multi-molecule (e.g. in a PCR well plate) formats always correlated for the same conditions. Also, digital efficiency correlated with the analytical sensitivity of the same reaction performed in a multi-molecule format. Our finding was supported experimentally with examples of primer design, the use or exclusion of loop primers in different combinations, and the use of different enzyme mixtures in one-step reverse-transcription loop-mediated amplification (RT-LAMP). Our results show that measuring the digital efficiency of amplification of single-template molecules allows quick, reliable comparisons of the analytical sensitivity of reactions under any two tested conditions, independent of the speeds of the isothermal amplification reactions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. LISA Pathfinder: OPD loop characterisation

    Science.gov (United States)

    Born, Michael; LPF Collaboration

    2017-05-01

    The optical metrology system (OMS) of the LISA Pathfinder mission is measuring the distance between two free-floating test masses with unprecedented precision. One of the four OMS heterodyne interferometers reads out the phase difference between the reference and the measurement laser beam. This phase from the reference interferometer is common to all other longitudinal interferometer read outs and therefore subtracted. In addition, the phase is fed back via the digital optical pathlength difference (OPD) control loop to keep it close to zero. Here, we analyse the loop parameters and compare them to on-ground measurement results.

  8. Fermions and loops on graphs: I. Loop calculus for determinants

    International Nuclear Information System (INIS)

    Chernyak, Vladimir Y; Chertkov, Michael

    2008-01-01

    This paper is the first in a series devoted to evaluation of the partition function in statistical models on graphs with loops in terms of the Berezin/fermion integrals. The paper focuses on a representation of the determinant of a square matrix in terms of a finite series, where each term corresponds to a loop on the graph. The representation is based on a fermion version of the loop calculus, previously introduced by the authors for graphical models with finite alphabets. Our construction contains two levels. First, we represent the determinant in terms of an integral over anti-commuting Grassmann variables, with some reparametrization/gauge freedom hidden in the formulation. Second, we show that a special choice of the gauge, called the BP (Bethe–Peierls or belief propagation) gauge, yields the desired loop representation. The set of gauge fixing BP conditions is equivalent to the Gaussian BP equations, discussed in the past as efficient (linear scaling) heuristics for estimating the covariance of a sparse positive matrix

  9. Does the Arctic Amplification peak this decade?

    Science.gov (United States)

    Martin, Torge; Haine, Thomas W. N.

    2017-04-01

    Temperatures rise faster in the Arctic than on global average, a phenomenon known as Arctic Amplification. While this is well established from observations and model simulations, projections of future climate (here: RCP8.5) with models of the Coupled Model Intercomparison Project phase 5 (CMIP5) also indicate that the Arctic Amplification has a maximum. We show this by means of an Arctic Amplification factor (AAF), which we define as the ratio of Arctic mean to global mean surface air temperature (SAT) anomalies. The SAT anomalies are referenced to the period 1960-1980 and smoothed by a 30-year running mean. For October, the multi-model ensemble-mean AAF reaches a maximum in 2017. The maximum moves however to later years as Arctic winter progresses: for the autumn mean SAT (September to November) the maximum AAF is found in 2028 and for winter (December to February) in 2060. Arctic Amplification is driven, amongst others, by the ice-albedo feedback (IAF) as part of the more general surface albedo feedback (involving clouds, snow cover, vegetation changes) and temperature effects (Planck and lapse-rate feedbacks). We note that sea ice retreat and the associated warming of the summer Arctic Ocean are not only an integral part of the IAF but are also involved in the other drivers. In the CMIP5 simulations, the timing of the AAF maximum coincides with the period of fastest ice retreat for the respective month. Presence of at least some sea ice is crucial for the IAF to be effective because of the contrast in surface albedo between ice and open water and the need to turn ocean warming into ice melt. Once large areas of the Arctic Ocean are ice-free, the IAF should be less effective. We thus hypothesize that the ice retreat significantly affects AAF variability and forces a decline of its magnitude after at least half of the Arctic Ocean is ice-free and the ice cover becomes basically seasonal.

