WorldWideScience

Sample records for autocatalytic loop amplification

  1. One New Method of Nucleic Acid Amplification-Loop-mediated Isothermal Amplification of DNA

    Institute of Scientific and Technical Information of China (English)

    Xue-en FANG; Jian LI; Qin CHEN

    2008-01-01

    Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of four specially designed primers and a Bst DNA polymerase with strand displacement activity. The basic principle, characteristics, development of LAMP and its applications are summarized in this article.

  2. Reactive Oxygen Species Mediated Activation of a Dormant Singlet Oxygen Photosensitizer: From Autocatalytic Singlet Oxygen Amplification to Chemicontrolled Photodynamic Therapy.

    Science.gov (United States)

    Durantini, Andrés M; Greene, Lana E; Lincoln, Richard; Martínez, Sol R; Cosa, Gonzalo

    2016-02-01

    Here we show the design, preparation, and characterization of a dormant singlet oxygen ((1)O2) photosensitizer that is activated upon its reaction with reactive oxygen species (ROS), including (1)O2 itself, in what constitutes an autocatalytic process. The compound is based on a two segment photosensitizer-trap molecule where the photosensitizer segment consists of a Br-substituted boron-dipyrromethene (BODIPY) dye. The trap segment consists of the chromanol ring of α-tocopherol, the most potent naturally occurring lipid soluble antioxidant. Time-resolved absorption, fluorescence, and (1)O2 phosphorescence studies together with fluorescence and (1)O2 phosphorescence emission quantum yields collected on Br2B-PMHC and related bromo and iodo-substituted BODIPY dyes show that the trap segment provides a total of three layers of intramolecular suppression of (1)O2 production. Oxidation of the trap segment with ROS restores the sensitizing properties of the photosensitizer segment resulting in ∼40-fold enhancement in (1)O2 production. The juxtaposed antioxidant (chromanol) and prooxidant (Br-BODIPY) antagonistic chemical activities of the two-segment compound enable the autocatalytic, and in general ROS-mediated, activation of (1)O2 sensitization providing a chemical cue for the spatiotemporal control of (1)O2.The usefulness of this approach to selectively photoactivate the production of singlet oxygen in ROS stressed vs regular cells was successfully tested via the photodynamic inactivation of a ROS stressed Gram negative Escherichia coli strain. PMID:26789198

  3. Loop-Mediated Amplification Accelerated by Stem Primers

    Directory of Open Access Journals (Sweden)

    Laurence Tisi

    2011-12-01

    Full Text Available Isothermal nucleic acid amplifications (iNAATs have become an important alternative to PCR for in vitro molecular diagnostics in all fields. Amongst iNAATs Loop-mediated amplification (LAMP has gained much attention over the last decade because of the simplicity of hardware requirements. LAMP demonstrates performance equivalent to that of PCR, but its application has been limited by the challenging primer design. The design of six primers in LAMP requires a selection of eight priming sites with significant restrictions imposed on their respective positioning and orientation. In order to relieve primer design constraints we propose an alternative approach which uses Stem primers instead of Loop primers and demonstrate the application of STEM-LAMP in assaying for Clostridium difficile, Listeria monocytogenes and HIV. Stem primers used in LAMP in combination with loop-generating and displacement primers gave significant benefits in speed and sensitivity, similar to those offered by Loop primers, while offering additional options of forward and reverse orientations, multiplexing, use in conjunction with Loop primers or even omission of one or two displacement primers, where necessary. Stem primers represent a valuable alternative to Loop primers and an additional tool for IVD assay development by offering more choices for primer design at the same time increasing assay speed, sensitivity, and reproducibility.

  4. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases.

    Science.gov (United States)

    Sahoo, Pravas Ranjan; Sethy, Kamadev; Mohapatra, Swagat; Panda, Debasis

    2016-05-01

    India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease. PMID:27284221

  5. Loop-mediated isothermal amplification of single pollen grains

    Institute of Scientific and Technical Information of China (English)

    Ali Bektaş; Ignacio Chapela

    2014-01-01

    The polymerase chain reaction (PCR) has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental require-ments of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplifica-tion methods provides an alternative to PCR, which circumvents key limitations of PCR for direct amplification in the field. Being able to analyze DNA in the pol en cloud of an ecosystem would provide very useful ecological information, yet would require a field-enabled, high-throughput method for this potential to be realized. Here, we demonstrate the applicability of the loop-mediated DNA amplification method (LAMP), an isothermal DNA amplification technique, to be used in pol en analysis. We demonstrate that LAMP can provide a reliable method to identify species from the pol en cloud, and that it can amplify successful y with sensitivity down to single pol en grains, thus opening the possibility of field-based, high-throughput analysis.

  6. Diagnosis of brugian filariasis by loop-mediated isothermal amplification.

    Directory of Open Access Journals (Sweden)

    Catherine B Poole

    Full Text Available In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis.

  7. Diagnosis of brugian filariasis by loop-mediated isothermal amplification.

    Science.gov (United States)

    Poole, Catherine B; Tanner, Nathan A; Zhang, Yinhua; Evans, Thomas C; Carlow, Clotilde K S

    2012-01-01

    In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis.

  8. Diagnosis of brugian filariasis by loop-mediated isothermal amplification.

    Science.gov (United States)

    Poole, Catherine B; Tanner, Nathan A; Zhang, Yinhua; Evans, Thomas C; Carlow, Clotilde K S

    2012-01-01

    In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis. PMID:23272258

  9. Rapid Diagnosis of Human Herpesvirus 6 Infection by a Novel DNA Amplification Method, Loop-Mediated Isothermal Amplification

    OpenAIRE

    Ihira, Masaru; Yoshikawa, Tetsushi; Enomoto, Yoshihiko; Akimoto, Shiho; Ohashi, Masahiro; Suga, Sadao; Nishimura, Naoko; Ozaki, Takao; Nishiyama, Yukihiro; Notomi, Tsugunori; Ohta, Yoshinori; Asano, Yoshizo

    2004-01-01

    A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious agents. LAMP was developed for human herpesvirus 6 (HHV-6), and its reliability was evaluated in this study. Although LAMP products were detected in HHV-6 B and HHV-6 A DNA, they were not detected in HHV-7 and human cytomegalovirus DNA. The s...

  10. Amplification and Compression of Ultrashort Fundamental Solitons in An Erbium-Doped Nonlinear Amplifying Fiber Loop Mirror

    Institute of Scientific and Technical Information of China (English)

    P.; K.; A.; Wai

    2003-01-01

    A nonlinear amplifying loop mirror constructed from erbium-doped fiber is proposed for simultaneous amplification and compression of ultrashort fundamental solitons. Numerical simulations show that, the proposed device performs efficient high-quality amplification and compression of solitons.

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

    Institute of Scientific and Technical Information of China (English)

    WANG Yong; GAO Zhaoming; XU Ying; LI Guangyu; HE Lisheng; QIAN Peiyuan

    2016-01-01

    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.

  12. The detection of Plasmodiophora brassicae using loop-mediated isothermal DNA amplification

    OpenAIRE

    Joanna Kaczmarek; Witold Irzykowski; Adam Burzyński; Małgorzata Jędryczka

    2014-01-01

    Plasmodiophora brassicae, the cause of clubroot, is a very serious problem preventing from successful and profitable cultivation of oilseed rape in Poland. The pathogen was found in all main growing areas of oilseed rape; it also causes considerable problems in growing of vegetable brassicas. The aim of this work was to elaborate fast, cheap and reliable screening method to detect P. brassicae. To achieve this aim the Loop-mediated isothermal DNA amplification (LAMP) technique has been elabor...

  13. Towards rapid genotyping of resistant malaria parasites: could loop-mediated isothermal amplification be the solution?

    OpenAIRE

    Abdul-Ghani, Rashad

    2014-01-01

    Loop-mediated isothermal amplification (LAMP) is an innovative molecular technique that has been validated for point-of-care testing to diagnose malaria. Molecular detection and tracking of anti-malarial drug resistance is mainly based on highly sophisticated, costly and time-consuming techniques. With the validation of resistance-associated gene mutations in malaria parasites, there is a need to develop rapid, easy-to-use molecular tests for anti-malarial drug resistance genotyping. LAMP cou...

  14. Electrochemical genosensor for the rapid detection of GMO using loop-mediated isothermal amplification.

    Science.gov (United States)

    Ahmed, Minhaz Uddin; Saito, Masato; Hossain, M Mosharraf; Rao, S Ramachandara; Furui, Satoshi; Hino, Akihiro; Takamura, Yuzuru; Takagi, Masahiro; Tamiya, Eiichi

    2009-05-01

    In this study, we are reporting for the first time an efficient, accurate and inexpensive rapid detection system which employs the integration of isothermal amplification and subsequent analysis of unpurified amplicons by an electrochemical system. In our experiments, loop-mediated isothermal amplification (LAMP) with its higher efficiency than PCR was performed at a constant temperature (65 degrees C). Amplification products were combined with a redox active molecule Hoechst 33258 [H33258, 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi(1H-benzimidazole)] and analyzed by a DNA stick (DS) which is integrated with a disposable electrochemical printed (DEP) chip using linear sweep voltammetry (LSV). The DNA minor groove binding of the H33258 molecule causes a significant drop in the peak current intensity of the H33258 oxidation. The phenomenon of DNA binding induced by H33258, in addition to changes in the anodic current peak, was used to detect maize CBH 351 variety (StarLink). Since laborious probe immobilization was not required, and amplification and detection were performed on a single device, our biosensor eliminates potential cross-contamination. We believe that this type of sensor will have an unprecedented impact for environmental protection.

  15. 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.

  16. Rapid detection of Brucella spp. using loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Chen, Shouyi; Li, Xunde; Li, Juntao; Atwill, Edward R

    2013-01-01

    Brucella spp. are facultative intracellular bacteria that cause zoonotic disease of brucellosis worldwide. Livestock that are most vulnerable to brucellosis include cattle, goats, and pigs. Brucella spp. cause serious health problems to humans and animals and economic losses to the livestock industry. Traditional methods for detection of Brucella spp. take 48-72 h (Kumar et al., J Commun Dis 29:131-137, 1997; Barrouin-Melo et al., Res Vet Sci 83:340-346, 2007) that do not meet the food industry's need of rapid detection. Therefore, there is an urgent need of fast, specific, sensitive, and inexpensive method for diagnosing of Brucella spp. Loop-mediated isothermal amplification (LAMP) is a method to amplify nucleic acid at constant temperatures. Amplification can be detected by visual detection, fluorescent stain, turbidity, and electrophoresis. We targeted at the Brucella-specific gene omp25 and designed LAMP primers for detection of Brucella spp. Amplification of DNA with Bst DNA polymerase can be completed at 65 °C in 60 min. Amplified products can be detected by SYBR Green I stain and 2.0% agarose gel electrophoresis. The LAMP method is feasible for detection of Brucella spp. from blood and milk samples.

  17. 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.

  18. Development of a Loop-Mediated Isothermal Amplification Assay for Porcine Circovirus Type 2

    Institute of Scientific and Technical Information of China (English)

    Ye-bing Liu; Lei Zhang; Qin-hong Xue; Yi-bao Ning; Zhi-gang Zhang

    2011-01-01

    In this study,the loop-mediated isothermal amplification(LAMP)method was used to develop a rapid and simple detection system for porcine circovirus type 2(PCV2).According to the PCV2 sequences published in GenBank,multiple LAMP primers were designed targeting conserved sequences of PCV2.Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template,LAMP reactions in a PCV2 LAMP system was performed,the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye.The results showed highly-efficient and specific amplification in 30 min at 63℃ with a LAMP real-time turbidimeter.Furthermore,PCV2 DNA templates,with a detection limit of 5.5×10-5ng of nucleic acid,indicated that this assay was highly sensitive.The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter,showing the potential simplicity of interpretation of the assay results.The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples.In addition it offers higher specificity and sensitivity,shorter reaction times and simpler procedures than the currently available methods of PCV2 detection.It is therefore a promising tool for the effective and efficient detection of PCV2.

  19. Detection of foot-and-mouth disease virus rna by reverse transcription loop-mediated isothermal amplification

    OpenAIRE

    Chen Hao-tai; Zhang Jie; Liu Yong-sheng; Liu Xiang-tao

    2011-01-01

    Abstract A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for foot-and-mouth disease virus (FMDV) RNA. The amplification was able to finish in 45 min under isothermal condition at 64°C by employing a set of four primers targeting FMDV 2B. The assay showed higher sensitivity than RT-PCR. No cross reactivity was observed from other RNA viruses including classical swine fever virus, swine vesicular disease, porcine reproductive and respiratory syndrome...

  20. Development of a loop-mediated isothermal amplification assay for detection of Trichomonas vaginalis.

    Science.gov (United States)

    Reyes, John Carlo B; Solon, Juan Antonio A; Rivera, Windell L

    2014-07-01

    A loop-mediated isothermal amplification (LAMP) assay targeting the 2-kbp repeated DNA species-specific sequence was developed for detection of Trichomonas vaginalis, the causative agent of trichomoniasis. The analytical sensitivity and specificity of the LAMP assay were evaluated using pooled genital swab and urine specimens, respectively, spiked with T. vaginalis trophozoites. Genital secretion and urine did not inhibit the detection of the parasite. The sensitivity of the LAMP was 10-1000 times higher than the PCR performed. The detection limit of LAMP was 1 trichomonad for both spiked genital swab and urine specimens. Also, LAMP did not exhibit cross-reactivity with closely-related trichomonads, Trichomonas tenax and Pentatrichomonas hominis, and other enteric and urogenital microorganisms, Entamoeba histolytica, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. This is the first report of a LAMP assay for the detection of T. vaginalis and has prospective application for rapid diagnosis and control of trichomoniasis.

  1. A simple identification method of saliva by detecting Streptococcus salivarius using loop-mediated isothermal amplification.

    Science.gov (United States)

    Nakanishi, Hiroaki; Ohmori, Takeshi; Hara, Masaaki; Takada, Aya; Shojo, Hideki; Adachi, Noboru; Saito, Kazuyuki

    2011-01-01

    We previously reported that detection of Streptococcus salivarius is feasible for proving the presence of saliva in a forensic sample. Here, a simple and rapid method for the detection of S. salivarius in forensic samples was developed that uses loop-mediated isothermal amplification (LAMP). The LAMP primer set was designed using S. salivarius-specific sequences of glucosyltransferase K. To simplify the procedure, the sample was prepared by boiling and mutanolysin treatment only, and the entire analytical process was completed within 2.5 h. The cut-off value was set at 0.1 absorbance units, measured at 660 nm, upon termination of the reaction. S. salivarius was identified in all saliva samples, but was not detected in other body fluids or on the skin surface. Using this method, S. salivarius was successfully detected in various mock forensic samples. We therefore suggest that this approach is useful for the identification of saliva in forensic practice. PMID:21198609

  2. Development of a loop-mediated isothermal amplification assay for rapid detection of Burkholderia mallei.

    Science.gov (United States)

    Mirzai, S; Safi, S; Mossavari, N; Afshar, D; Bolourchian, M

    2016-01-01

    The present study was conducted to establish a Loop-mediated isothermal amplification (LAMP) technique for the rapid detection of B. mallei the etiologic agent of glanders, a highly contagious disease of equines. A set of six specific primers targeting integrase gene cluster were designed for the LAMP test. The reaction was optimized using different temperatures and time intervals. The specificity of the assay was evaluated using DNA from B.pseudomallei and Pseudomonas aeruginosa. The LAMP products were analyzed both visually and under UV light after electrophoresis. The optimized conditions were found to be at 63ºC for 60 min. The assay showed high specificity and sensitivity. It was concluded that the established LAMP assay is a rapid, sensitive and practical tool for detection of B. mallei and early diagnosis of glanders. PMID:27609471

  3. Detection of Staphylococcus aureus in Milk Using Real-time Fluorescence Loop-mediated Isothermal Amplification

    Directory of Open Access Journals (Sweden)

    Ying Yu

    2015-07-01

    Full Text Available Staphylococcus aureus is a kind of worldwide food-borne pathogen. Recently, S. aureus has gained considerable attention because of the increasing alimentary toxicosis incidence. In this study, a Real-Time fluorescence Loop-Mediated isothermal Amplification (RT-LAMP was developed to detect S. aureus rapidly. The heat-stable nuclease (nuc gene of S. aureus, the target sequence, was selected to design four special primers. A rapid detection method for S. aureus was initially established under optimum reaction conditions. The assay, performed for 40 min at 61°C, did not show cross reactivity with other bacterial species. The specificity and sensitivity of RT-LAMP for detecting S. aureus were 100% and 8.0 CFU/mL, respectively. Results indicated that RT-LAMP was a potential field-usable molecular tool for detecting S. aureus This method can be an alternative to conventional LAMP in clinical applications and operational programs.

  4. Real-time fluorescence loop-mediated isothermal amplification for the diagnosis of hemorrhagic enteritis virus.

    Science.gov (United States)

    Liu, Xuemei; Li, Yuhao; Xu, Chenggang; Qin, Jianru; Hao, Jianyong; Feng, Min; Tan, Liqiang; Jia, Weixin; Liao, Ming; Cao, Weisheng

    2014-04-01

    Suspected cases of hemorrhagic enteritis associated with hemorrhagic enteritis virus (HEV) are becoming more frequent among yellow chickens in the Guangdong Province of China. In this study, we have developed a one-step, ecumenical, real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay for the rapid diagnosis of HEV. The RealAmp assay was performed at 63°C and reduced the assay time to 15min, using a simple and portable device, the ESE-Quant Tube Scanner. The detection limit of DNA was 1fg/μl, and the detection was specific only to HEV. We also used nested PCR to evaluate the application of the RealAmp assay. The coincidence rate of the two methods was 100%. Our data indicated that the RealAmp assay provides a sensitive, specific, and user-friendly diagnostic tool for the identification and quantification of HEV for field diagnosis and in laboratory research.