  10. Amplification Effects and Unconventional Monetary Policies

    Directory of Open Access Journals (Sweden)

    Cécile BASTIDON GILLES

    2012-02-01

    Full Text Available Global financial crises trigger off amplification effects, which allow relatively small shocks to propagate through the whole financial system. For this reason, the range of Central banks policies is now widening beyond conventional monetary policies and lending of last resort. The aim of this paper is to establish a rule for this practice. The model is based on the formalization of funding conditions in various types of markets. We conduct a comprehensive analysis of the “unconventional monetary policies”, and especially quantify government bonds purchases by the Central bank.

  11. Feedback - closing the loop digitally

    International Nuclear Information System (INIS)

    Zagel, J.; Chase, B.

    1992-01-01

    Many feedback and feedforward systems are now using microprocessors within the loop. We describe the wide range of possibilities and problems that arise. We also propose some ideas for analysis and testing, including examples of motion control in the Flying Wire systems in Main Ring and Tevatron and Low Level RF control now being built for the Fermilab Linac upgrade. (author)

  12. Closing the Loop with Exercises

    Science.gov (United States)

    Altizer, Andy

    2008-01-01

    Conducting exercises provides a critical bridge between the theory of an Emergency Action Plan and its effective implementation. When conducted properly, exercises can fill the gap between training and after-action review to close the preparedness loop--before an actual emergency occurs. Often exercises are planned and conducted on campus based on…

  13. Two loops in eleven dimensions

    CERN Document Server

    Green, Michael B.; Vanhove, Pierre; Green, Michael B.; Kwon, Hwang-h.; Vanhove, Pierre

    2000-01-01

    The two-loop Feynman diagram contribution to the four-graviton amplitude of eleven-dimensional supergravity compactified on a two-torus, T^2, is analyzed in detail. The Schwinger parameter integrations are re-expressed as integration over the moduli space of a second torus, \\hat T^2, which enables the leading low-momentum contribution to be evaluated in terms of maps of \\hat T^2 into T^2. The ultraviolet divergences associated with boundaries of moduli space are regularized in a manner that is consistent with the expected duality symmetries of string theory. This leads to an exact expression for terms of order contraction of four Weyl tensors), thereby extending earlier results for the R^4 term that were based on the one-loop eleven-dimensional amplitude. Precise agreement is found with terms in type IIA and IIB superstring theory that arise from the low energy expansion of the tree-level and one-loop string amplitudes and predictions are made for the coefficients of certain two-loop string theory terms as we...

  14. Loop quantum cosmology and singularities.

    Science.gov (United States)

    Struyve, Ward

    2017-08-15

    Loop quantum gravity is believed to eliminate singularities such as the big bang and big crunch singularity. This belief is based on studies of so-called loop quantum cosmology which concerns symmetry-reduced models of quantum gravity. In this paper, the problem of singularities is analysed in the context of the Bohmian formulation of loop quantum cosmology. In this formulation there is an actual metric in addition to the wave function, which evolves stochastically (rather than deterministically as the case of the particle evolution in non-relativistic Bohmian mechanics). Thus a singularity occurs whenever this actual metric is singular. It is shown that in the loop quantum cosmology for a homogeneous and isotropic Friedmann-Lemaître-Robertson-Walker space-time with arbitrary constant spatial curvature and cosmological constant, coupled to a massless homogeneous scalar field, a big bang or big crunch singularity is never obtained. This should be contrasted with the fact that in the Bohmian formulation of the Wheeler-DeWitt theory singularities may exist.

  15. PONDEROMOTIVE ACCELERATION IN CORONAL LOOPS

    Energy Technology Data Exchange (ETDEWEB)

    Dahlburg, R. B.; Obenschain, K. [LCP and FD, Naval Research Laboratory, Washington, DC 20375 (United States); Laming, J. M. [Space Science Division, Naval Research Laboratory, Washington, DC 20375 (United States); Taylor, B. D. [AFRL Eglin AFB, Pensacola, FL 32542 (United States)

    2016-11-10

    Ponderomotive acceleration has been asserted to be a cause of the first ionization potential (FIP) effect, the well-known enhancement in abundance by a factor of 3–4 over photospheric values of elements in the solar corona with FIP less than about 10 eV. It is shown here by means of numerical simulations that ponderomotive acceleration occurs in solar coronal loops, with the appropriate magnitude and direction, as a “by-product” of coronal heating. The numerical simulations are performed with the HYPERION code, which solves the fully compressible three-dimensional magnetohydrodynamic equations including nonlinear thermal conduction and optically thin radiation. Numerical simulations of coronal loops with an axial magnetic field from 0.005 to 0.02 T and lengths from 25,000 to 75,000 km are presented. In the simulations the footpoints of the axial loop magnetic field are convected by random, large-scale motions. There is a continuous formation and dissipation of field-aligned current sheets, which act to heat the loop. As a consequence of coronal magnetic reconnection, small-scale, high-speed jets form. The familiar vortex quadrupoles form at reconnection sites. Between the magnetic footpoints and the corona the reconnection flow merges with the boundary flow. It is in this region that the ponderomotive acceleration occurs. Mirroring the character of the coronal reconnection, the ponderomotive acceleration is also found to be intermittent.

  16. Morbidity of temporary loop ileostomies

    NARCIS (Netherlands)

    Bakx, R.; Busch, O. R. C.; Bemelman, W. A.; Veldink, G. J.; Slors, J. F. M.; van Lanschot, J. J. B.

    2004-01-01

    Background/Aims: A temporary loop ileostomy is constructed to protect a distal colonic anastomosis. Closure is usually performed not earlier than 8 - 12 weeks after the primary operation. During this period, stoma-related complications can occur and enhance the adverse effect on quality of life. The

  17. Parametric amplification in MoS2drum resonator.

    Science.gov (United States)

    Prasad, Parmeshwar; Arora, Nishta; Naik, A K

    2017-11-30

    Parametric amplification is widely used in diverse areas from optics to electronic circuits to enhance low level signals by varying relevant system parameters. Parametric amplification has also been performed in several micro-nano resonators including nano-electromechanical system (NEMS) resonators based on a two-dimensional (2D) material. Here, we report the enhancement of mechanical response in a MoS 2 drum resonator using degenerate parametric amplification. We use parametric pumping to modulate the spring constant of the MoS 2 resonator and achieve a 10 dB amplitude gain. We also demonstrate quality factor enhancement in the resonator with parametric amplification. We investigate the effect of cubic nonlinearity on parametric amplification and show that it limits the gain of the mechanical resonator. Amplifying ultra-small displacements at room temperature and understanding the limitations of the amplification in these devices is key for using these devices for practical applications.

  18. Development of Capillary Loop Convective Polymerase Chain Reaction Platform with Real-Time Fluorescence Detection

    Directory of Open Access Journals (Sweden)

    Wen-Pin Chou

    2017-02-01

    Full Text Available Polymerase chain reaction (PCR has been one of the principal techniques of molecular biology and diagnosis for decades. Conventional PCR platforms, which work by rapidly heating and cooling the whole vessel, need complicated hardware designs, and cause energy waste and high cost. On the other hand, partial heating on the various locations of vessels to induce convective solution flows by buoyancy have been used for DNA amplification in recent years. In this research, we develop a new convective PCR platform, capillary loop convective polymerase chain reaction (clcPCR, which can generate one direction flow and make the PCR reaction more stable. The U-shaped loop capillaries with 1.6 mm inner diameter are designed as PCR reagent containers. The clcPCR platform utilizes one isothermal heater for heating the bottom of the loop capillary and a CCD device for detecting real-time amplifying fluorescence signals. The stable flow was generated in the U-shaped container and the amplification process could be finished in 25 min. Our experiments with different initial concentrations of DNA templates demonstrate that clcPCR can be applied for precise quantification. Multiple sample testing and real-time quantification will be achieved in future studies.