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

    Science.gov (United States)

    Reyes, John Carlo B; Solon, Juan Antonio A; Rivera, Windell L

    2014-07-01

    A loop-mediated isothermal amplification (LAMP) assay targeting the 2-kbp repeated DNA species-specific sequence was developed for detection of Trichomonas vaginalis, the causative agent of trichomoniasis. The analytical sensitivity and specificity of the LAMP assay were evaluated using pooled genital swab and urine specimens, respectively, spiked with T. vaginalis trophozoites. Genital secretion and urine did not inhibit the detection of the parasite. The sensitivity of the LAMP was 10-1000 times higher than the PCR performed. The detection limit of LAMP was 1 trichomonad for both spiked genital swab and urine specimens. Also, LAMP did not exhibit cross-reactivity with closely-related trichomonads, Trichomonas tenax and Pentatrichomonas hominis, and other enteric and urogenital microorganisms, Entamoeba histolytica, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. This is the first report of a LAMP assay for the detection of T. vaginalis and has prospective application for rapid diagnosis and control of trichomoniasis. PMID:24792836

  6. Detection of Clostridium perfringens alpha toxin gene in lambs by loop mediated isothermal amplification

    Directory of Open Access Journals (Sweden)

    B. Radhika

    2016-01-01

    Full Text Available Aim: The loop mediated isothermal amplification (LAMP was standardized for rapid detection of Clostridium perfringens. Materials and Methods: A total of 120 fecal samples were collected from enterotoxemia suspected lambs were used for screening of C. perfringens cpa gene by LAMP. The specificity of the LAMP amplified products was tested by digesting with restriction enzyme XmnI for alpha toxin gene. Results: Out of 120 samples screened 112 (93.3% samples were positive by both LAMP and polymerase chain reaction (PCR for detection of cpa gene which indicated the equal sensitivity of both the tests. The enzyme produced single cut in 162 base pair amplified product of alpha toxin gene at 81 base pair resulting in a single band in gel electrophoresis. Conclusion: Both LAMP and PCR for detection of cpa gene indicated the equal sensitivity of both the tests. Standardization of LAMP reaction for amplification of epsilon and beta toxin genes will help to identify the C. perfringens toxin types from the clinical samples. The test could be a suitable alternative to the PCR in detection of toxin types without the help of sophisticated machinery like thermal cycler. Considering its simplicity in operation and high sensitivity, there is the potential use of this technique in clinical diagnosis and surveillance of infectious diseases.

  7. 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 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 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. PMID:27014303

  8. 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.

  9. 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. PMID:27014303

  10. Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay

    Science.gov (United States)

    Yao, Xiefeng; Li, Pingfang; Xu, Jinghua; Zhang, Man; Ren, Runsheng; Liu, Guang; Yang, Xingping

    2016-01-01

    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.

  11. Development and application of loop-mediated isothermal amplification (LAMP) for detection of Plasmopara viticola.

    Science.gov (United States)

    Kong, Xiangjiu; Qin, Wentao; Huang, Xiaoqing; Kong, Fanfang; Schoen, Cor D; Feng, Jie; Wang, Zhongyue; Zhang, Hao

    2016-01-01

    A rapid LAMP (loop-mediated isothermal amplification) detection method was developed on the basis of the ITS sequence of P. viticola, the major causal agent of grape downy mildew. Among the 38 fungal and oomycete species tested, DNA isolated exclusively from P. viticola resulted in a specific product after LAMP amplification. This assay had high sensitivity and was able to detect the presence of less than 33 fg of genomic DNA per 25-μL reaction within 30 min. The infected leaves may produce sporangia that serve as a secondary inoculum. The developed LAMP assay is efficient for estimating the latent infection of grape leaves by P. viticola. When combined with the rapid and simple DNA extraction method, this assay's total detection time is shortened to approximately one hour; therefore it is suitable for on-site detection of latent infection in the field. The sporangia levels in the air are strongly associated with disease severity. The LAMP method was also demonstrated to be able to estimate the level of sporangia released in the air in a certain period. This assay should make disease forecasting more accurate and rapid and should be helpful in decision-making regarding the control of grape downy mildew. PMID:27363943

  12. Development of loop-mediated isothermal amplification for rapid detection of Entamoeba histolytica

    Institute of Scientific and Technical Information of China (English)

    Windell L Rivera; Vanissa A Ong

    2013-01-01

    Objective: To develop a loop-mediated isothermal amplification (LAMP) assay for the detection of Entamoeba histolytica (E. histolytica), the causative agent of amebiasis. Methods: The LAMP primer set was designed from E. histolytica hemolysin gene HLY6. Genomic DNA of E. histolytica trophozoites strain HK9 was used to optimize the LAMP mixture and conditions. Amplification of DNA in the LAMP mixture was monitored through visual inspection for turbidity of the LAMP mix as well as addition of fluorescent dye. Results: Positive LAMP reactions turned turbid while negative ones remained clear. Upon addition of a fluorescent dye, all positive reactions turned green while the negative control remained orange under ambient light. After elecrophoresis in 1.5%agarose gels, a ladder of multiple bands of different sizes can be observed in positive samples while no bands were detected in the negative control. The sensitivity of the assay was found to be 5 parasites per reaction which corresponds to approximately 15.8 ng/μL DNA. The specificity of the assay was verified by the absence of amplified products when DNA from other gastrointestinal parasites such as the morphologically similar but non-pathogenic species, Entamoeba dispar, and other diarrhea-causing organisms such as Blastocystis hominis and Escherichia coli were used. Conclusions: The LAMP assay we have developed enables the detection of E. histolytica with rapidity and ease, therefore rendering it is suitable for laboratory and field diagnosis of amebiasis.

  13. 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 vi

  14. AECL passive autocatalytic recombiners

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, L.B.; Marcinkowska, K. [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada)

    2012-03-15

    Atomic Energy of Canada Limited's (AECL) Passive Autocatalytic Recombiner (PAR) is a passive device used for hydrogen mitigation under post-accident conditions in nuclear reactor containment. The PAR employs a proprietary AECL catalyst which promotes the exothermal reaction between hydrogen and oxygen to form water vapour. The heat of reaction combined with the PAR geometry establishes a convective flow through the recombiner, where ambient hydrogen-rich gas enters the PAR inlet and hot, humid, hydrogen-depleted gas exits the outlet. AECL's PAR has been extensively qualified for CANDU and light water reactors (LWRs), and has been supplied to France, Finland, Ukraine, South Korea and is currently being deployed in Canadian nuclear power plants. (author)

  15. AECL passive autocatalytic recombiners

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, L.B.; Marcinkowska, K. [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada)

    2011-07-01

    Atomic Energy of Canada Limited's (AECL) Passive Autocatalytic Recombiner (PAR) is a passive device used for hydrogen mitigation under post-accident conditions in nuclear reactor containment. The PAR employs a proprietary AECL catalyst which promotes the exothermal reaction between hydrogen and oxygen to form water vapour. The heat of reaction combined with the PAR geometry establishes a convective flow through the recombiner, where ambient hydrogen-rich gas enters the PAR inlet and hot, humid, hydrogen-depleted gas exits the outlet. AECL's PAR has been extensively qualified for CANDU and light water reactors (LWRs), and has been supplied to France, Finland, Ukraine, South Korea and is currently being deployed in Canadian nuclear power plants. (author)

  16. Autocatalytic chemical smoke rings

    CERN Document Server

    Rogers, M C; Rogers, Michael C.; Morris, Stephen W.

    2005-01-01

    Buoyant plumes, evolving free of boundary constraints, may develop well-defined mushroom shaped heads. In normal plumes, overturning flow in the head entrains less buoyant fluid from the surroundings as the head rises, robbing the plume of its driving force. We consider here a new type of plume in which the source of buoyancy is an autocatalytic chemical reaction. The reaction occurs at a sharp front which separates reactants from less dense products. In this type of plume, entrainment assists the reaction, producing new buoyancy which fuels an accelerating plume head. When the head has grown to a critical size, it detaches from the upwelling conduit, forming an accelerating, buoyant vortex ring. This vortex is analogous to a rising smoke ring. A second-generation head then develops at the point of detachment.Multiple generations of chemical vortex rings can detach from a single triggering event.

  17. Loop-mediated isothermal amplification (LAMP method for rapid detection of Trypanosoma brucei rhodesiense.

    Directory of Open Access Journals (Sweden)

    Zablon Kithinji Njiru

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

  18. Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay.

    Science.gov (United States)

    Yao, Xiefeng; Li, Pingfang; Xu, Jinghua; Zhang, Man; Ren, Runsheng; Liu, Guang; Yang, Xingping

    2016-01-01

    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. PMID:27625648

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

    Directory of Open Access Journals (Sweden)

    Yee-Ling Lau

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

  20. Development of double loop-mediated isothermal amplification to detect Listeria monocytogenes in food.

    Science.gov (United States)

    Wu, Rina; Liu, Xiang; Guo, Bangcheng; Chen, Fusheng; Wang, Xiaohong

    2014-12-01

    In this study, a double loop-mediated isothermal amplification (dLAMP) based on two target genes hlyA and iap was developed for the rapid detection of Listeria monocytogenes in food. The results revealed that the detection time and temperature of our dLAMP assay for L. monocytogenes were 15 min and 63 °C respectively, with a sensitivity of 10 fg DNA of L. monocytogenes per tube. While normal LAMP (nLAMP) of hlyA or iap was 100 fg DNA of L. monocytogenes per tube for 45 min and 63 °C. Furthermore, mineral oil and GoldViewII nucleic acid stain were chosen as the basic materials to develop a simple visualized identification of the positive samples. A total of 450 food samples were tested for L. monocytogenes using the dLAMP protocol developed in this study. The results showed that the accuracy of the dLAMP and the "gold standard" culture-biotechnical method were 100 % identical, suggesting that the modified dLAMP assay would provide a potential for detection of L. monocytogenes in food products. PMID:25086581

  1. Highly Sensitive Loop-Mediated Isothermal Amplification for the Detection of Leptospira

    Directory of Open Access Journals (Sweden)

    Hua-Wei Chen

    2015-01-01

    Full Text Available Leptospirosis is a worldwide zoonosis caused by an infection with the pathogenic species of Leptospira. We have developed a loop-mediated isothermal amplification (LAMP assay to detect the DNA of Leptospira spp. Six sets of primers targeting the gene of the subsurface protein, lipL32, were evaluated for their detection sensitivity. The best primer set detected less than 25 copies of lipL32 per reaction of both plasmid DNA template and purified leptospiral genomic DNA. By combining primers targeting lipL32 with the previously published primer set targeting lipL41, the sensitivity of the assay was improved to 12 copies of L. interrogans. The specificity of the LAMP assay was evaluated by using the genomic DNA from other clinically encountered bacterial species such as different strains of Orientia tsutsugamushi, Rickettsia typhi, Rickettsia conorii, Rickettsia rickettsii, Coxiella burnetii, and Bartonella bacilliformis. These genomic DNA samples were all negative in our LAMP assay. The sensitivity of the LAMP assay was very similar to that of quantitative real time PCR. Several detection methods for the amplified product of LAMP assay were performed to demonstrate the simplicity of the assay. In summary, our results have suggested that this assay is rapid, robust, and easy to perform and has the potential to be used in endemic locations.

  2. The detection of Plasmodiophora brassicae using loop-mediated isothermal DNA amplification

    Directory of Open Access Journals (Sweden)

    Joanna Kaczmarek

    2014-12-01

    Full Text Available Plasmodiophora brassicae, the cause of clubroot, is a very serious problem preventing from successful and profitable cultivation of oilseed rape in Poland. The pathogen was found in all main growing areas of oilseed rape; it also causes considerable problems in growing of vegetable brassicas. The aim of this work was to elaborate fast, cheap and reliable screening method to detect P. brassicae. To achieve this aim the Loop-mediated isothermal DNA amplification (LAMP technique has been elaborated. The set of three primer pairs was designed using LAMP software. The detection was performed with the GspSSD polymerase, isolated from bacteria Geobacillus sp., with strand displacement activity. DNA extraction from clubbed roots obtained from farmers’ fields of oilseed rape infected by P. brassicae was done using a modified CTAB method. The reaction was performed for 60 min at 62oC. The visual detection was done using CFX96 Real Time PCR Detection System (BioRad or Gerie II Amplicatior (Optigen. The detection with LAMP proved its usefulness; it was easy, fast and accurate and independent of plant age. The detection limit was 5 spores per 1 µl of the spore suspension, so LAMP was less sensitive than quantitative PCR tests reported in the literature. However, the method is cheap and simple, so it is a good alternative, when it comes to practical use and the assessment of numerous samples.

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

    Directory of Open Access Journals (Sweden)

    Qing Zhu

    2012-11-01

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

  4. 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.

  5. Fluorogenic Detection of Duck Tembusu Virus( DTMUV ) by Loop-mediated Isothermal Amplification(LAMP)

    Institute of Scientific and Technical Information of China (English)

    Zhang; Lin; Wang; Bin; Zhang; Wei; Zhang; Xiumei

    2014-01-01

    This study was to develop an efficient and simple method for the detection of duck Tembusu virus( DTMUV) by loop-mediated isothermal amplification( LAMP). Six pairs of LAMP primers were designed according to the conserved region of the DTMUV E gene sequence in Gen Bank,which were then used for the optimization of various reaction components and reaction system of specific LAMP for DTMUV. Further the fluorescent reagent SYBR Green I and a certain proportion of calcium and manganese ion were used to determin the color development of products for visible analysis instead of agarose gel electrophoresis. The results showed that the sensitivity SYBR Green I as the fluorescent reagent was 10 copies viruses per μL,which is 100 times higher than normal PCR method,while the detection limit of combined use of calcium and manganese ion was 1 000 copies viruses per μL. Although the sensitivity of mixture of calcium and manganese ion is lower than SYBR Green I,it can avoid the aerosol contamination. The fluorogenic analysis-based LAMP system established in our study has a high sensitivity and avoid the cross contamination,which is of huge potential in research institutions,grass-roots laboratories and field testing and can provide effective means to completely curb the occurrence and spreading of DTMUV.

  6. Loop-Mediated Isothermal Amplification Targeting Actin DNA of Trichomonas vaginalis

    Science.gov (United States)

    Goo, Youn-Kyoung; Shin, Won-Sik; Yang, Hye-Won; Joo, So-Young; Song, Su-Min; Ryu, Jae-Sook; Kong, Hyun-Hee; Lee, Won-Ki; Chung, Dong-Il; Hong, Yeonchul

    2016-01-01

    Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications. PMID:27417089

  7. Development of Lyophilized Loop-Mediated Isothermal Amplification Reagents for the Detection of Leptospira.

    Science.gov (United States)

    Chen, Hua-Wei; Weissenberger, Giulia; Ching, Wei-Mei

    2016-05-01

    Leptospirosis is considered to be the most widespread zoonosis. This worldwide emerging infectious disease is caused by the pathogenic species belonging to the genus Leptospira. Polymerase chain reaction (PCR)-based diagnostic assays have been developed for detecting Leptospira DNA in cell cultures and clinical samples. Because PCR requires specialized equipment and extensive end-user training, it is not suitable for routine work in resource-limited areas. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of Leptospira in patient, animal and environmental samples using lyophilized reagents at a single temperature of around 63°C with a heating block. The sensitivity of this LAMP assay is very similar to the PCR method. The amplified DNA products can be visualized with the naked eyes using hydroxy naphthol blue or detected by the fluorescence signal of SYBR green dye in the reaction when an ultraviolet lamp or compact fluorescence tube scanner is used. This LAMP assay is simple, rapid, and can be performed with a water bath or heating block. The lyophilized LAMP reagents are stable for 3 months when stored at 4°C and 1 month when stored at 25°C, respectively. It is ideal for resource-limited settings where leptospirosis is endemic. PMID:27168577

  8. Elevated OPN, IP-10, and Neutrophilia in Loop-Mediated Isothermal Amplification Confirmed Tuberculosis Patients

    Directory of Open Access Journals (Sweden)

    Beata Shiratori

    2014-01-01

    Full Text Available Tuberculosis (TB is the second most common cause of death from infectious diseases and results in high socioeconomic losses to many countries. Proper diagnosis is the first step in TB eradication. To develop a rapid, simple, and accurate diagnostic TB test and to characterize the prevalence of Mycobacterium tuberculosis (MTB genotypes and immune profiles of TB patients, a total of 37 TB patients and 30 healthy control (HC from Metro Manila were enrolled. Loop-mediated isothermal amplification (LAMP reliably detected MTB infection. Manila genotype was identified by spoligotyping method in all TB patients. Osteopontin (OPN, interferon-γ-induced protein 10 kDa (IP-10, and neutrophil counts were found to reflect the acute stage of MTB infection. The sensitivity and specificity were 94.6% and 93.3%, respectively, for both OPN and IP-10, and they were 83.8% and 78.6%, respectively, for neutrophils. The combination of OPN, IP-10, neutrophil count, IL-6, IL-8, TNF-α, MCP-1, platelets, galectin-9, and leukocyte count correctly identifies all the HC and 96.3% of TB patients. LAMP method may serve as a rapid, supportive method in addition to time-consuming culture methods. OPN, IP-10, and neutrophil counts are useful in detecting MTB infection and may have utility in monitoring the course of the disease.