  19. Space Optical Communications Using Laser Beam Amplification

    Science.gov (United States)

    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.

  20. Primordial magnetic field amplification from turbulent reheating

    International Nuclear Information System (INIS)

    Calzetta, Esteban; Kandus, Alejandra

    2010-01-01

    We analyze the possibility of primordial magnetic field amplification by a stochastic large scale kinematic dynamo during reheating. We consider a charged scalar field minimally coupled to gravity. During inflation this field is assumed to be in its vacuum state. At the transition to reheating the state of the field changes to a many particle/anti-particle state. We characterize that state as a fluid flow of zero mean velocity but with a stochastic velocity field. We compute the scale-dependent Reynolds number Re(k), and the characteristic times for decay of turbulence, t d and pair annihilation t a , finding t a d . We calculate the rms value of the kinetic helicity of the flow over a scale L and show that it does not vanish. We use this result to estimate the amplification factor of a seed field from the stochastic kinematic dynamo equations. Although this effect is weak, it shows that the evolution of the cosmic magnetic field from reheating to galaxy formation may well be more complex than as dictated by simple flux freezing

  1. Electronic cyclotron radiation amplification in thermonuclear plasmas

    International Nuclear Information System (INIS)

    Ziebell, L.F.

    1983-01-01

    The amplified emission of electron cyclotron radiation near the fundamental frequency from an inhomogeneous, anisotropic plasma slab is investigated in a linear theory. Plasma polarization effects are consistently included. Expressions are developed in the WKB approximation for emission in the ordinary and the extraordinary modes, for propagation perpendicular to the magnetic field. Numerical results are given for the extraordinary mode, for which effects are strongest. For the case of a loss-cone-type electron momentum distribution, it is shown that the amplification is sensitively dependent on the ratio of parallel-to-perpendicular temperature and on inhomogeneities in the magnetic field. The dependence of the amplification on the distribution is further investigated by considering superpositions of loss-cone and Maxwellian components. It is show that the presence of a Maxwellian component in general reduces the emission relative to the pure loss-cone case, and situations occur in which a layer in the slab very effectively absorbs all the radiation amplified elsewhere. A peculiar behaviour of the refractive index, which occurs in the transition from the pure loss-cone to the pure Maxwellian case, is discussed. (author)

  2. Earthquake acceleration amplification based on single microtremor test

    Science.gov (United States)

    Jaya Syahbana, Arifan; Kurniawan, Rahmat; Soebowo, Eko

    2018-02-01

    Understanding soil dynamics is needed to understand soil behaviour, including the parameters of earthquake acceleration amplification. Many researchers now conduct single microtremor tests to obtain amplification of velocity and natural periods of soil at test sites. However, these amplification parameters are rarely used, so a method is needed to convert the velocity amplification to acceleration amplification. This paper will discuss the proposed process of changing the value of amplification. The proposed method is to integrate the time histories of the synthetic earthquake acceleration of the soil surface under the deaggregation at that location so the time histories of the velocity earthquake will be obtained. Next is to conduct a “fitting curve” between amplification by a single microtremor test with amplification of the synthetic earthquake velocity time histories. After obtaining the fitting curve time histories of velocity, differentiation will be conducted to obtain fitting curve acceleration time histories. The final step after obtaining the fitting curve is to compare the acceleration of the “fitting curve” against the histories time of the acceleration of synthetic earthquake at bedrocks to obtain single microtremor acceleration amplification factor.

  3. Improved PCR Amplification of Broad Spectrum GC DNA Templates.

    Science.gov (United States)

    Guido, Nicholas; Starostina, Elena; Leake, Devin; Saaem, Ishtiaq

    2016-01-01

    Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate incomplete or nonspecific products that hamper downstream applications. In this study, we address multiplexed PCR amplification of DNA segments containing a wide range of GC content. In order to mitigate amplification complications due to high or low GC regions, we tested a combination of different PCR cycling conditions and chemical additives. To assess the fate of specific oligonucleotide (oligo) species with varying GC content in a multiplexed PCR, we developed a novel method of sequence analysis. Here we show that subcycling during the amplification process significantly improved amplification of short template pools (~200 bp), particularly when the template contained a low percent of GC. Furthermore, the combination of subcycling and 7-deaza-dGTP achieved efficient amplification of short templates ranging from 10-90% GC composition. Moreover, we found that 7-deaza-dGTP improved the amplification of longer products (~1000 bp). These methods provide an updated approach for PCR amplification of DNA segments containing a broad range of GC content.

  4. C-MET overexpression and amplification in gliomas.

    Science.gov (United States)

    Kwak, Yoonjin; Kim, Seong-Ik; Park, Chul-Kee; Paek, Sun Ha; Lee, Soon-Tae; Park, Sung-Hye

    2015-01-01

    We investigated c-Met overexpression and MET gene amplification in gliomas to determine their incidence and prognostic significance. c-Met immunohistochemistry and MET gene fluorescence in situ hybridization were carried out on tissue microarrays from 250 patients with gliomas (137 grade IV GBMs and 113 grade II and III diffuse gliomas). Clinicopathological features of these cases were reviewed. c-Met overexpression and MET gene amplification were detected in 13.1% and 5.1% of the GBMs, respectively. All the MET-amplified cases showed c-Met overexpression, but MET amplification was not always concordant with c-Met overexpression. None of grade II and III gliomas demonstrated c-Met overexpression or MET gene amplification. Mean survival of the GBM patients with MET amplification was not significantly different from patients without MET amplification (P=0.155). However, GBM patients with c-Met overexpression survived longer than patients without c-Met overexpression (P=0.035). Although MET amplification was not related to poor GBM prognosis, it is partially associated with the aggressiveness of gliomas, as MET amplification was found only in grade IV, not in grade II and III gliomas. We suggest that MET inhibitor therapy may be beneficial in about 5% GBMs, which was the incidence of MET gene amplification found in the patients included in this study.

  5. Vertically Polarized Omnidirectional Printed Slot Loop Antenna

    DEFF Research Database (Denmark)

    Kammersgaard, Nikolaj Peter Iversen; Kvist, Søren H.; Thaysen, Jesper

    2015-01-01

    A novel vertically polarized omnidirectional printed slot loop antenna has been designed, simulated, fabricated and measured. The slot loop works as a magnetic loop. The loop is loaded with inductors to insure uniform and in-phase fields in the slot in order to obtain an omnidirectional radiation...... pattern. The antenna is designed for the 2.45 GHz Industrial, Scientific and Medical band. Applications of the antenna are many. One is for on-body applications since it is ideal for launching a creeping waves due to the polarization.......A novel vertically polarized omnidirectional printed slot loop antenna has been designed, simulated, fabricated and measured. The slot loop works as a magnetic loop. The loop is loaded with inductors to insure uniform and in-phase fields in the slot in order to obtain an omnidirectional radiation...

  6. Evaluation of Nucleic Acid Isothermal Amplification Methods for Human Clinical Microbial Infection Detection

    Directory of Open Access Journals (Sweden)