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

    Science.gov (United States)

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

    2016-01-15

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

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

    Directory of Open Access Journals (Sweden)

    Jing Liu

    2013-01-01

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

  11. Loop-Mediated Isothermal Amplification for Detection of Staphylococcus aureus in Dairy Cow Suffering from Mastitis

    Directory of Open Access Journals (Sweden)

    Zhang Tie

    2012-01-01

    Full Text Available To develop a rapid detection method of Staphylococcus aureus using loop-mediated isothermal amplification (LAMP, four specific primers were designed according to six distinct sequences of the nuc gene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was 1×102 CFU/mL and that of PCR was 1×104 CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection of Staphylococcus aureus has many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection of Staphylococcus aureus.

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

    Science.gov (United States)

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

    2015-01-01

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

  13. Rapid and Sensitive Detection of PRRSV by a Reverse Transcription-Loop-mediated Isothermal Amplification Assay

    Institute of Scientific and Technical Information of China (English)

    Lei Zhang; Ye-bing Liu; Lei Chen; Jian-huan Wang; Yi-bao Ning

    2011-01-01

    A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic,prevalent North American porcine reproductive and respiratory syndrome virus (PRRSV)strains.As a higher sensitivity and specificity method than reverse transcription polymerase chain reaction (RT-PCR),the RT-LAMP method only used a turbidimeter,exhibited a detection limit corresponding to a 10-4 dilution of template RNA extracted from 250 μL of 105 of the 50% tissue culture infective dose (TCID50) of PRRSV containing cells,and no cross-reactivity was observed with other related viruses including porcine circovirus type 2,swine influenza virus,porcine rotavirus and classical swine fever virus.From forty-two field samples,33 samples in the RT-LAMP assay was detected positive,whereas three of which were not detected by RT-PCR.Furthermore,in 33 strains of PRRSV,an identical detection rate was observed with the RT-LAMP assay to what were isolated using porcine alveolar macrophages.These findings demonstrated that the RT-LAMP assay has potential clinical applications for the detection of highly pathogenic PRRSV isolates,especially in developing countries.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Simple detection of Pythium irregulare using loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Feng, Wenzhuo; Ishiguro, Yasushi; Hotta, Keisuke; Watanabe, Hideki; Suga, Haruhisa; Kageyama, Koji

    2015-11-01

    Pythium irregulare is an important soil-borne pathogen that causes seed, stem and root rot, and seedling damping-off in various crops. Here, we have developed a rapid and reliable approach for detecting the pathogen using loop-mediated isothermal amplification (LAMP) in combination with primers designed from the sequences of the P. irregulare ribosomal DNA internal transcribed spacer region. The specificity of the primers for P. irregulare was tested using 50 isolates of 40 Pythium species, 11 Phytophthora isolates and 8 isolates of 7 other soil-borne pathogens. The assay showed that the limit of sensitivity of the LAMP method was 100 fg of pure DNA, a similar level to that of a polymerase chain reaction. LAMP detected P. irregulare from the supernatant after mixing culture medium (template DNA source) with distilled water. Similarly, positive results were obtained using a 'Plant-LAMP' method applied to a suspension rotted roots in water. A 'Bait-LAMP' method using the supernatant of autoclaved perilla seeds incubated in a soil/water mixture for 1 week at 25°C successfully detected P. irregulare from the soil. The LAMP assay described in this study is therefore a simple and effective way for practical detection of P. irregulare. PMID:26394643

  16. Detection of Goss's Wilt Pathogen Clavibacter michiganensis subsp. nebraskensis in Maize by Loop-Mediated Amplification.

    Science.gov (United States)

    Yasuhara-Bell, Jarred; de Silva, Asoka; Heuchelin, Scott A; Chaky, Jennifer L; Alvarez, Anne M

    2016-03-01

    The Goss's wilt pathogen, Clavibacter michiganensis subsp. nebraskensis, can cause considerable losses in maize (Zea mays) production. Diagnosis of Goss's wilt currently is based on symptomology and identification of C. michiganensis subsp. nebraskensis, following isolation on a semiselective medium and/or serological testing. In an effort to provide a more efficient identification method, a loop-mediated amplification (LAMP) assay was developed to detect the tripartite ATP-independent periplasmic (TRAP)-type C4-dicarboxylate transport system large permease component and tested using strains of C. michiganensis subsp. nebraskensis, all other C. michiganensis subspecies and several genera of nontarget bacteria. Only strains of C. michiganensis subsp. nebraskensis reacted positively with the LAMP assay. The LAMP assay was then used to identify bacterial isolates from diseased maize. 16S rDNA and dnaA sequence analyses were used to confirm the identity of the maize isolates and validate assay specificity. The Cmm ImmunoStrip assay was included as a presumptive identification test of C. michiganensis subsp. nebraskensis at the species level. The Cmn-LAMP assay was further tested using symptomatic leaf tissue. The Cmn-LAMP assay was run in a hand-held real-time monitoring device (SMART-DART) and performed equally to in-lab quantitative polymerase chain reaction equipment. The Cmn-LAMP assay accurately identified C. michiganensis subsp. nebraskensis and has potential as a field test. The targeted sequence also has potential application in other molecular detection platforms. PMID:26595113

  17. Screening of vibriosis in Asian seabass, Lates calcarifer using loop-mediated isothermal amplification (LAMP assay

    Directory of Open Access Journals (Sweden)

    Christopher Marlowe A. Caipang

    2012-09-01

    Full Text Available The aim of this study was to standardize a loop-mediated isothermal amplification (LAMP assay for the detection of Vibrio harveyi,the causative agent of vibriosis in Asian seabass, Lates calcarifer. The dnaJ gene of the bacterial pathogen was used as the target gene for theLAMP assay. It was optimized at an incubation time of 1 h at 630C. The assay was highly specific for V. harveyiand did not cross-react withother bacterial pathogens of fish. However, the assay was able to detect V. harveyithat was isolated from infected shrimps. The limit of detectionof the LAMP assay was 40 pg of DNA mL-1 or 40 fg of the genomic DNA per LAMP reaction and was 10 times more sensitive than conventionalPCR in detecting the bacterial pathogen from infected samples. The LAMP products can be quantified spectrophotometrically usinghydroxynaphthol blue (HNB dye and showed positive correlation with the amount of the pathogen. These results demonstrated that LAMP isa simple and sensitive detection technique that has potential application for routine diagnosis of vibrosis caused by V. harveyiin Asian seabassand other aquatic species.

  18. 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. PMID:24769198

  19. Loop-Mediated Isothermal Amplification Targeting Actin DNA of Trichomonas vaginalis.

    Science.gov (United States)

    Goo, Youn-Kyoung; Shin, Won-Sik; Yang, Hye-Won; Joo, So-Young; Song, Su-Min; Ryu, Jae-Sook; Kong, Hyun-Hee; Lee, Won-Ki; Chung, Dong-Il; Hong, Yeonchul

    2016-06-01

    Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications. PMID:27417089

  20. 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.

  1. Rapid detection of squash leaf curl virus by loop-mediated isothermal amplification.

    Science.gov (United States)

    Kuan, Cheng-Ping; Wu, Min-Tze; Lu, Yi-Lin; Huang, Hung-Chang

    2010-10-01

    A loop-mediated isothermal amplification (LAMP) assay was employed to develop a simple and efficient system for the detection of squash leaf curl virus (SLCV) in diseased plants of squash (Cucurbita pepo) and melon (Cucumis melo). Completion of LAMP assay required 30-60 min under isothermal conditions at 65 degrees C by employing a set of four primers targeting SLCV. Although the sensitivity of the LAMP assay and the polymerase chain reaction (PCR) assay was comparable at high virus concentrations, the LAMP assay was by a 10-fold dilution factor more sensitive than the PCR assay for the detection of SLCV in diseased plants. No reaction was detected in the tissues of healthy plants by either the LAMP or the PCR. The LAMP products can be visualized by staining directly in the tube with SYBR Safe DNA gel stain dye. The sensitivity of the SYBR Safe DNA gel stain is similar to analysis by gel electrophoresis. Although both the LAMP and the PCR methods were capable of detecting SLCV in infected tissues of squash and melon, the LAMP method would be more useful than the PCR method for detection of SLCV infection in cucurbitaceous plants because it is more rapid, simple, accurate and sensitive.

  2. 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...

  3. Rapid typing of foot-and-mouth disease serotype Asia 1 by reverse transcription loop-mediated isothermal amplification

    OpenAIRE

    Chen Hao-tai; Zhang Jie; Liu Yong-sheng; Liu Xiang-tao

    2011-01-01

    Abstract A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was rapidly used to detect serotype Asia 1 of foot-and-mouth disease virus (FMDV) within 45 min at 61°C. All FMDV serotype Asia 1 reference strains were positive by RT-LAMP, while other viruses such as FMDV serotypes O, C, A and classical swine fever virus, swine vesicular disease virus, porcine reproductive and respiratory syndrome virus and Japanese encephalitis virus remained negative. Furthermore, FMDV...

  4. 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.

  5. 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)

    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 ...

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Rapid detection of Streptococcus uberis in raw milk by loop-mediated isothermal amplification.

    Science.gov (United States)

    Cornelissen, J B W J; De Greeff, A; Heuvelink, A E; Swarts, M; Smith, H E; Van der Wal, F J

    2016-06-01

    A loop-mediated isothermal amplification (LAMP) method to detect Streptococcus uberis in raw milk was developed and evaluated. Three genes (sodA, pauA, cpn60) were assessed for their suitability as targets in LAMP. The analytical sensitivity was 120, 120, and 12 fg per assay for the sodA, pauA, and cpn60 assays, respectively, with a detectable signal within 8 min for the highest concentration (12ng/assay) and ~60 min for the lowest concentrations. The LAMP assays correctly identified 7 Strep. uberis strains among a set of 83 mastitis pathogens. To enable DNA isolation from raw milk, a new method was used in which a pretreatment with a cocktail of lysing enzymes was performed before an established procedure. This method resulted in an analytical sensitivity of 48 cfu/assay for the sodA LAMP assay using raw milk spiked with Strep. uberis, corresponding to 2.4×10(4) cfu/mL milk. For raw milk samples from cows experimentally infected with Strep. uberis, results of enumeration were largely reflected by results of LAMP. Evaluation of the sodA LAMP assay with 100 raw milk field samples, of which 50 were Strep. uberis culture-negative and 50 Strep. uberis culture-positive, showed that the assay had a diagnostic sensitivity of 96.0% and a diagnostic specificity of 96.0%. In conclusion, the described LAMP assay may offer a simple alternative for convenient and sensitive detection of S. uberis in raw milk, provided a compatible rapid DNA isolation procedure is available. PMID:27060835

  8. Loop-mediated isothermal amplification for rapid and semiquantitative detection of Loa loa infection.

    Science.gov (United States)

    Drame, Papa M; Fink, Doran L; Kamgno, Joseph; Herrick, Jesica A; Nutman, Thomas B

    2014-06-01

    Rapid and accurate tests are currently needed to identify individuals with high levels of Loa loa microfilaria (mf), so that these individuals may be excluded from mass ivermectin administration campaigns against onchocerciasis and lymphatic filariasis being conducted in areas where Onchocerca volvulus, Wuchereria bancrofti, and L. loa are coendemic. To address this need, colorimetric loop-mediated isothermal amplification (LAMP) assays targeting the L. loa-specific gene sequences LLMF72 and LLMF342 were developed for the detection and quantification of L. loa microfilaremia. Both LAMP assays were highly specific (100%) for L. loa infection compared to the absence of infection or infection with related filarial pathogens. The LLMF72-based LAMP assay showed greater analytic sensitivity (limit of detection, 0.1 pg/ml of genomic DNA [gDNA] and/or 5 mf/ml) than the LLMF342-based LAMP assay (10 pg/ml of gDNA and/or 50 mf/ml), and its analytic sensitivity was similar to that of LLMF72-based quantitative PCR (qPCR). A high level of correlation was observed between microfilaria counts as determined by LLMF72-based qPCR and time to positivity by the LAMP assay, and performance measures of sensitivity, specificity, and positive and negative predictive values were similar for both assays when applied to field-collected clinical samples. By simply varying the run time, the LAMP assay was able to accurately distinguish individuals at risk for serious adverse events (SAEs) after exposure to ivermectin, using thresholds of >5,000 mf/ml and >30,000 mf/ml as indicators of increasing levels of risk. In summary, LLMF72 LAMP represents a new molecular diagnostic tool that is readily applicable as a point-of-care method for L. loa microfilarial detection and quantification in resource-limited countries where L. loa infection is endemic. PMID:24696020

  9. 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.

  10. 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. PMID:27063408

  11. Detection of foot-and-mouth disease virus rna by reverse transcription loop-mediated isothermal amplification

    Directory of Open Access Journals (Sweden)

    Chen Hao-tai

    2011-11-01

    Full Text Available Abstract A reverse transcription loop-mediated isothermal amplification (RT-LAMP assay was developed for foot-and-mouth disease virus (FMDV RNA. The amplification was able to finish in 45 min under isothermal condition at 64°C by employing a set of four primers targeting FMDV 2B. The assay showed higher sensitivity than RT-PCR. No cross reactivity was observed from other RNA viruses including classical swine fever virus, swine vesicular disease, porcine reproductive and respiratory syndrome virus, Japanese encephalitis virus. Furthermore, the assay correctly detected 84 FMDV positive samples but not 65 FMDV negative specimens. The result indicated the potential usefulness of the technique as a simple and rapid procedure for the detection of FMDV infection.

  12. A one-step reverse transcription loop-mediated isothermal amplification for detection and discrimination of infectious bursal disease virus

    Directory of Open Access Journals (Sweden)

    Qi Xiaole

    2011-03-01

    Full Text Available Abstract Background Infectious bursal disease (IBD is a highly contagious immunosuppressive disease in young chickens caused by infectious bursal disease virus (IBDV. It causes huge economic losses to the poultry industry. The objective of this study is to develop a loop-mediated isothermal amplification (LAMP method for the detection and discrimination of IBDV. Results In this study, we applied reverse transcription loop-mediated isothermal amplification (RT-LAMP to detect IBDV in one simple step and further identified the very virulent strain from non-vvIBDVs with a simply post-amplification restriction enzyme analysis. Based on sequence analysis, a set of two inner, two outer and two loop primers were designed to target the VP5 gene and they showed great specificity with no cross reaction to the other common avian pathogens. The detection limit determined by both color change inspection and agarose gel electrophoresis was 28 copies viral RNA, which was almost as sensitive as a real-time RT-PCR previous developed in our laboratory. We also identified a unique Tfi I restriction site located exclusively in non-vvIBDVs, so very virulent strain could be distinguished from current vaccine strains. By screening a panel of clinical specimens, results showed that this method is high feasible in clinical settings, and it obtained results 100% correlated with real-time RT-PCR. Conclusion RT-LAMP is a rapid, simple and sensitive assay. In combination with the Tfi I restriction analysis, this method holds great promises not only in laboratory detection and discrimination of IBDV but also in large scale field and clinical studies.

  13. A reverse transcription loop-mediated isothermal amplification assay to rapidly diagnose foot-and-mouth disease virus C

    OpenAIRE

    Ding, Yao-zhong; Zhou, Jian-Hua; Ma, Li-na; Qi, Yan-ni; Wei, Gang; Zhang, Jie; Zhang, Yong-guang

    2014-01-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to rapidly detect foot-and-mouth disease virus serotype C (FMDV C). By testing 10-fold serial dilutions of FMDV C samples, sensitivity of the FMDV C RT-LAMP was found to be 10 times higher than that of conventional reverse transcription-PCR (RT-PCR). No cross-reactivity with A, Asia 1, or O FMDV or swine vesicular disease virus (SVDV) indicated that FMDV C RT-LAMP may be an exciting novel method for d...

  14. Spatial model of autocatalytic reactions

    Science.gov (United States)

    de Anna, Pietro; di Patti, Francesca; Fanelli, Duccio; McKane, Alan J.; Dauxois, Thierry

    2010-05-01

    Biological cells with all of their surface structure and complex interior stripped away are essentially vesicles—membranes composed of lipid bilayers which form closed sacs. Vesicles are thought to be relevant as models of primitive protocells, and they could have provided the ideal environment for prebiotic reactions to occur. In this paper, we investigate the stochastic dynamics of a set of autocatalytic reactions, within a spatially bounded domain, so as to mimic a primordial cell. The discreteness of the constituents of the autocatalytic reactions gives rise to large sustained oscillations even when the number of constituents is quite large. These oscillations are spatiotemporal in nature, unlike those found in previous studies, which consisted only of temporal oscillations. We speculate that these oscillations may have a role in seeding membrane instabilities which lead to vesicle division. In this way synchronization could be achieved between protocell growth and the reproduction rate of the constituents (the protogenetic material) in simple protocells.

  15. Spatial model of autocatalytic reactions

    OpenAIRE

    De Anna, Pietro; Di Patti, Francesca; Fanelli, Duccio; McKane, Alan J.; Dauxois, Thierry

    2010-01-01

    Biological cells with all of their surface structure and complex interior stripped away are essentially vesicles - membranes composed of lipid bilayers which form closed sacs. Vesicles are thought to be relevant as models of primitive protocells, and they could have provided the ideal environment for pre-biotic reactions to occur. In this paper, we investigate the stochastic dynamics of a set of autocatalytic reactions, within a spatially bounded domain, so as to mimic a primordial cell. The ...

  16. 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.

  17. A loop-mediated isothermal amplification (LAMP) method for the identification of species within the Echinococcus granulosus complex.

    Science.gov (United States)

    Wassermann, Marion; Mackenstedt, Ute; Romig, Thomas

    2014-02-24

    To facilitate the specific identification of Echinococcus spp. isolates in endemic countries, a LAMP (loop-mediated isothermal amplification) assay was developed to detect the various agents known to cause cystic echinococcosis (E. granulosus s.s., E. equinus, E. ortleppi, E. canadensis and E. felidis). The infectivity of the different species and the severity of the disease in humans and livestock vary significantly among those species, and correct molecular identification of large numbers of field isolates is crucial to understand their epidemiology. However, funding constraints in many CE endemic countries often prevent PCR-based screening of field isolates. The LAMP method allows the amplification of DNA fragments under isothermal conditions which can be achieved using an ordinary waterbath, and the detection of amplification products only requires a UV light source. In the present study a LAMP assay was developed which allows the detection and differentiation of the 5 CE causing Echinococcus species. The diagnostic power was adjusted to species level, i.e. intraspecific strains (G1-3 within E. granulosus s.s., G6-10 within E. canadensis) are not discriminated. Wherever this would be necessary for epidemiological purposes, the method can be adjusted according to local requirements. The sensitivity of the assay was tested down to one fiftieth of a single protoscolex or egg, respectively. The present study describes a fast and simple method for the differentiation of CE causing Echinococcus species which can facilitate epidemiological studies in endemic countries.

  18. Loop-mediated isothermal amplification assay for the detection of Ehrlichia canis DNA in blood samples from dogs

    Directory of Open Access Journals (Sweden)

    SA Faggion

    2013-01-01

    Full Text Available The rickettsial bacterium Ehrlichia canis is the etiological agent of canine monocytic ehrlichiosis, one of the most important canine tick-borne diseases in the world. In this study, a loop-mediated isothermal amplification (LAMP assay was developed for detection of E. canis DNA using LAMP primers targeting the groESL operon. Reactions were performed at 60°C for 60 min and the results were visualized by gel electrophoresis. Successful amplification was obtained using plasmid DNA containing a fragment of the groESL operon and DNA extracted from blood samples that tested positive for E. canis by real-time PCR. The specificity of amplification was confirmed by EcoRI restriction of internal sites in the LAMP primers and no cross-reactivity with blood samples positive for Babesia spp., another common tick-borne pathogen, was observed. The high cost of nucleic acid tests (NAT is one of the disadvantages for their large-scale use as routine diagnostic tests. The E. canis LAMP assay developed here is an interesting alternative to PCR since it does not require a thermocycler, thus reducing costs for the veterinary clinical laboratory.

  19. 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.

  20. Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods

    Directory of Open Access Journals (Sweden)

    Vojnov Adrian A

    2010-06-01

    Full Text Available Abstract Background Citrus Bacterial Canker (CBC is a major, highly contagious disease of citrus plants present in many countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker, named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative agent for type A CBC is Xanthomonas citri subsp. citri, while Xanthomonas fuscans subsp. aurantifolii, strain B causes type B CBC and Xanthomonas fuscans subsp. aurantifolii strain C causes CBC type C. The early and accurate identification of those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation, antibodies or polymerase chain reaction (PCR have been developed previously; however, these approaches may be time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated isothermal amplification (LAMP, which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and requires no specialized laboratory equipment. Results A loop-mediated isothermal amplification assay for the diagnosis of Citrus Bacterial Canker (CBC-LAMP was developed and evaluated. DNA samples were obtained from infected plants or cultured bacteria. A typical ladder-like pattern on gel electrophoresis was observed in all positive samples in contrast to the negative controls. In addition, amplification products were detected by visual inspection using SYBRGreen and using a lateral flow dipstick, eliminating the need for gel electrophoresis. The sensitivity and specificity of the assay were evaluated in different conditions and using several sample sources which included purified DNA, bacterium culture and infected plant tissue. The sensitivity of the CBC-LAMP was 10 fg of pure Xcc DNA, 5 CFU in culture samples and 18 CFU in samples of infected plant tissue. No cross reaction was observed with DNA

  1. Linear nonequilibrium thermodynamics describes the dynamics of an autocatalytic system.

    Science.gov (United States)

    Cortassa, S; Aon, M A; Westerhoff, H V

    1991-01-01

    A model simulating oscillations in glycolysis was formulated in terms of nonequilibrium thermodynamics. In the kinetic rate equations every metabolite concentration was replaced with an exponential function of its chemical potential. This led to nonlinear relations between rates and chemical potentials. Each chemical potential was then expanded around its steady-state value as a Taylor series. The linear (first order) term of the Taylor series sufficed to simulate the dynamic behavior of the system, including the damped and even sustained oscillations at low substrate input or high free-energy load. The glycolytic system is autocatalytic in the first half. Because oscillations were obtained only in the presence of that autocatalytic feed-back loop we conclude that this type of kinetic nonlinearity was sufficient to account for the oscillatory behavior. The matrix of phenomenological coefficients of the system is nonsymmetric. Our results indicate that this is the symmetry property and not the linearity of the flow-force relations in the near equilibrium domain that precludes oscillations. Given autocatalytic properties, a system exhibiting liner flow-force relations and being outside the near equilibrium domain may show bifurcations, leading to self-organized behavior. Images FIGURE 5 PMID:1742453

  2. Autocatalytic plume pinch-off

    CERN Document Server

    Rogers, Michael C; Morris, Stephen W

    2010-01-01

    A localized source of buoyancy flux in a non-reactive fluid medium creates a plume. The flux can be provided by either heat, a compositional difference between the fluid comprising the plume and its surroundings, or a combination of both. For autocatalytic plumes produced by the iodate-arsenous acid reaction, however, buoyancy is produced along the entire reacting interface between the plume and its surroundings. Buoyancy production at the moving interface drives fluid motion, which in turn generates flow that advects the reaction front. As a consequence of this interplay between fluid flow and chemical reaction, autocatalytic plumes exhibit a rich dynamics during their ascent through the reactant medium. One of the more interesting dynamical features is the production of an accelerating vortical plume head that in certain cases pinches-off and detaches from the upwelling conduit. After pinch-off, a new plume head forms in the conduit below, and this can lead to multiple generations of plume heads for a singl...

  3. Development of loop-mediated isothermal amplification method for detecting Wuchereria bancrofti DNA in human blood and vector mosquitoes.

    Science.gov (United States)

    Takagi, Hidekazu; Itoh, Makoto; Kasai, Shinji; Yahathugoda, Thishan C; Weerasooriya, Mirani V; Kimura, Eisaku

    2011-12-01

    We have developed loop-mediated isothermal amplification (LAMP) method to detect Wuchereria bancrofti DNA. The sensitivity and specificity of LAMP method were equivalent to those of PCR method which detects SspI repeat sequence in W. bancrofti genomic DNA: both methods detected one thousandth of W. bancrofti DNA from one microfilaria (Mf), and did not cross-react with DNAs of Brugia malayi, B. pahangi, Dirofilaria immitis, human and Culex quinquefasciatus. We also examined the sensitivity of LAMP using the mimic samples of patient's blood or blood-fed mosquitoes containing one W. bancrofti Mf per sample. The LAMP method was able to detect W. bancrofti DNA in 1000 μl of blood or in a pool of 60 mosquitoes, indicating its usefulness in detecting/monitoring W. bancrofti infection in humans and vector mosquitoes in endemic areas. PMID:21930238

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

    Directory of Open Access Journals (Sweden)

    Guan-Hua Lai

    2015-01-01

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

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

    Science.gov (United States)

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

    2014-07-15

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

  6. Rapid typing of foot-and-mouth disease serotype Asia 1 by reverse transcription loop-mediated isothermal amplification

    Directory of Open Access Journals (Sweden)

    Chen Hao-tai

    2011-10-01

    Full Text Available Abstract A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP assay was rapidly used to detect serotype Asia 1 of foot-and-mouth disease virus (FMDV within 45 min at 61°C. All FMDV serotype Asia 1 reference strains were positive by RT-LAMP, while other viruses such as FMDV serotypes O, C, A and classical swine fever virus, swine vesicular disease virus, porcine reproductive and respiratory syndrome virus and Japanese encephalitis virus remained negative. Furthermore, FMDV sreotype Asia 1 positive samples were able to detect by RT-LAMP assay. This RT-LAMP assay may be suitable particularly for diagnosis of FMDV serotype Asia 1 infection in field stations.

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

    Directory of Open Access Journals (Sweden)

    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

  8. Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification.

    Science.gov (United States)

    Itonaga, Masahiro; Matsuzaki, Ibu; Warigaya, Kenji; Tamura, Takaaki; Shimizu, Yuki; Fujimoto, Masakazu; Kojima, Fumiyoshi; Ichinose, Masao; Murata, Shin-Ichi

    2016-01-01

    Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation. PMID:26999437

  9. Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification.

    Directory of Open Access Journals (Sweden)

    Masahiro Itonaga

    Full Text Available Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP, for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation.

  10. Real-time loop-mediated isothermal amplification (RealAmp for the species-specific identification of Plasmodium vivax.

    Directory of Open Access Journals (Sweden)

    Jaymin C Patel

    Full Text Available Plasmodium vivax infections remain a major source of malaria-related morbidity and mortality. Early and accurate diagnosis is an integral component of effective malaria control programs. Conventional molecular diagnostic methods provide accurate results but are often resource-intensive, expensive, have a long turnaround time and are beyond the capacity of most malaria-endemic countries. Our laboratory has recently developed a new platform called RealAmp, which combines loop-mediated isothermal amplification (LAMP with a portable tube scanner real-time isothermal instrument for the rapid detection of malaria parasites. Here we describe new primers for the detection of P. vivax using the RealAmp method. Three pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the P. vivax genome. The amplification was carried out at 64°C using SYBR Green or SYTO-9 intercalating dyes for 90 minutes with the tube scanner set to collect fluorescence signals at 1-minute intervals. Clinical samples of P. vivax and other human-infecting malaria parasite species were used to determine the sensitivity and specificity of the primers by comparing with an 18S ribosomal RNA-based nested PCR as the gold standard. The new set of primers consistently detected laboratory-maintained isolates of P. vivax from different parts of the world. The primers detected P. vivax in the clinical samples with 94.59% sensitivity (95% CI: 87.48-98.26% and 100% specificity (95% CI: 90.40-100% compared to the gold standard nested-PCR method. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting P. vivax.

  11. Adaptation of a visualized loop-mediated isothermal amplification technique for field detection of Plasmodium vivax infection

    Directory of Open Access Journals (Sweden)

    Wang Wei-Ming

    2011-06-01

    Full Text Available Abstract Background Loop-mediated isothermal amplification (LAMP is a high performance method for detecting DNA and holds promise for use in the molecular detection of infectious pathogens, including Plasmodium spp. However, in most malaria-endemic areas, which are often resource-limited, current LAMP methods are not feasible for diagnosis due to difficulties in accurately interpreting results with problems of sensitive visualization of amplified products, and the risk of contamination resulting from the high quantity of amplified DNA produced. In this study, we establish a novel visualized LAMP method in a closed-tube system, and validate it for the diagnosis of malaria under simulated field conditions. Methods A visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction. A total of 89 blood samples were collected on filter paper and processed using a simple boiling method for DNA extraction, and then tested by the visualized LAMP method for Plasmodium vivax infection. Results The wax capsule remained intact during isothermal amplification, and released the DNA dye to the reaction mixture only when the temperature was raised to the melting point following amplification. Soon after cooling down, the solidified wax sealed the reaction mix at the bottom of the tube, thus minimizing the risk of aerosol contamination. Compared to microscopy, the sensitivity and specificity of LAMP were 98.3% (95% confidence interval (CI: 91.1-99.7% and 100% (95% CI: 88.3-100%, and were in close agreement with a nested polymerase chain reaction method. Conclusions This novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis in resource-limited field settings.

  12. Detection of shrimp Taura syndrome virus by loop-mediated isothermal amplification using a designed portable multi-channel turbidimeter.

    Science.gov (United States)

    Sappat, Assawapong; Jaroenram, Wansadaj; Puthawibool, Teeranart; Lomas, Tanom; Tuantranont, Adisorn; Kiatpathomchai, Wansika

    2011-08-01

    In this study, a portable turbidimetric end-point detection method was devised and tested for the detection of Taura syndrome virus (TSV) using spectroscopic measurement of a loop-mediated isothermal amplification (LAMP) by-product: magnesium pyrophosphate (Mg(2)P(2)O(7)). The device incorporated a heating block that maintained an optimal temperature of 63°C for the duration of the RT-LAMP reaction. Turbidity of the RT-LAMP by-product was measured when light from a light-emitting diode (LED) passed through the tube to reach a light dependent resistance (LDR) detector. Results revealed that turbidity measurement of the RT-LAMP reactions using this device provided the same detection sensitivity as the agarose gel electrophoresis detection of RT-LAMP and nested RT-PCR (IQ2000™) products. Cross reactions with other shrimp viruses were not found, indicating that the RT-LAMP-turbidity measurement was highly specific to TSV. The combination of 10 min for rapid RNA preparation with 30 min for RT-LAMP amplification followed by turbidity measurement resulted in a total assay time of less than 1h compared to 4-8h for the nested RT-PCR method. RT-LAMP plus turbidity measurement constitutes a platform for the development of more rapid and user-friendly detection of TSV in the field.

  13. Rapid and sensitive detection of Listeria ivanovii by loop-mediated isothermal amplification of the smcL gene.

    Directory of Open Access Journals (Sweden)

    Yi Wang

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

  14. Rapid identification and differentiation of Theileria sergenti and Theileria sinensis using a loop-mediated isothermal amplification (LAMP) assay.

    Science.gov (United States)

    Liu, Aihong; Guan, Guiquan; Du, Pengfei; Gou, Huitian; Zhang, Jun; Liu, Zhijie; Ma, Milin; Ren, Qiaoyun; Liu, Junlong; Yang, Jifei; Li, Youquan; Niu, Qinli; Bai, Qi; Yin, Hong; Luo, Jianxun

    2013-01-16

    The present study developed and validated a species-specific loop-mediated isothermal amplification (LAMP) assay for the rapid detection and discrimination of two benign bovine Theileria species -T. sergenti and T. sinensis. The LAMP assay is inexpensive and easy to perform and involves a rapid reaction-the amplification can be performed in 55 min or 50 min under isothermal conditions of 61°C or 63°C, respectively, by employing a set of four species-specific primer mixtures. The results can be checked using agarose gels. The optimal assay conditions, under which the assay exhibited with no cross-reaction with other closely related tick-borne parasites (T. annulata, Babesia bovis, B. bigemina, B. major, B. ovata, B. U. sp., Anaplasma marginale) or between the two Theileria species of interest, was established. The assay is approximately 10-fold more sensitive than the conventional specific PCR assay. The LAMP assay was validated using DNA from 6 standard stocks in the laboratory and was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infection cattle or yaks in China. These findings indicate that this Theileria species-specific LAMP assay may have potential clinical applications for the detection and differentiation of two benign bovine Theileria species -T. sergenti and T. sinensis, especially in endemic countries.

  15. 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. PMID:27288706

  16. Development of a rapid assay to detect the jellyfish Cyanea nozakii using a loop-mediated isothermal amplification method.

    Science.gov (United States)

    Liu, Zhongyuan; Dong, Zhijun; Liu, Dongyan

    2016-07-01

    Blooms of the harmful jellyfish Cyanea nozakii, which are a severe nuisance to fisheries and tourisms, frequently occur in the northern East China Sea, Yellow Sea, and Bohai Sea. To provide early warning of this species, a simple and effective molecular method for identifying C. nozakii was developed using the loop-mediated isothermal amplification method (LAMP). The LAMP assay is highly specific and uses a set of four primers that target six different regions on the mitochondrial cytochrome c oxidase subunit I (COI) gene of C. nozakii. The amplification conditions, including the dNTP and betaine concentrations, the inner primer to outer primer concentration ratio, reaction time and temperature, were optimized. The LAMP assay amplified DNA extracted from tissue samples of C. nozakii but did not amplify DNA from other common scyphozoans and hydrozoans collected in the same region. In addition, the LAMP assay was more sensitive than conventional PCR. Therefore, the established LAMP assay is a sensitive, specific, fast, and easily performed method for detection of C. nozakii at different stages in their life cycle.

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  19. A Loop-Mediated Isothermal Amplification Assay and Sample Preparation Procedure for Sensitive Detection of Xanthomonas fragariae in Strawberry.