    Brett E. Etchebarne

    2017-12-01

    Full Text Available Battling infection is a major healthcare objective. Untreated infections can rapidly evolve toward the condition of sepsis in which the body begins to fail and resuscitation becomes critical and tenuous. Identification of infection followed by rapid antimicrobial treatment are primary goals of medical care, but precise identification of offending organisms by current methods is slow and broad spectrum empirical therapy is employed to cover most potential pathogens. Current methods for identification of bacterial pathogens in a clinical setting typically require days of time, or a 4- to 8-h growth phase followed by DNA extraction, purification and PCR-based amplification. We demonstrate rapid (70–120 min genetic diagnostics methods utilizing loop-mediated isothermal amplification (LAMP to test for 15 common infection pathogen targets, called the Infection Diagnosis Panel (In-Dx. The method utilizes filtration to rapidly concentrate bacteria in sample matrices with lower bacterial loads and direct LAMP amplification without DNA purification from clinical blood, urine, wound, sputum and stool samples. The In-Dx panel was tested using two methods of detection: (1 real-time thermocycler fluorescent detection of LAMP amplification and (2 visual discrimination of color change in the presence of Eriochrome Black T (EBT dye following amplification. In total, 239 duplicate samples were collected (31 blood, 122 urine, 73 mucocutaneous wound/swab, 11 sputum and two stool from 229 prospectively enrolled hospital patients with suspected clinical infection and analyzed both at the hospital and by In-Dx. Sensitivity (Se of the In-Dx panel targets pathogens from urine samples by In-Dx was 91.1% and specificity (Sp was 97.3%, with a positive predictive value (PPV of 53.7% and a negative predictive value (NPV of 99.7% as compared to clinical microbial detection methods. Sensitivity of detection of the In-Dx panel from mucocutaneous swab samples was 65.5% with a

  7. Estimation of complex permittivity using loop antenna

    DEFF Research Database (Denmark)

    Lenler-Eriksen, Hans-Rudolph; Meincke, Peter

    2004-01-01

    A method for estimating the complex permittivity of materials in the vicinity of a loop antenna is proposed. The method is based on comparing measured and numerically calculated input admittances for the loop antenna.......A method for estimating the complex permittivity of materials in the vicinity of a loop antenna is proposed. The method is based on comparing measured and numerically calculated input admittances for the loop antenna....

  8. Pattern effect reduction scheme for high-speed all-optical amplification system

    Science.gov (United States)

    Razaghi, Mohammad; Jafari, Omid; Das, Narottam K.

    2014-07-01

    A method for high-bit-rate optical pulse amplification without a pattern effect (PE) phenomenon is numerically analyzed and presented. In the proposed new scheme, the input signals are applied to a series of 1×2 optical switches and semiconductor optical amplifiers (SOAs). Based on the input signal bit rate and the desired PE reduction rate, it is shown that the number of these devices can be easily optimized. For reducing the SOA nonlinearities on the output signal, a high-birefringent fiber loop mirror is used as an optical Gaussian filter. The achieved results depict that symmetry and the time-bandwidth product of the output signal obtained by this filter are significantly improved. The simulations are performed for high bit-rate signals (>50 Gbps) therefore, in the SOA model, all relevant nonlinear effects that occur in the subpicosecond regimes are taken into account.

  9. Verifying parallel loops with separation logic

    NARCIS (Netherlands)

    Blom, Stefan; Darabi, Saeed; Huisman, Marieke

    2014-01-01

    This paper proposes a technique to specify and verify whether a loop can be parallelised. Our approach can be used as an additional step in a parallelising compiler to verify user annotations about loop dependences. Essentially, our technique requires each loop iteration to be specified with the

  10. In, On, or Out of the Loop?

    DEFF Research Database (Denmark)

    Schaub Jr, Gary John; Kristoffersen, Jens Wenzel

    being the degree of sophist ication of weapon responses to external stimuli. Such weapons can be controlled directly with a “man-in-the-loop,” managed by a “man-on-the-loop,” or supervised by a “man-out-of-the-loop.” Although all uses of force by Western militaries take place within an institution...