    Directory of Open Access Journals (Sweden)

    Hehe Wang

    Full Text Available Xanthomonas fragariae is a bacterium that causes angular leaf spot of strawberry. Asymptomatic infection is common and contributes to the difficulties in disease management. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP assay as an efficient method for detection of asymptomatic infections of X. fragariae. In addition, a new method of sample preparation was developed that allows sampling of a larger amount of plant tissue, hence increasing the detection rate in real-life samples. The sample preparation procedure includes an overnight incubation of strawberry tissues in phosphate-buffered saline (PBS, followed by a quick sample concentration and a boiling step to extract DNA for amplification. The detection limit of the LAMP assay was approximately 2×10(3 CFU/mL for pure bacteria culture and 300 CFU/mL for bacteria spiked strawberry leaf and petiole samples. LAMP provided a 2-3 fold lower detection limit than the standard qPCR assay but was faster, and more user-friendly. The LAMP assay should serve as a rapid, sensitive and cost-effective tool for detecting asymptomatic infections of X. fragariae in strawberry nursery stock and contribute to improved disease management.

  20. Development of a rapid and specific loop-mediated isothermal amplification detection method that targets Marek's disease virus meq gene.

    Science.gov (United States)

    Wei, Xiuying; Shi, Xingming; Zhao, Yan; Zhang, Jing; Wang, Mei; Liu, Changjun; Cui, Hongyu; Hu, Shunlei; Quan, Yanming; Chen, Hongyan; Wang, Yunfeng

    2012-08-01

    A rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) method was developed and evaluated for the detection of Marek's disease virus (MDV) by amplification of conserved MDV meq gene sequences. LAMP is an innovative technique that allows the rapid detection of targeted nucleic acid sequences under isothermal conditions without the need for complex instrumentation. In this study, meq gene sequences were amplified successfully from different MDV strains by LAMP within 60min and no cross-reactivity was observed in a panel of related viruses that were associated with diseases of chickens. The detection limit of LAMP was 3.2 copies/million cells compared with 320 copies/million cells required for conventional PCR. Positive detection rates were assessed using either LAMP or PCR by examination of feather follicles that were collected from chickens infected experimentally with either strain J-1 (n=20) or strain Md5 (n=17), In addition to these samples, three isolates that were suspected to have been infected in the clinic were also tested. Results showed that the positive detection rate for LAMP was 95% (38/40), compared with 87.5% (35/40) and 90% (38/40) for strains J-1 and Md5 by PCR, respectively. These results indicated that the LAMP assay was more sensitive, rapid and specific than conventional PCR for the detection of MDV. This easy-to-perform technique will be useful for the detection of MDV and will aid in the establishment of disease control protocols.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2013-11-01

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

  3. Detection of Puccinia kuehnii Causing Sugarcane Orange Rust with a Loop-Mediated Isothermal Amplification-Based Assay.

    Science.gov (United States)

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

    2016-03-01

    Puccinia kuehnii is a fungal pathogen that causes orange rust in sugarcane, which is now prevalent in many countries. At the early stage of disease, it is almost indistinguishable from brown rust, which is caused by Puccinia melanocephala. Although several PCR assays are available to detect these diseases, the loop-mediated isothermal amplification (LAMP)-based assay has been reported to be more economical and easier to perform. Under isothermal conditions, DNA is amplified with high specificity and rapidity. Moreover, visual judgment of color change without further post-amplification processing makes the method convenient. The present study was undertaken to detect P. kuehnii genomic DNA using four primers corresponding to a unique DNA sequence of P. kuehnii. The LAMP assay was found to be optimal when 8 mM MgSO4 was used and the reaction was incubated at 63 °C for 90 min. Positive samples showed a color change from orange to green upon SYBR Green I dye addition. Specificity of the LAMP test was checked with DNA of P. melanocephala, which showed no reaction. Sensitivity of the LAMP method was observed to be the same as real-time PCR at 0.1 ng, thus providing a rapid and more affordable option for early disease detection. PMID:26837389

  4. Development of a loop-mediated isothermal amplification assay for the detection of Streptococcus agalactiae in bovine milk.

    Science.gov (United States)

    Bosward, Katrina L; House, John K; Deveridge, Amber; Mathews, Karen; Sheehy, Paul A

    2016-03-01

    Streptococcus agalactiae is a well-characterized bovine mastitis pathogen that is known to be highly contagious and capable of spreading rapidly in affected dairy herds. Loop-mediated isothermal amplification (LAMP) is a novel molecular diagnostic method that has the capability to provide rapid, cost-effective screening for pathogens to support on-farm disease control and eradication programs. In the current study, a LAMP test was developed to detect S. agalactiae in milk. The assay was validated on a bank of existing clinical mastitis milk samples that had previously been identified as S. agalactiae positive via traditional microbiological culture techniques and PCR. The LAMP assay was conducted on bacterial colonies and DNA extracted from milk in tube- and plate-based formats using multiple detection platforms. The 1-h assay conducted at 64 °C exhibited repeatability (coefficient of variation) of 2.07% (tube) and 8.3% (plate), sensitivity to ~20 pg of extracted DNA/reaction, and specificity against a panel of known bacterial mastitis pathogens. Of the 109 known S. agalactiae isolates assessed by LAMP directly from bacterial cells in culture, 108 were identified as positive, in accordance with PCR analysis. The LAMP analysis from the corresponding milk samples indicated that 104 of these milks exhibited a positive amplification curve. Although exhibiting some limitations, this assay provides an opportunity for rapid screening of milk samples to facilitate on-farm management of this pathogen. PMID:26778303

  5. Development and evaluation of loop-mediated isothermal amplification (LAMP) for the rapid diagnosis of Penicillium marneffei in archived tissue samples

    NARCIS (Netherlands)

    Sun, J.; Li, X.; Zeng, H.X.; Xie, Z.; Lu, C.; Xi, L.; de Hoog, G.S.

    2010-01-01

    Penicillium marneffei is the etiologic agent of a severe systemic disease in immunocompromised hosts in Southeast Asia. In the present study, a novel method, known as loop-mediated isothermal amplification (LAMP), is described for the rapid and specific detection of the species, using a primer set d

  6. 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.

  7. Autocatalytic activation of influenza hemagglutinin.

    Science.gov (United States)

    Lee, Jeong H; Goulian, Mark; Boder, Eric T

    2006-12-01

    Enveloped viruses contain surface proteins that mediate fusion between the viral and target cell membranes following an activating stimulus. Acidic pH induces the influenza virus fusion protein hemagglutinin (HA) via irreversible refolding of a trimeric conformational state leading to exposure of hydrophobic fusion peptides on each trimer subunit. Herein, we show that cells expressing fowl plague virus HA demonstrate discrete switching behavior with respect to the HA conformational change. Partially activated states do not exist at the scale of the cell, activation of HA leads to aggregation of cell surface trimers, and newly synthesized HA refold spontaneously in the presence of previously activated HA. These observations imply a feedback mechanism involving self-catalyzed refolding of HA and thus suggest a mechanism similar to the autocatalytic refolding and aggregation of prions.

  8. Weber's Law in Autocatalytic Reaction Networks

    CERN Document Server

    Inoue, Masayo

    2011-01-01

    Biological responses often obey Weber's law, according to which the magnitude of the response depends only on the fold change in the external input. In this study, we demonstrate that a system involving a simple autocatalytic reaction shows such response when a chemical is slowly synthesized by the reaction from a faster influx process. We also show that an autocatalytic reaction process occurring in series or in parallel can obey Weber's law with an oscillatory adaptive response. Considering the simplicity and ubiquity of the autocatalytic process, our proposed mechanism is thought to be commonly observed in biological reactions.

  9. Challenging loop-mediated isothermal amplification(LAMP) technique for molecular detection of Toxoplasma gondii

    Institute of Scientific and Technical Information of China (English)

    Shirzad; Fallahi; Zahra; Arab; Mazar; Mehrdad; Ghasemian; Ali; Haghighi

    2015-01-01

    Objective:To compare analytical sensitivity and specificity of a newly described DNA amplification technique.LAMP and nested PCR assay targeting the RE and Bl genes for the detection of Toxoplasma gondii(T.gondii) DNA.Methods:The analytical sensitivity of LAMP and ncstcd-PCR was obtained against 10-fold serial dilutions of T.gondii DNA ranging from 1 ng to 0.01 fg.DNA samples of other parasites and human chromosomal DNA were used to determine the specificity of molecular assays.Results:After testing LAMP and nesled-PCR in duplicate,the detection limit of RE-LAMP.B1-LAMP,RE-nested PCR and B1-nested PCR assays was one fg.100 fg,1 pg and 10 pg of T.gondii DNA respectively.All the LAMP assays and nested PCRs were 100% specific.The RE-LAMP assay revealed the most sensitivity for the detection of T.gondii DNA.Conclusions:The obtained results demonstrate that the LAMP technique has a greater sensitivity for detection of T.gondii.Furthermore,these findings indicate that primers based on the RE are more suitable than those based on the B1 gene.However,the B1-LAMP assay has potential as a diagnostic tool for detection of T.gondii.

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

    Science.gov (United States)

    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. PMID:24193953

  11. Open-loop control of noise amplification in a separated boundary layer flow

    CERN Document Server

    Boujo, Edouard; Gallaire, François

    2014-01-01

    Linear optimal gains are computed for the subcritical two-dimensional separated boundary-layer flow past a bump. Very large optimal gain values are found, making it possible for small-amplitude noise to be strongly amplified and to destabilize the flow. The optimal forcing is located close to the summit of the bump, while the optimal response is the largest in the shear layer. The largest amplification occurs at frequencies corresponding to eigenvalues which first become unstable at higher Reynolds number. Nonlinear direct numerical simulations show that a low level of noise is indeed sufficient to trigger random flow unsteadiness, characterized here by large-scale vortex shedding. Next, a variational technique is used to compute efficiently the sensitivity of optimal gains to steady control (through source of momentum in the flow, or blowing/suction at the wall). A systematic analysis at several frequencies identifies the bump summit as the most sensitive region for control with wall actuation. Based on thes...

  12. Loop mediated isothermal amplification (LAMP) assay for detection of coconut root wilt disease and arecanut yellow leaf disease phytoplasma.

    Science.gov (United States)

    Nair, Smita; Manimekalai, Ramaswamy; Ganga Raj, Palliyath; Hegde, Vinayaka

    2016-07-01

    The coconut root wilt disease (RWD) and the arecanut yellow leaf disease (YLD) are two major phytoplasma associated diseases affecting palms in South India. Greatly debilitating the palm health, these diseases cause substantial yield reduction and economic loss to farmers. A rapid and robust diagnostic technique is crucial in efficient disease management. We established phytoplasma 16S rDNA targeted loop mediated isothermal amplification (LAMP) and real time LAMP based diagnostics for coconut RWD and arecanut YLD. The LAMP reaction was set at 65 °C and end point detection made using hydroxynaphthol blue (HNB) and agarose gel electrophoresis. Molecular typing of LAMP products were made with restriction enzyme HpyCH4 V. Conventional PCR with LAMP external primers and sequencing of amplicons was carried out. Real time LAMP was performed on the Genei II platform (Optigene Ltd., UK). An annealing curve analysis was programmed at the end of the incubation to check the fidelity of the amplicons. The phytoplasma positive samples produced typical ladder like bands on agarose gel, showed colour change from violet to blue with HNB and produced unique annealing peak at 85 ± 0.5 °C in the real time detection. Restriction digestion produced predicted size fragments. Sequencing and BLASTN analysis confirmed that the amplification corresponded to phytoplasma 16S rRNA gene. LAMP method devised here was found to be more robust compared to conventional nested PCR and hence has potential applications in detection of phytoplasma from symptomatic palm samples and in rapid screening of healthy seedlings. PMID:27263003

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

    Directory of Open Access Journals (Sweden)

    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

  14. Development of mitochondrial loop-mediated isothermal amplification for detection of the small liver fluke Opisthorchis viverrini (Opisthorchiidae; Trematoda; Platyhelminthes).

    Science.gov (United States)

    Le, Thanh Hoa; Nguyen, Nga Thi Bich; Truong, Nam Hai; De, Nguyen Van

    2012-04-01

    Mitochondrial DNA sequences offer major advantages over the more usual nuclear targets for loop-mediated isothermal amplification approaches (mito-LAMP) because multiple copies occur in every cell. Four LAMP primers [F3, FIP(F1c+F2), BIP(B1c+B2), and B3] were designed based on the mitochondrial nad1 sequence of Opisthorchis viverrini and used for a highly specific assay (mito-OvLAMP) to distinguish DNA of O. viverrini from that of another opisthorchiid (Clonorchis sinensis) and other trematodes (Haplorchis pumilio, Haplorchis taichui, Fasciola hepatica, and Fasciola gigantica). Conventional PCR was applied using F3/B3 primer pairs to verify the specificity of the primers for O. viverrini DNA templates. All LAMP-positive samples could be detected with the naked eye in sunlight, by gel electrophoresis (stained with ethidium bromide), and by addition of SYBR green I to the product in sunlight or under UV light. Only DNA from O. viverrini yielded amplification products by LAMP (and by PCR verification), and the LAMP limit of detection was as little as 100 fg (10(-4) ng DNA), indicating that this assay is 10 to 100 times more sensitive than PCR. Field testing was done using representative egg and metacercarial samples collected from localities where the fluke is endemic. With the advantages of simplicity, rapidity, sensitivity, and cost effectiveness, mito-OvLAMP is a good tool for molecular detection and epidemiology studies in regions or countries where O. viverrini is endemic, which can lead to more effective control of opisthorchiasis and trematodiasis.

  15. Loop mediated isothermal amplification (LAMP) assay for detection of coconut root wilt disease and arecanut yellow leaf disease phytoplasma.

    Science.gov (United States)

    Nair, Smita; Manimekalai, Ramaswamy; Ganga Raj, Palliyath; Hegde, Vinayaka

    2016-07-01

    The coconut root wilt disease (RWD) and the arecanut yellow leaf disease (YLD) are two major phytoplasma associated diseases affecting palms in South India. Greatly debilitating the palm health, these diseases cause substantial yield reduction and economic loss to farmers. A rapid and robust diagnostic technique is crucial in efficient disease management. We established phytoplasma 16S rDNA targeted loop mediated isothermal amplification (LAMP) and real time LAMP based diagnostics for coconut RWD and arecanut YLD. The LAMP reaction was set at 65 °C and end point detection made using hydroxynaphthol blue (HNB) and agarose gel electrophoresis. Molecular typing of LAMP products were made with restriction enzyme HpyCH4 V. Conventional PCR with LAMP external primers and sequencing of amplicons was carried out. Real time LAMP was performed on the Genei II platform (Optigene Ltd., UK). An annealing curve analysis was programmed at the end of the incubation to check the fidelity of the amplicons. The phytoplasma positive samples produced typical ladder like bands on agarose gel, showed colour change from violet to blue with HNB and produced unique annealing peak at 85 ± 0.5 °C in the real time detection. Restriction digestion produced predicted size fragments. Sequencing and BLASTN analysis confirmed that the amplification corresponded to phytoplasma 16S rRNA gene. LAMP method devised here was found to be more robust compared to conventional nested PCR and hence has potential applications in detection of phytoplasma from symptomatic palm samples and in rapid screening of healthy seedlings.

  16. Specific, sensitive and rapid detection of human plasmodium knowlesi infection by loop-mediated isothermal amplification (LAMP in blood samples

    Directory of Open Access Journals (Sweden)

    Anthony Claudia N

    2011-07-01

    Full Text Available Abstract Background The emergence of Plasmodium knowlesi in humans, which is in many cases misdiagnosed by microscopy as Plasmodium malariae due to the morphological similarity has contributed to the needs of detection and differentiation of malaria parasites. At present, nested PCR targeted on Plasmodium ssrRNA genes has been described as the most sensitive and specific method for Plasmodium detection. However, this method is costly and requires trained personnel for its implementation. Loop-mediated isothermal amplification (LAMP, a novel nucleic acid amplification method was developed for the clinical detection of P. knowlesi. The sensitivity and specificity of LAMP was evaluated in comparison to the results obtained via microscopic examination and nested PCR. Methods LAMP assay was developed based on P. knowlesi genetic material targeting the apical membrane antigen-1 (AMA-1 gene. The method uses six primers that recognize eight regions of the target DNA and it amplifies DNA within an hour under isothermal conditions (65°C in a water-bath. Results LAMP is highly sensitive with the detection limit as low as ten copies for AMA-1. LAMP detected malaria parasites in all confirm cases (n = 13 of P. knowlesi infection (sensitivity, 100% and none of the negative samples (specificity, 100% within an hour. LAMP demonstrated higher sensitivity compared to nested PCR by successfully detecting a sample with very low parasitaemia ( Conclusion With continuous efforts in the optimization of this assay, LAMP may provide a simple and reliable test for detecting P. knowlesi malaria parasites in areas where malaria is prevalent.

  17. Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria.

    Science.gov (United States)

    Nagasawa, Mitsuaki; Kaku, Mitsuo; Kamachi, Kazunari; Shibayama, Keigo; Arakawa, Yoshichika; Yamaguchi, Keizo; Ishii, Yoshikazu

    2014-10-01

    Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings.

  18. Quality-factor amplification in piezoelectric MEMS resonators applying an all-electrical feedback loop

    International Nuclear Information System (INIS)

    An all-electrical velocity feedback control to enhance the quality factor of piezoelectric aluminium nitride (AlN)-based microcantilevers and microbridges was implemented. Two alternatives to obtain a velocity-proportional signal were demonstrated depending on the top electrode configuration. For a straightforward electrode design in one-port configuration (i.e. self-actuation and self-sensing), a velocity signal, proportional to the piezoelectric current, was used in the feedback loop by cancelling out the dielectric current electronically. For top electrodes allowing a two-port configuration (i.e. one for actuation and one for sensing), the piezoelectric current is directly extracted and its relationship with velocity is analysed taking the symmetry of the modal shape into account. Standard operational amplifier-based configurations for the feedback circuits were implemented on a printed circuit board. Quality factors were determined from the transient electrical response of the devices. Comparable results were obtained from the displacement spectrum applying a laser Doppler vibrometer. Quality factors as high as 2 × 105, corresponding to an enhancement factor of about 200, were achieved in air for the lowest gain margin achievable before the circuit becomes unstable, making this kind of device more competitive for mass sensor applications due to enhanced spectral resolution.

  19. Loop-mediated isothermal amplification applied to filarial parasites detection in the mosquito vectors: Dirofilaria immitis as a study model

    Directory of Open Access Journals (Sweden)

    Nelson Bryce

    2009-03-01

    Full Text Available Abstract Background Despite recent advances in our understanding of the basic biology behind transmission of zoonotic infectious diseases harbored by arthropod vectors these diseases remain threatening public health concerns. For effective control of vector and treatment, precise sampling indicating the prevalence of such diseases is essential. With an aim to develop a quick and simple method to survey zoonotic pathogen-transmitting vectors, LAMP (loop-mediated isothermal amplification was applied to the detection of filarial parasites using a filarial parasite-transmitting experimental model that included one of the mosquito vectors, Aedes aegypti, and the canine heartworm, Dirofilaria immitis. Results LAMP reactions amplifying the cytochrome oxidase subunit I gene demonstrated high sensitivity when a single purified D. immitis microfilaria was detected. Importantly, the robustness of the LAMP reaction was revealed upon identification of an infected mosquito carrying just a single parasite, a level easily overlooked using conventional microscopic analysis. Furthermore, successful detection of D. immitis in wild-caught mosquitoes demonstrated its applicability to field surveys. Conclusion Due to its simplicity, sensitivity, and reliability, LAMP is suggested as an appropriate diagnostic method for routine diagnosis of mosquito vectors carrying filarial parasites. This method can be applied to the survey of not only canine filariasis but also lymphatic filariasis, another major public health problem. Therefore, this method offers great promise as a useful diagnostic method for filarial parasite detection in endemic filariasis regions.

  20. Loop-mediated isothermal amplification (LAMP assays for the species-specific detection of Eimeria that infect chickens

    Directory of Open Access Journals (Sweden)

    Blake Damer P

    2011-11-01

    Full Text Available Abstract Background Eimeria parasites can cause the disease coccidiosis in poultry and even subclinical infection can incur economic loss. Diagnosis of infection predominantly relies on traditional techniques including lesion scoring and faecal microscopy despite the availability of sensitive molecular assays, largely due to cost and the requirement for specialist equipment. Despite longstanding proven efficacy these traditional techniques demand time and expertise, can be highly subjective and may under-diagnose subclinical disease. Recognition of the tight economic margins prevailing in modern poultry production and the impact of avian coccidiosis on poverty in many parts of the world has highlighted a requirement for a panel of straightforward and sensitive, but cost-effective, Eimeria species-specific diagnostic assays. Results Loop-mediated isothermal amplification (LAMP is an uncomplicated, quick and relatively inexpensive diagnostic tool. In this study we have developed a panel of species-specific LAMP assays targeting the seven Eimeria species that infect the chicken. Each assay has been shown to be genuinely species-specific with the capacity to detect between one and ten eimerian genomes, equivalent to less than a single mature schizont. Development of a simple protocol for template DNA preparation from tissue collected post mortem with no requirement for specialist laboratory equipment supports the use of these assays in routine diagnosis of eimerian infection. Preliminary field testing supports this hypothesis. Conclusions Development of a panel of sensitive species-specific LAMP assays introduces a valuable new cost-effective tool for use in poultry husbandry.

  1. 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.

  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. 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. PMID:27339262

  5. Loop-mediated isothermal amplification (LAMP) assay for the rapid detection of the sexually-transmitted parasite, Trichomonas vaginalis.

    Science.gov (United States)

    Adao, Davin Edric V; Rivera, Windell L

    2016-01-01

    A loop-mediated isothermal amplification (LAMP) assay was developed to detect the sexually-transmitted parasite, Trichomonas vaginalis in vaginal swabs. The presence of T. vaginalis was detected from 121 female sex workers attending a social hygiene clinic in Balibago, Angeles City, Pampanga, Philippines using culture, polymerase chain reaction (PCR), and the developed LAMP assay. The high analytical sensitivity of LAMP detected a higher prevalence of T. vaginalis (42.06%) compared to culture (8.26%) and PCR (7.44%). Additionally, this assay did not cross-react with DNAs of other trichomonads that can infect humans such as Trichomonas tenax and Pentatrichomonas hominis as well as the pathogens, Candida albicans and Staphylococcus aureus. The LAMP assay developed had a limit of detection (0.036 ng/μl) lower than that of PCR using the primers TvK3 and TvK7 (0.36 ng/μl). Prevalence of T. vaginalis in female sex workers in this area of the Philippines may be higher than previously estimated. Discordant results of PCR and LAMP may be due to different reactions to different kinds of inhibitors in the vaginal swabs. PMID:27262954

  6. Efficient detection of pathogen virus in sand dabs,Paralichthys olivaceus using loop-mediated isothermal amplification (LAMP)

    Institute of Scientific and Technical Information of China (English)

    HWANG Jinik; PARK So Yun; SUH Sung-Suk; PARK Mirye; LEE Sukchan; LEE Taek-Kyun

    2016-01-01

    Viral hemorrhagic septicemia virus (VHSV) and marine birnavirus (MABV) are the causative pathogens for some of the most explosive epidemics of emerging viral diseases in many Asian countries, leading to huge economic losses in aquaculture. Rapid molecular detection for surveillance or diagnosis has been a critical component in reducing the prevalence of pathogen infection. The loop-mediated isothermal amplification (LAMP) of DNA is currently one of the most commonly used molecular diagnostic tools, as it is simple, quick, and easy to amplify target DNA under isothermal conditions. In the present study, a novel and highly specific LAMP assay for the sensitive and rapid detection of VHSV and MABV infection in fish was developed. Using a set of synthesized primers matching a specific region of the genome, the efficiency and specificity of the LAMP assay were optimized in terms of the reaction temperature and DNA polymerase concentration, as they are the main determinants of the sensitivity and specificity of the LAMP assay. In particular, we demonstrated that our assay could be applied to efficient detection of VHSV and MABV infection in the wild fish,Paralichthys olivaceus. Our results demonstrate the simplicity and convenience of this method for the detection of viral infection in aquatic organisms.

  7. Meningococcal carriage rates in healthy individuals in Japan determined using Loop-Mediated Isothermal Amplification and oral throat wash specimens.

    Science.gov (United States)

    Takahashi, Hideyuki; Haga, Masae; Sunagawa, Tomimasa; Saitoh, Takehito; Kitahara, Takeru; Matsumoto, Sohkichi; Ohnishi, Makoto

    2016-07-01

    The detailed epidemiology of meningococcal diseases in Japan has yet to be determined and, moreover, the healthy carriage rate is also unknown. In this study, to obtain insight into the carriage rate of Neisseria meningitidis in healthy individuals in Japan, we developed a new method to detect the N. meningitidis-specific ctrB gene, one of the genes encoding enzymes for capsule synthesis, by Loop-Mediated Isothermal Amplification (LAMP) and examined the meningococcal carriage rate by using self-collected oral throat wash specimens from 836 students at a university. Examination by LAMP showed that 7 out of 836 samples were positive for N. meningitidis DNA, and the results were also verified by the nested PCR method for the meningococcus specific ggt gene. The N. meningitidis carriage rate in healthy individuals was estimated to be 0.84%. Moreover, we further confirmed by the nested-PCR-based serogroup typing method that 5 of the positive samples belonged to serogroup Y, 1 belonged to group B and 1 was unidentifiable. Considering the epidemiology for meningococcal diseases in Japan, the carriage rate and the serogroup profile seem to be consistent with low incidence of meningococcal diseases and serogroup distribution of clinical meningococcal isolates in Japan, respectively. PMID:26895673

  8. Detection and identification of Trichophyton tonsurans from clinical isolates and hairbrush samples by loop-mediated isothermal amplification system.

    Science.gov (United States)

    Yo, Ayaka; Yamamoto, Mikachi; Nakayama, Takako; Ishikawa, Jun; Makimura, Koichi

    2016-09-01

    Since the 1990s, there have been reports of the spread of dermatophytosis caused by Trichophyton tonsurans among contact sports athletes in several countries, including Japan. This study was performed to develop a loop-mediated isothermal amplification (LAMP) system for rapid and accurate detection and identification of T. tonsurans from clinical isolates or hairbrush samples for diagnosis and to prevent the spread of infection. A specific primer set was prepared by comparing the whole genome sequence of T. tonsurans with those of six other closely related dermatophytes. After confirming the sensitivity and specificity of this system, LAMP assay was performed using 37 clinical samples obtained from three healthy volunteers and 24 judo athletes. A total of 155 fungal isolates (56 strains of various standard fungi, 96 identified T. tonsurans isolates, three hairbrush-cultured isolates from judo athletes) and 37 hairbrush samples (34 samples from 24 judo athletes, and three samples from three healthy volunteers) were used for culture and LAMP assay, respectively. The assay showed no cross-reactivity to standard strains other than T. tonsurans. The detection limit was 100 copies of DNA template per tube. All of the 96 T. tonsurans isolates were amplified, and all samples from healthy volunteers showed negative results. Four of the 34 hairbrush samples obtained from judo athletes showed positive results in LAMP assay, and two of the four were positive in both culture and LAMP assay. We developed a rapid LAMP system with high specificity and sensitivity for diagnosis of T. tonsurans infection. PMID:26892741

  9. Loop-mediated isothermal amplification (LAMP) assays for detection and identification of aquaculture pathogens: current state and perspectives.

    Science.gov (United States)

    Biswas, Gouranga; Sakai, Masahiro

    2014-04-01

    Since its invention in 2000, loop-mediated isothermal amplification (LAMP) assay has been one of the most extensively used molecular diagnostic tools in bio-medical fields due to the rapidity, accuracy, and cost-effectiveness of the technique. This technique has also earned popularity in aquaculture disease diagnosis. Aquaculture, as a result of its rapid intensification and expansion, experiences increased infectious disease occurrences. For maintenance of economic viability, rapid, sensitive and efficient diagnosis of disease causing agents is an important step prior to undertaking effective prevention and control measures in aquaculture. Constraints on time and expertise required for conventional biochemical, serological and polymerase chain reaction (PCR)-based techniques offer avenues in adoption of the LAMP by the aquaculturists at field conditions. This assay has been successfully applied in detection of several bacterial, viral and parasitic pathogens causing serious diseases in aquaculture. In this review, we endeavored to accommodate the LAMP methodology with its different recent improvements and an overview of its application for the detection of aquaculture-associated pathogens.

  10. Rapid and Sensitive Detection of RNA Viruses Based on Reverse Transcription Loop-Mediated Isothermal Amplification, Magnetic Nanoparticles, and Chemiluminescence.

    Science.gov (United States)

    Wang, Jiuhai; Lu, Peng; Yan, Jieni; Zhang, Yufan; Huang, Lanye; Ali, Zeeshan; Li, Zhiyang; He, Nongyue

    2016-04-01

    RNA viruses, particularly, the highly pathogenic avian influenza (HPAI) virus, pose serious health concerns, and cause huge economic losses worldwide. Diagnostic tools for the early detection of these deadly RNA viruses are urgently needed to implement treatment and disease control strategies. Conventional reverse transcription polymerase chain reaction (RT-PCR)-based chemiluminescent (RT-PCR-CL) detection is frequently used for the diagnosis of viral infections. However, the requirements for expensive PCR machines and longer thermocycling times are significant drawbacks. In this study, we propose a method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with chemiluminescence (CL) to detect H7N9 virus. The proposed method does not require any expensive instruments, and processing time is remarkably shortened compared to that of RT-PCR-CL. Since several factors including RT-LAMP temperature, probe concentration, hybridization temperature, and hybridization duration might affect the CL signal, each of these parameters was investigated and optimized. One thousand copies/mL of H7N9 RNA were detectable using the optimized RT-LAMP-CL method. The detection time was significantly reduced by using RT-LAMP, in comparison with conventional RT-PCR-CL. This technique holds great promise for viral detection and diagnosis, especially with regard to avian influenza virus. PMID:27301197

  11. Detection of Salmonella and several common Salmonella serotypes in food by loop-mediated isothermal amplification method

    Directory of Open Access Journals (Sweden)

    Zhongqiang Chen

    2015-06-01

    Full Text Available Salmonella Choleraesuis, S. Enteritidis and S. Typhimurium are the main pathogens that contaminate animal products and cause human Salmonella food poisoning. To establish the loop-mediated isothermal amplification (LAMP method for the rapid detection of Salmonella and 3 common Salmonella serotypes, inner and outer primer sets targeting Salmonella invE gene and 3 serotype-specific genes fliC, lygD and STM4495 were designed. The LAMP reaction conditions were optimized. The specificity of LAMP primers was identified by testing 10 different bacterial strains including S. Choleraesuis, S. Enteritidis and S. Typhimurium. Take S. Choleraesuis as example, the detection limit of LAMP assay was 1.33 × 101 CFU/mL for bacteria culture and 2.0 × 101 CFU/mL for simulated pork sample. The results show that LAMP is a rapid, sensitive and specific method for Salmonella detection and can be used for the rapid detection of Salmonella in food.

  12. Detection and identification of Trichophyton tonsurans from clinical isolates and hairbrush samples by loop-mediated isothermal amplification system.

    Science.gov (United States)

    Yo, Ayaka; Yamamoto, Mikachi; Nakayama, Takako; Ishikawa, Jun; Makimura, Koichi

    2016-09-01

    Since the 1990s, there have been reports of the spread of dermatophytosis caused by Trichophyton tonsurans among contact sports athletes in several countries, including Japan. This study was performed to develop a loop-mediated isothermal amplification (LAMP) system for rapid and accurate detection and identification of T. tonsurans from clinical isolates or hairbrush samples for diagnosis and to prevent the spread of infection. A specific primer set was prepared by comparing the whole genome sequence of T. tonsurans with those of six other closely related dermatophytes. After confirming the sensitivity and specificity of this system, LAMP assay was performed using 37 clinical samples obtained from three healthy volunteers and 24 judo athletes. A total of 155 fungal isolates (56 strains of various standard fungi, 96 identified T. tonsurans isolates, three hairbrush-cultured isolates from judo athletes) and 37 hairbrush samples (34 samples from 24 judo athletes, and three samples from three healthy volunteers) were used for culture and LAMP assay, respectively. The assay showed no cross-reactivity to standard strains other than T. tonsurans. The detection limit was 100 copies of DNA template per tube. All of the 96 T. tonsurans isolates were amplified, and all samples from healthy volunteers showed negative results. Four of the 34 hairbrush samples obtained from judo athletes showed positive results in LAMP assay, and two of the four were positive in both culture and LAMP assay. We developed a rapid LAMP system with high specificity and sensitivity for diagnosis of T. tonsurans infection.

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

    Science.gov (United States)

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

    2015-01-01

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

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Mevaree Srisawat

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2015-01-01

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

  18. Application of novel loop-mediated isothermal amplification (LAMP) for rapid authentication of the herbal tea ingredient Hedyotis diffusa Willd.

    Science.gov (United States)

    Li, Ming; Wong, Yuk-Lau; Jiang, Li-Li; Wong, Ka-Lok; Wong, Yuen-Ting; Lau, Clara Bik-San; Shaw, Pang-Chui

    2013-12-01

    Hedyotis diffusa Willd. (Baihuasheshecao) is an ingredient of herbal teas commonly consumed in the Orient and tropical Asia for cancer treatment and health maintenance. In the market, this ingredient is frequently adulterated by the related species Hedyotis corymbosa (L.) Lam. The objective of this study is to develop a novel loop-mediated isothermal amplification (LAMP) technique to differentiate H. diffusa from its adulterant H. corymbosa. A set of four internal control primers (F3, FIP, BIP and B3) were designed based on six loci in the internal transcribed spacer (ITS) for LAMP of both H. diffusa and H. corymbosa. Two specific primers (S_F3 and S_FIP) were designed for specific LAMP detection of H. diffusa only. Our data showed that LAMP was successful for both H. diffusa and H. corymbosa in internal control. In contrast, only H. diffusa was detected in specific LAMP using the specific primers S_F3 and S_FIP. This study showed that LAMP was useful to differentiate H. diffusa from its adulterant H. corymbosa. This study is significant for the verification of the authenticity for better quality control of this common herbal tea ingredient. The strategy of including an internal control assures the quality of the concerned DNA region for LAMP. PMID:23870990

  19. Loop-mediated isothermal amplification of specific endoglucanase gene sequence for detection of the bacterial wilt pathogen Ralstonia solanacearum.

    Directory of Open Access Journals (Sweden)

    Rok Lenarčič

    Full Text Available The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes.

  20. Development of loop-mediated isothermal amplification (LAMP assay for rapid and sensitive identification of ostrich meat.

    Directory of Open Access Journals (Sweden)

    Amir Abdulmawjood

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

  1. Real-time fluorescence Loop-Mediated Isothermal Amplification (LAMP) for rapid and reliable diagnosis of pulmonary tuberculosis.