  11. Theory of K-loops

    CERN Document Server

    Kiechle, Hubert

    2002-01-01

    The book contains the first systematic exposition of the current known theory of K-loops, as well as some new material. In particular, big classes of examples are constructed. The theory for sharply 2-transitive groups is generalized to the theory of Frobenius groups with many involutions. A detailed discussion of the relativistic velocity addition based on the author's construction of K-loops from classical groups is also included. The first chapters of the book can be used as a text, the later chapters are research notes, and only partially suitable for the classroom. The style is concise, but complete proofs are given. The prerequisites are a basic knowledge of algebra such as groups, fields, and vector spaces with forms.

  12. Loop connectors in dentogenic diastema

    Directory of Open Access Journals (Sweden)

    Sanjna Nayar

    2015-01-01

    Full Text Available Patients with a missing tooth along with diastema have limited treatment options to restore the edentulous space. The use of a conventional fixed partial denture (FPD to replace the missing tooth may result in too wide anterior teeth leading to poor esthetics. Loss of anterior teeth with existing diastema may result in excess space available for pontic. This condition presents great esthetic challenge for prosthodontist. If implant supported prosthesis is not possible because of inadequate bone support, FPD along with loop connector may be a treatment option to maintain the diastema and provide optimal esthetic restoration. Here, we report a clinical case where FPD along with loop connector was used to achieve esthetic rehabilitation in maxillary anterior region in which midline diastema has been maintained.

  13. Closed-Loop Neuromorphic Benchmarks

    Directory of Open Access Journals (Sweden)

    Terrence C Stewart

    2015-12-01

    Full Text Available Evaluating the effectiveness and performance of neuromorphic hardware is difficult. It is evenmore difficult when the task of interest is a closed-loop task; that is, a task where the outputfrom the neuromorphic hardware affects some environment, which then in turn affects thehardware’s future input. However, closed-loop situations are one of the primary potential uses ofneuromorphic hardware. To address this, we present a methodology for generating closed-loopbenchmarks that makes use of a hybrid of real physical embodiment and a type of minimalsimulation. Minimal simulation has been shown to lead to robust real-world performance, whilestill maintaining the practical advantages of simulation, such as making it easy for the samebenchmark to be used by many researchers. This method is flexible enough to allow researchersto explicitly modify the benchmarks to identify specific task domains where particular hardwareexcels. To demonstrate the method, we present a set of novel benchmarks that focus on motorcontrol for an arbitrary system with unknown external forces. Using these benchmarks, we showthat an error-driven learning rule can consistently improve motor control performance across arandomly generated family of closed-loop simulations, even when there are up to 15 interactingjoints to be controlled.

  14. Raman amplification in optical communication systems

    DEFF Research Database (Denmark)

    Kjær, Rasmus

    2008-01-01

    Fiber Raman amplifiers are investigated with the purpose of identifying new applications and limitations for their use in optical communication systems. Three main topics are investigated, namely: New applications of dispersion compensating Raman amplifiers, the use Raman amplification to increase...... støjtal under 4,5 dB og en samlet udgangseffekt på 22 dBm. Med henblik på at forlænge rækkevidden af fremtidige access-netværk foreslås en ny arkitektur for såkaldte langdistance passive optiske netværk (PON). Dette system evalueres både teoretisk og eksperimentelt. Distribueret Raman-forstærkning bruges...

  15. Parametric Amplification of a Superconducting Plasma Wave.

    Science.gov (United States)

    Rajasekaran, S; Casandruc, E; Laplace, Y; Nicoletti, D; Gu, G D; Clark, S R; Jaksch, D; Cavalleri, A

    2016-11-01

    Many applications in photonics require all-optical manipulation of plasma waves1, which can concentrate electromagnetic energy on sub-wavelength length scales. This is difficult in metallic plasmas because of their small optical nonlinearities. Some layered superconductors support Josephson plasma waves (JPWs)2,3, involving oscillatory tunneling of the superfluid between capacitively coupled planes. Josephson plasma waves are also highly nonlinear4, and exhibit striking phenomena like cooperative emission of coherent terahertz radiation5,6, superconductor-metal oscillations7 and soliton formation8. We show here that terahertz JPWs can be parametrically amplified through the cubic tunneling nonlinearity in a cuprate superconductor. Parametric amplification is sensitive to the relative phase between pump and seed waves and may be optimized to achieve squeezing of the order parameter phase fluctuations9 or single terahertz-photon devices.

  16. Parametric amplification by coupled flux qubits

    Energy Technology Data Exchange (ETDEWEB)

    Rehák, M.; Neilinger, P.; Grajcar, M. [Department of Experimental Physics, Comenius University, SK-84248 Bratislava (Slovakia); Institute of Physics, Slovak Academy of Science, 845 11 Bratislava (Slovakia); Oelsner, G.; Hübner, U.; Meyer, H.-G. [Leibniz Institute of Photonic Technology, P.O. Box 100239, D-07702 Jena (Germany); Il' ichev, E. [Leibniz Institute of Photonic Technology, P.O. Box 100239, D-07702 Jena (Germany); Novosibirsk State Technical University, 20 K. Marx Ave., 630092 Novosibirsk (Russian Federation)

    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.

  17. Heralded amplification of path entangled quantum states

    Science.gov (United States)

    Monteiro, F.; Verbanis, E.; Caprara Vivoli, V.; Martin, A.; Gisin, N.; Zbinden, H.; Thew, R. T.

    2017-06-01

    Device-independent quantum key distribution (DI-QKD) represents one of the most fascinating challenges in quantum communication, exploiting concepts of fundamental physics, namely Bell tests of nonlocality, to ensure the security of a communication link. This requires the loophole-free violation of a Bell inequality, which is intrinsically difficult due to losses in fibre optic transmission channels. Heralded photon amplification (HPA) is a teleportation-based protocol that has been proposed as a means to overcome transmission loss for DI-QKD. Here we demonstrate HPA for path entangled states and characterise the entanglement before and after loss by exploiting a recently developed displacement-based detection scheme. We demonstrate that by exploiting HPA we are able to reliably maintain high fidelity entangled states over loss-equivalent distances of more than 50 km.

  18. Introduction to quantum noise, measurement, and amplification

    Science.gov (United States)

    Clerk, A. A.; Devoret, M. H.; Girvin, S. M.; Marquardt, Florian; Schoelkopf, R. J.

    2010-04-01

    The topic of quantum noise has become extremely timely due to the rise of quantum information physics and the resulting interchange of ideas between the condensed matter and atomic, molecular, optical-quantum optics communities. This review gives a pedagogical introduction to the physics of quantum noise and its connections to quantum measurement and quantum amplification. After introducing quantum noise spectra and methods for their detection, the basics of weak continuous measurements are described. Particular attention is given to the treatment of the standard quantum limit on linear amplifiers and position detectors within a general linear-response framework. This approach is shown how it relates to the standard Haus-Caves quantum limit for a bosonic amplifier known in quantum optics and its application to the case of electrical circuits is illustrated, including mesoscopic detectors and resonant cavity detectors.

  19. Controlled Microwave Heating Accelerates Rolling Circle Amplification.

    Science.gov (United States)

    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.

  20. Raman Amplification with a Flying Focus

    Science.gov (United States)

    Turnbull, D.; Bucht, S.; Davies, A.; Haberberger, D.; Kessler, T.; Shaw, J. L.; Froula, D. H.

    2018-01-01

    We propose a new laser amplifier scheme utilizing stimulated Raman scattering in plasma in conjunction with a "flying focus"—a chromatic focusing system combined with a chirped pump beam that provides spatiotemporal control over the pump's focal spot. Pump intensity isosurfaces are made to propagate at v =-c so as to be in sync with the injected counterpropagating seed pulse. By setting the pump intensity in the interaction region to be just above the ionization threshold of the background gas, an ionization wave is produced that travels at a fixed distance ahead of the seed. Simulations show that this will make it possible to optimize the plasma temperature and mitigate many of the issues that are known to have impacted previous Raman amplification experiments, in particular, the growth of precursors.