    Science.gov (United States)

    Cao, Donglin; Hu, Liangshan; Lin, Maorui; Li, Mingyou; Ye, Zebing; Sun, Hongtao; Huang, Jiwei; Yang, Huawen; Tian, Junzhang

    2015-02-01

    A reliable, simple and rapid diagnostic method that can be helpful in pulmonary tuberculosis diagnosis is urgently needed. Loop-mediated Isothermal Amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. In this study, real-time fluorescence LAMP was evaluated to rapidly detect Mycobacterium tuberculosis in sputum and was compared to the performance of real-time fluorescence quantitative PCR (Q-PCR). All the standard MTB strains were successfully detected and limit of detection (LOD) was 10(2)CFU/mL by real-time fluorescence LAMP within 20min. In light of MTB in sputum, the real-time fluorescence LAMP method yielded a sensitivity of 98.0% and a specificity of 78.3%, compared to Q-PCR assay, which yielded a sensitivity of 96.0% and a specificity of 82.6% for PTB diagnosis. There was an excellent overall agreement between LAMP and Q-PCR for PTB (κ=0.315) and non-PTB (κ=0.862). Therefore, the real-time fluorescence LAMP assay is a rapid, sensitive, and specific method to detect pulmonary tuberculosis.

  2. An Ultrasensitive Chemiluminescence Biosensor for Carcinoembryonic Antigen Based on Autocatalytic Enlargement of Immunogold Nanoprobes

    OpenAIRE

    2012-01-01

    A sensitive flow injection chemiluminescence assay for carcinoembryonic antigen (CEA) detection based on signal amplification with gold nanoparticles (NPs) is reported in the present work. The sandwich system of CEA/anti-CEA/goat-anti-mouse IgG functionalized Au nanoparticles was used as the sensing platform. In order to improve detection sensitivity, a further gold enlargement step was developed based on the autocatalytic Au deposition of gold nanoprobes via the reduction of AuCl4 − to Au0 o...

  3. Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence

    OpenAIRE

    Mingming Zhao; Yuhua Shi; Lan Wu; Licheng Guo; Wei Liu; Chao Xiong; Song Yan; Wei Sun; Shilin Chen

    2016-01-01

    Saffron is one of the most expensive species of Chinese herbs and has been subjected to various types of adulteration because of its high price and limited production. The present study introduces a loop-mediated isothermal amplification (LAMP) technique for the differentiation of saffron from its adulterants. This novel technique is sensitive, efficient and simple. Six specific LAMP primers were designed on the basis of the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nucl...

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

    OpenAIRE

    Deguo Wang; Yanhong Liu

    2015-01-01

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactia...

  5. An inexpensive and rapid diagnostic method of Koi Herpesvirus (KHV infection by loop-mediated isothermal amplification

    Directory of Open Access Journals (Sweden)

    El-Matbouli Mansour

    2005-10-01

    Full Text Available Abstract Background Koi Herpesvirus (KHV affects both juvenile and adult common carp and koi, and is especially lethal to fry. The high mortalities caused by the disease have had a negative impact on the international koi trade. Different diagnostic techniques have been used to detect KHV, including: isolation of the virus in cell culture, electron microscopy, several PCR tests, ELISA and in situ hybridisation. All of these methods are time consuming, laborious and require specialised equipment. Results A rapid field diagnosis of KHV in common and koi carp was developed using loop-mediated isothermal amplification (LAMP. The LAMP reaction rapidly amplified nucleic acid with high specificity and efficiency under isothermal conditions using a simple water bath. Two methods of extracting DNA from host tissue were compared: extraction by boiling and by using a commercial extraction kit. A set of six primers – two inner primers, two outer primers and two loop primers – was designed from a KHV amplicon. The reaction conditions were optimised for detection of KHV in 60 min at 65°C using Bst (Bacillus stearothermophilus DNA polymerase. When visualised by gel electrophoresis, the products of the KHV LAMP assay appeared as a ladder pattern, with many bands of different sizes from 50 base-pairs (bp up to the loading well. The KHV LAMP product could also be simply detected visually by adding SYBR Green I to the reaction tube and observing a colour change from orange to green. All samples positive for KHV by visual detection were confirmed positive by gel electrophoresis. The KHV LAMP had the same sensitivity as a standard PCR assay for the detection of KHV. Conclusion This paper describes an accelerated LAMP assay for diagnosis of KHV. The entire procedure took only 90 minutes to produce a result: 15 minutes for DNA extraction; 60 min for the LAMP reaction; 2 min for visual detection using SYBR Green I. The test can be used under field conditions

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2015-09-01

    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.

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

    Science.gov (United States)

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

    2013-09-01

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

  9. A Pilot Study of Quantitative Loop-mediated Isothermal Amplification-guided Target Therapies for Hospital-acquired Pneumonia

    Institute of Scientific and Technical Information of China (English)

    Fang Wang; Ran Li; Ying Shang; Can Wang; Guo-Qing Wang; De-Xun Zhou; Dong-Hong Yang

    2016-01-01

    Background:It is important to achieve the definitive pathogen identification in hospital-acquired pneumonia (HAP),but the traditional culture results always delay the target antibiotic therapy.We assessed the method called quantitative loop-mediated isothermal amplification (qLAMP) as a new implement for steering of the antibiotic decision-making in HAP.Methods:Totally,76 respiratory tract aspiration samples were prospectively collected from 60 HAP patients.DNA was isolated from these samples.Specific DNA fragments for identifying 11 pneumonia-related bacteria were amplified by qLAMP assay.Culture results of these patients were compared with the qLAMP results.Clinical data and treatment strategies were analyzed to evaluate the effects of qLAMP results on clinical data.McNemar test and Fisher's exact test were used for statistical analysis.Results:The detection of Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa,Klebsiella pneumonia,Stenotrophomonas maltophilia,Streptococcus pneumonia,and Acinetobacter baumannii by qLAMP was consistent with sputum culture (P > 0.05).The qLAMP results of 4 samples for Haemophilus influenzae,Legionella pneumophila,or Mycoplasma pneumonia (MP) were inconsistent with culture results;however,clinical data revealed that the qLAMP results were all reliable except 1 MP positive sample due to the lack of specific species identified in the final diagnosis.The improvement of clinical condition was more significant (P < 0.00l) in patients with pathogen target-driven therapy based on qLAMP results than those with empirical therapy.Conclusion:qLAMP is a more promising method for detection of pathogens in an early,rapid,sensitive,and specific manner than culture.

  10. Performance of reversed transcription loop-mediated isothermal amplification technique detecting EV71: a systematic review with meta-analysis.

    Science.gov (United States)

    Lei, Xiaoying; Wen, Hongling; Zhao, Li; Yu, Xuejie

    2014-04-01

    Human enterovirus 71 (EV71) is the major etiological agent of hand, foot and mouth disease (HFMD), which is a common infectious disease in young children. Studies in the past have shown that reversed transcription loop-mediated isothermal amplification (RT-LAMP) was a rapid approach for the detection of EV71 in HFMD. This meta-analysis study is to evaluate the diagnostic role of RT-LAMP in detecting EV71 infection. A comprehensive literature research of PubMed, Embase, Wan Fang Data, and Chinese National Knowledge Infrastructure databases was conducted on articles aiming at the diagnostic performance of RT-LAMP in EV71 detection published before February 10, 2014. Data from selected studies were pooled to yield the summary sensitivity, specificity, positive and negative likelihood ratio (PLR, NLR), diagnostic odds ratio (DOR), and receiver operating characteristic (SROC) curve by using STATA VERSION 12.0 software. Ten studies including a total of 907 clinical samples were of high quality in this meta-analysis. Overall, the pooled sensitivity, specificity, PLR, NLR, DOR, and the area under the SROC curve was 0.99 (0.97, 1.00), 0.97 (0.94, 1.00), 5.90 (95% CI: 3.90-8.94), 0.20 (95% CI: 0.14-0.29), and 1.00 (95% CI: 0.99-1.00), respectively. The univariate analysis of potential variables showed some changes in the diagnostic performance, but none of the differences reached statistical significance. Despite inter-study variability, the test performance of RT-LAMP was consistent with real-time RT-PCR in detecting EV71. This meta-analysis suggests that RT-LAMP is a useful diagnostic tool with high sensitivity and specificity for detecting EV71. PMID:24815384

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

    Directory of Open Access Journals (Sweden)

    Catherine B Poole

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

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

    Science.gov (United States)

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

    2015-01-01

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

  13. Visual Detection of Brucella spp. in Spiked Bovine Semen Using Loop-Mediated Isothermal Amplification (LAMP) Assay.

    Science.gov (United States)

    Prusty, Bikash R; Chaudhuri, Pallab; Chaturvedi, V K; Saini, Mohini; Mishra, B P; Gupta, Praveen K

    2016-06-01

    Several pathogens including Brucella spp. are shed in semen of infected bulls and can be transmitted to cows through contaminated semen during artificial insemination. The present study reports omp2a and bcsp31 gene based loop-mediated isothermal amplification (LAMP) assays for detection of Brucella genomic DNA in semen from infected bulls. The positive results could be interpreted visually by change in colour of reaction mixture containing hydroxyl naphthol blue (HNB) dye from violet to sky blue. LAMP assays based on omp2a and bcsp31 could detect as little as 10 and 100 fg of B. abortus S19 genomic DNA, respectively. Sensitivity of omp2a and bcsp31 LAMP assays for direct detection of organisms in bovine semen was 2.28 × 10(1) CFU and 2.28 × 10(2) CFU of B. abortus S19 in spiked bovine semen, respectively. The omp2a LAMP assay was found equally sensitive to TaqMan probe based real-time PCR and 100 times more sensitive than conventional PCR in identifying Brucella in spiked semen. The diagnostic applicability of the omp2a LAMP assay was evaluated with seventy-nine bovine semen samples and results were re-evaluated through TaqMan probe based real-time PCR and conventional PCR. Taken together, the omp2a LAMP assay is easy to perform, rapid and sensitive in diagnosis of Brucella spp. in bovine semen. PMID:27570305

  14. Performance of reversed transcription loop-mediated isothermal amplification technique detecting EV71: a systematic review with meta-analysis.

    Science.gov (United States)

    Lei, Xiaoying; Wen, Hongling; Zhao, Li; Yu, Xuejie

    2014-04-01

    Human enterovirus 71 (EV71) is the major etiological agent of hand, foot and mouth disease (HFMD), which is a common infectious disease in young children. Studies in the past have shown that reversed transcription loop-mediated isothermal amplification (RT-LAMP) was a rapid approach for the detection of EV71 in HFMD. This meta-analysis study is to evaluate the diagnostic role of RT-LAMP in detecting EV71 infection. A comprehensive literature research of PubMed, Embase, Wan Fang Data, and Chinese National Knowledge Infrastructure databases was conducted on articles aiming at the diagnostic performance of RT-LAMP in EV71 detection published before February 10, 2014. Data from selected studies were pooled to yield the summary sensitivity, specificity, positive and negative likelihood ratio (PLR, NLR), diagnostic odds ratio (DOR), and receiver operating characteristic (SROC) curve by using STATA VERSION 12.0 software. Ten studies including a total of 907 clinical samples were of high quality in this meta-analysis. Overall, the pooled sensitivity, specificity, PLR, NLR, DOR, and the area under the SROC curve was 0.99 (0.97, 1.00), 0.97 (0.94, 1.00), 5.90 (95% CI: 3.90-8.94), 0.20 (95% CI: 0.14-0.29), and 1.00 (95% CI: 0.99-1.00), respectively. The univariate analysis of potential variables showed some changes in the diagnostic performance, but none of the differences reached statistical significance. Despite inter-study variability, the test performance of RT-LAMP was consistent with real-time RT-PCR in detecting EV71. This meta-analysis suggests that RT-LAMP is a useful diagnostic tool with high sensitivity and specificity for detecting EV71.

  15. Detection of Mycobacterium tuberculosis by Using Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick in Clinical Samples

    Directory of Open Access Journals (Sweden)

    Thongchai Kaewphinit

    2013-01-01

    Full Text Available Tuberculosis (TB is a communicable disease caused by the bacterium Mycobacterium tuberculosis (MTB and is a persistent problem in the developing countries. Loop-mediated isothermal amplification (LAMP allows DNA to be amplified rapidly at a constant temperature. Here, a LAMP method was combined with a chromatographic lateral-flow dipstick (LFD to detect IS6110 gene of M. tuberculosis specifically and rapidly. The reaction was optimized at 63°C for 60 min, and the amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5 min was detected at the LFD test line 5 min after application. Excluding the step of DNA extraction, the test results could be generated approximately within 1 h. In addition to the advantage of short assay time, this technique could avoid the contact of carcinogenic ethidium bromide due to the exclusion of the electrophoresis analysis step. Furthermore, the data indicated that LAMP-LFD could detect M. tuberculosis genomic DNA as little as 5 pg. The technique showed a significant specificity since no cross-hybridization to M. intracellulare (MIC, M. fortuitum (MFT, M. avium (MAV, M. kansasii (MKS, and M. gordonae (MGD genomic DNAs was observed. In the clinical unknown samples test, the sensitivity of LAMP-LFD was 98.92 % and the specificity was 100 % compared to those of the standard culture assay. Based on its sensitivity, specificity, rapidity, low cost, and convenience, LAMP-LFD could be applicable for use in both laboratories and epidemiological surveys of MTB.

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

    Science.gov (United States)

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

    2015-01-01

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

  17. [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.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Principle and Application of Loop-mediated Isothermal Amplification%环介导等温扩增反应的原理及应用

    Institute of Scientific and Technical Information of China (English)

    黄思佳; 解庭波; 严家新

    2011-01-01

    环介导等温扩增反应(Loop-mediated isothermal amplification,LAMP)是日本学者Notomi于2000年在NucleicAcids Res杂志上公开的一种新的基因诊断技术,该法操作简便,扩增效率及灵敏度高,已广泛应用于食品、临床、农业等领域,有望成为主流的基因诊断方法.本文对LAMP的反应特征及原理、优缺点、临床应用作一综述.%Loop-mediated isothermal amplification (LAMP) is a novel technique for genetic diagnosis, which was published on Nucleic Acids Res in 2000 by Notomi, a Japanese scientist. It is a simple, efficient and sensitive DNA amplification method which has been widely used in various fields including food, clinic and agriculture, and might be a mainstream method for genetic diagnosis. This paper reviews the reaction characters, principle, advantage, disadvantage as well as clinical application of LAMP.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    P K Balne

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

  4. 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...... and arctic-like strains of classical rabies virus due to the primer design and is therefore expected to be universally applicable independent of region of the world where the virus is isolated. Of the samples tested all were found to be positive after incubation for 25 to 30 minutes. The method made...

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

    Science.gov (United States)

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

    2015-01-01

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

  6. An autocatalytic network model for stock markets

    Science.gov (United States)

    Caetano, Marco Antonio Leonel; Yoneyama, Takashi

    2015-02-01

    The stock prices of companies with businesses that are closely related within a specific sector of economy might exhibit movement patterns and correlations in their dynamics. The idea in this work is to use the concept of autocatalytic network to model such correlations and patterns in the trends exhibited by the expected returns. The trends are expressed in terms of positive or negative returns within each fixed time interval. The time series derived from these trends is then used to represent the movement patterns by a probabilistic boolean network with transitions modeled as an autocatalytic network. The proposed method might be of value in short term forecasting and identification of dependencies. The method is illustrated with a case study based on four stocks of companies in the field of natural resource and technology.

  7. 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.

  8. 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.

  9. Loop-mediated isothermal amplification (RT-LAMP): a new approach for the detection of foot-and-mouth disease virus and its sero-types in Pakistan.

    Science.gov (United States)

    Farooq, U; Latif, A; Irshad, H; Ullah, A; Zahur, A B; Naeem, K; Khan, S U H; Ahmed, Z; Rodriguez, L L; Smoliga, G

    2015-01-01

    Successful disease management requires a rapid and sensitive diagnosis method that can recognize early infection even before the manifestation of its clinical signs. The only available field diagnostic tests for foot-and-mouth disease (FMD) are lateral flow devices, commonly known as chromatographic strips. Low sensitivity and inability to detect FMD virus (FMDV) at the serotype level are limitations of lateral flow devices. Therefore, a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) was standardized using universal and sero-type specific genes in a single tube. This test does not require sophisticated equipment and can detect FMDV at serotype level in about 60 min. In addition, the sensitivity and specificity of this test is comparable to conventional reverse transcriptase PCR and real time PCR (rRT-PCR). PMID:27175198

  10. 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.

  11. Giardia and Cryptosporidium spp. dissemination during wastewater treatment and comparative detection via immunofluorescence assay (IFA), nested polymerase chain reaction (nested PCR) and loop mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Gallas-Lindemann, Carmen; Sotiriadou, Isaia; Plutzer, Judit; Noack, Michael J; Mahmoudi, Mohammad Reza; Karanis, Panagiotis

    2016-06-01

    Environmental water samples from the Lower Rhine area in Germany were investigated via immunofluorescence assays (IFAs), nested polymerase chain reaction (nested PCR) and loop-mediated isothermal amplification (LAMP) to detect the presence of Giardia spp. (n=185) and Cryptosporidium spp. (n=227). The samples were concentrated through filtration or flocculation, and oocysts were purified via centrifugation through a sucrose density gradient. For all samples, IFA was performed first, followed by DNA extraction for the nested PCR and LAMP assays. Giardia cysts were detected in 105 samples (56.8%) by IFA, 62 samples (33.5%) by nested PCR and 79 samples (42.7%) by LAMP. Cryptosporidium spp. were detected in 69 samples (30.4%) by IFA, 95 samples (41.9%) by nested PCR and 99 samples (43.6%) by LAMP. According to these results, the three detection methods are complementary for monitoring Giardia and Cryptosporidium in environmental waters.

  12. 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.

  13. 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.

  14. 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.

  15. Loop-mediated isothermal amplification as an emerging technology for detection of Yersinia ruckeri the causative agent of enteric red mouth disease in fish

    Directory of Open Access Journals (Sweden)

    Soliman Hatem

    2008-08-01

    Full Text Available Abstract Background Enteric Redmouth (ERM disease also known as Yersiniosis is a contagious disease affecting salmonids, mainly rainbow trout. The causative agent is the gram-negative bacterium Yersinia ruckeri. The disease can be diagnosed by isolation and identification of the causative agent, or detection of the Pathogen using fluorescent antibody tests, ELISA and PCR assays. These diagnostic methods are laborious, time consuming and need well trained personnel. Results A loop-mediated isothermal amplification (LAMP assay was developed and evaluated for detection of Y. ruckeri the etiological agent of enteric red mouth (ERM disease in salmonids. The assay was optimised to amplify the yruI/yruR gene, which encodes Y. ruckeri quorum sensing system, in the presence of a specific primer set and Bst DNA polymerase at an isothermal temperature of 63°C for one hour. Amplification products were detected by visual inspection, agarose gel electrophoresis and by real-time monitoring of turbidity resulted by formation of LAMP amplicons. Digestion with HphI restriction enzyme demonstrated that the amplified product was unique. The specificity of the assay was verified by the absence of amplification products when tested against related bacteria. The assay had 10-fold higher sensitivity compared with conventional PCR and successfully detected Y. ruckeri not only in pure bacterial culture but also in tissue homogenates of infected fish. Conclusion The ERM-LAMP assay represents a practical alternative to the microbiological approach for rapid, sensitive and specific detection of Y. ruckeri in fish farms. The assay is carried out in one hour and needs only a heating block or water bath as laboratory furniture. The advantages of the ERM-LAMP assay make it a promising tool for molecular detection of enteric red mouth disease in fish farms.

  16. Feasibility and Operational Performance of Tuberculosis Detection by Loop-Mediated Isothermal Amplification Platform in Decentralized Settings: Results from a Multicenter Study.

    Science.gov (United States)

    Gray, Christen M; Katamba, Achilles; Narang, Pratibha; Giraldo, Jorge; Zamudio, Carlos; Joloba, Moses; Narang, Rahul; Paramasivan, C N; Hillemann, Doris; Nabeta, Pamela; Amisano, Danielle; Alland, David; Cobelens, Frank; Boehme, Catharina C

    2016-08-01

    Currently available nucleic acid amplification platforms for tuberculosis (TB) detection are not designed to be simple or inexpensive enough to implement in decentralized settings in countries with a high burden of disease. The loop-mediated isothermal amplification platform (LAMP) may change this. We conducted a study in adults with symptoms suggestive of TB in India, Uganda, and Peru to establish the feasibility of using TB-LAMP (Eiken Chemical Co.) in microscopy laboratories compared with using smear microscopy against a reference standard of solid and liquid cultures. Operational characteristics were evaluated as well. A total of 1,777 participants met the eligibility criteria and were included for analysis. Overall, TB-LAMP sensitivities among culture-positive samples were 97.2% (243/250; 95% confidence interval [CI], 94.3% to 98.2%) and 62.0% (88/142; 95% CI, 53.5% to 70.0%) for smear-positive and smear-negative TB, respectively, but varied widely by country and operator. Specificities ranged from 94.5% (446/472; 95% CI, 92.0% to 96.4%) to 98.0% (350/357; 95% CI, 96.0% to 99.2%) by country. A root cause analysis identified high temperatures, high humidity, and/or low reaction volumes as possible causes for false-positive results, as they may result in nonspecific amplification. The study was repeated in India with training focused on vulnerable steps and an updated protocol; 580 participants were included for analysis. Specificity in the repeat trial was 96.6% (515/533; 95% CI, 94.7% to 97.9%). To achieve acceptable performance of LAMP at the microscopy center level, significant training and infrastructure requirements are necessary. PMID:27194691

  17. Comparative study of sensitivity, linearity, and resistance to inhibition of digital and nondigital polymerase chain reaction and loop mediated isothermal amplification assays for quantification of human cytomegalovirus.

    Science.gov (United States)

    Nixon, Gavin; Garson, Jeremy A; Grant, Paul; Nastouli, Eleni; Foy, Carole A; Huggett, Jim F

    2014-05-01

    Performing nucleic acid amplification techniques (NAATs) in digital format using limiting dilution provides potential advantages that have recently been demonstrated with digital polymerase chain reaction (dPCR). Key benefits that have been claimed are the ability to quantify nucleic acids without the need of an external calibrator and a greater resistance to inhibitors than real-time quantitative PCR (qPCR). In this study, we evaluated the performance of four NAATs, qPCR, dPCR, real-time quantitative loop mediated isothermal amplification (qLAMP), and digital LAMP (dLAMP), for the detection and quantification of human cytomegalovirus (hCMV). We used various DNA templates and inhibitors to compare the performance of these methods using a conventional real-time thermocycler platform (Bio-Rad CFX96) and a chip based digital platform (Fluidigm Biomark 12.765 Digital Array). dPCR performed well and demonstrated greater resistance to inhibitors than the other methods although this resistance did not apply equally to all inhibitors tested. dLAMP was found to be less sensitive than dPCR, but its quantitative performance was better than qLAMP, the latter being unable to quantify below 1000 copies. dLAMP was also more resistant to inhibitors than qLAMP. Unlike qPCR, both digital methods were able to quantify viral genomes without requiring a calibrator; however, neither can currently compete with the large reaction volumes, and thus the greater absolute sensitivity, of qPCR. With the introduction of digital instrumentation that will enable larger reaction volumes, digital amplification methods such as those evaluated in this study could potentially offer a robust alternative to qPCR for nucleic acid quantification. PMID:24684191

  18. 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. PMID:27044566

  19. Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence.

    Science.gov (United States)

    Zhao, Mingming; Shi, Yuhua; Wu, Lan; Guo, Licheng; Liu, Wei; Xiong, Chao; Yan, Song; Sun, Wei; Chen, Shilin

    2016-01-01

    Saffron is one of the most expensive species of Chinese herbs and has been subjected to various types of adulteration because of its high price and limited production. The present study introduces a loop-mediated isothermal amplification (LAMP) technique for the differentiation of saffron from its adulterants. This novel technique is sensitive, efficient and simple. Six specific LAMP primers were designed on the basis of the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA of Crocus sativus. All LAMP amplifications were performed successfully, and visual detection occurred within 60 min at isothermal conditions of 65 °C. The results indicated that the LAMP primers are accurate and highly specific for the discrimination of saffron from its adulterants. In particular, 10 fg of genomic DNA was determined to be the limit for template accuracy of LAMP in saffron. Thus, the proposed novel, simple, and sensitive LAMP assay is well suited for immediate on-site discrimination of herbal materials. Based on the study, a practical standard operating procedure (SOP) for utilizing the LAMP protocol for herbal authentication is provided. PMID:27146605

  20. 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.

  1. 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.

  2. 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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence

    Science.gov (United States)

    Zhao, Mingming; Shi, Yuhua; Wu, Lan; Guo, Licheng; Liu, Wei; Xiong, Chao; Yan, Song; Sun, Wei; Chen, Shilin

    2016-01-01

    Saffron is one of the most expensive species of Chinese herbs and has been subjected to various types of adulteration because of its high price and limited production. The present study introduces a loop-mediated isothermal amplification (LAMP) technique for the differentiation of saffron from its adulterants. This novel technique is sensitive, efficient and simple. Six specific LAMP primers were designed on the basis of the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA of Crocus sativus. All LAMP amplifications were performed successfully, and visual detection occurred within 60 min at isothermal conditions of 65 °C. The results indicated that the LAMP primers are accurate and highly specific for the discrimination of saffron from its adulterants. In particular, 10 fg of genomic DNA was determined to be the limit for template accuracy of LAMP in saffron. Thus, the proposed novel, simple, and sensitive LAMP assay is well suited for immediate on-site discrimination of herbal materials. Based on the study, a practical standard operating procedure (SOP) for utilizing the LAMP protocol for herbal authentication is provided. PMID:27146605

  5. 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

    2013-01-01

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

  6. 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.

  7. The Heads and Tails of Buoyant Autocatalytic Balls

    CERN Document Server

    Rogers, Michael C

    2012-01-01

    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 a large diameter, extended cylinder that is relatively free of boundary constraints, localized initiation of an iodate-arsenous acid (IAA) reaction front on the bottom boundary generates a rising autocatalytic plume. Such plumes have several differences from their non-reactive counterparts. Using numerical simulation, we have found that if reaction is initiated using a spherical ball of product solution well above the bottom boundary, the subsequent 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, except that the tail is disconnected from the boundary. In the limit of large initial autocatalytic balls, however, growth of a reacting tail is suppressed and the resemblance to plumes disappears. Conversely, very small bal...

  8. 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.

  9. 副溶血弧菌tdh基因LAMP检测技术的建立%Establishment of Loop-Mediated Isothermal Amplification for Detecting Vibrio Parahaemolyticus tdh Gene

    Institute of Scientific and Technical Information of China (English)

    程晓艳; 刘庆慧; 黄倢

    2012-01-01

    Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification technology which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. In this paper, LAMP was applied for rapid detection of Vibrio parahaemolyticus. According to the sequence of the specific tdh gene of Vibrio parahaemolyticus, a set of primers was designed to amplify the specifial DNA sequences by LAMP. Moreover, the reaction conditions were optimized. The results showed the optimum LAMP assay for the rapid detection of Vibrio paiakaemolyticus was performed at 60.8 ℃ for 45 min. No crossreaction was found with other common bacteria which showed a good specificity. The LAMP assay had an detection limit of 35.5 fg/tube, which was 10 times lower than that of PCR assay. The LAMP assay could be finished within an hour requiring only a regular laboratory water bath for reaction. The detection of the strains isolated from scallop also proved the LAMP assay reliability.%环等温扩增技术(Loop-mediated isothermal amplification,LAMP)是一种在等温条件下高特异性、高效、快速地扩增靶基因的DNA扩增技术.根据副溶血弧菌特异性的毒力基因tdh保守序列,本文设计1套特异性引物对该基因进行环等温扩增,同时对反应条件进行优化,建立携带tdh基因的致病性副溶血弧菌的LAMP快速检测技术.结果表明,LAMP最适反应在60.8℃恒温、45 min内完成,与其它常见的细菌无交叉反应.LAMP方法的细菌DNA最低检出限为35.5 fg/反应管,灵敏度较PCR方法高10倍.对扇贝样品中分离的菌株的检测表明,LAMP方法对检测致病性副溶血弧菌具有良好的可靠性.

  10. Rapid Identification of Officinal Akebiae Caulis and Its Toxic Adulterant Aristolochiae Manshuriensis Caulis (Aristolochia manshuriensis) by Loop-Mediated Isothermal Amplification

    Science.gov (United States)

    Wu, Lan; Wang, Bo; Zhao, Mingming; Liu, Wei; Zhang, Peng; Shi, Yuhua; Xiong, Chao; Wang, Ping; Sun, Wei; Chen, Shilin

    2016-01-01

    Mu-tong (Akebiae Caulis) is a traditional Chinese medicine commonly used as a diuretic and antiphlogistic. A common adulterant of Mu-tong is Guan-mu-tong (Aristolochiae Manshuriensis Caulis), which is derived from the stem of Aristolochia manshuriensis Komarov, and contains carcinogenic aristolochic acids. We used an alternative technique, loop-mediated isothermal amplification (LAMP), to differentiate Mu-tong from Guan-mu-tong because LAMP is quick, highly sensitive, and specific. We designed a set of four common primers (G-F3, G-B3, G-FIP, and G-BIP) and a loop primer (G-LB) for LAMP based on the internal transcribed spacer 2 sequence of Ar. manshuriensis. We successfully amplified the LAMP assays and visual detection occurred within 60 min at isothermal conditions of 65°C. The LAMP reaction exhibited a tenfold increase in detection (4.22 pg/μl DNA) over conventional polymerase chain reaction demonstrating that LAMP is a useful technique to detect Guan-mu-tong. We conclude that the LAMP technique is a potentially valuable safety control method for simple and efficient discrimination of Mu-tong from its adulterant Guan-mu-tong. PMID:27379153

  11. An improved method for detection of Edwardsiella tarda by loop-mediated isothermal amplification by targeting the EsrB gene

    Institute of Scientific and Technical Information of China (English)

    XIE Guosi; ZHANG Qingli; HAN Nana; SHI Chengyin; WANG Xiuhua; LIU Qinghui; HUANG Jie

    2012-01-01

    Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses.An improved method for quick and accurate detection of E.tarda by loop-mediated isothermal amplification (LAMP) with two additional loop primers was developed by targeting the EsrB gene (EsrBLAMP).In this method,the Mg2+ concentration,reaction temperature,and reaction time were optimized to 8 mmol/L,61℃,and 40 min,respectively.The detection limit with the EsrB gene was as low as 10 copies,which is 100 times more sensitive than that of conventional polymerase chain reaction (PCR).The EsrB-LAMP assay was shown more sensitive and rapid than previously reported LAMP assays targeting the hemolysin gene (hemolysin-LAMP) for detection ofE.tarda.The EsrB-LAMP was also highly specific to E.tarda and had no cross-reaction with 13 other strains of bacteria.The assay can be carried out in a simple heating device and the EsrB-LAMP products can be visually detected by adding fluorescent dye to the reaction mixture.Taken together,the improved EsrB-LAMP diagnostic protocol has the potential for detection ofE.tarda from indoor and outdoor samples.

  12. Discreteness-Induced Transitions in Autocatalytic Systems

    CERN Document Server

    Togashi, Y; Togashi, Yuichi; Kaneko, Kunihiko

    2005-01-01

    To study the dynamics of chemical processes, we often adopt rate equations to observe the change in chemical concentrations. However, when the number of the molecules is small, the fluctuations cannot be neglected. We often study the effects of fluctuations with the help of stochastic differential equations. Chemicals are composed of molecules on a microscopic level. In principle, the number of molecules must be an integer, which must only change discretely. However, in analysis using stochastic differential equations, the fluctuations are regarded as continuous changes. This approximation can only be valid if applied to fluctuations that involve a sufficiently large number of molecules. In the case of extremely rare chemical species, the actual discreteness of the molecules may critically affect the dynamics of the system. To elucidate the effects of the discreteness, we study an autocatalytic system consisting of several interacting chemical species with a small number of molecules through stochastic partic...

  13. 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.

  14. 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.

  15. General autocatalytic theory and simple model of financial markets

    Science.gov (United States)

    Thuy Anh, Chu; Lan, Nguyen Tri; Viet, Nguyen Ai

    2015-06-01

    The concept of autocatalytic theory has become a powerful tool in understanding evolutionary processes in complex systems. A generalization of autocatalytic theory was assumed by considering that the initial element now is being some distribution instead of a constant value as in traditional theory. This initial condition leads to that the final element might have some distribution too. A simple physics model for financial markets is proposed, using this general autocatalytic theory. Some general behaviours of evolution process and risk moment of a financial market also are investigated in framework of this simple model.

  16. A CCD-based fluorescence imaging system for real-time loop-mediated isothermal amplification-based rapid and sensitive detection of waterborne pathogens on microchips.

    Science.gov (United States)

    Ahmad, Farhan; Seyrig, Gregoire; Tourlousse, Dieter M; Stedtfeld, Robert D; Tiedje, James M; Hashsham, Syed A

    2011-10-01

    Rapid, sensitive, and low-cost pathogen diagnostic systems are needed for early disease diagnosis and treatment, especially in resource-limited settings. This study reports a low-cost charge-coupled device (CCD)-based fluorescence imaging system for rapid detection of waterborne pathogens by isothermal gene amplification in disposable microchips. Fluorescence imaging capability of this monochromatic CCD camera is evaluated by optimizing the gain, offset, and exposure time. This imaging system is validated for 12 virulence genes of major waterborne pathogens on cyclic olefin polymer (COP) microchips, using SYTO-82 dye and real time fluorescence loop-mediated isothermal amplification referred here as microRT(f)-LAMP. Signal-to-noise ratio (SNR) and threshold time (Tt) of microRT(f)-LAMP assays are compared with those from a commercial real-time polymerase chain reaction (PCR) instrument. Applying a CCD exposure of 5 s to 10(5) starting DNA copies of microRT(f)-LAMP assays increases the SNR by 8-fold and reduces the Tt by 9.8 min in comparison to a commercial real-time PCR instrument. Additionally, single copy level sensitivity for Campylobacter jejuni 0414 gene is obtained for microRT(f)-LAMP with a Tt of 19 min, which is half the time of the commercial real-time PCR instrument. Due to the control over the exposure time and the wide field imaging capability of CCD, this low-cost fluorescence imaging system has the potential for rapid and parallel detection of pathogenic microorganisms in high throughput microfluidic chips.

  17. 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

  18. Rapid and sensitive detection of Shigella spp. and Salmonella spp. by multiple endonuclease restriction real-time loop-mediated isothermal amplification technique

    Directory of Open Access Journals (Sweden)

    Yi eWang

    2015-12-01

    Full Text Available 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 minutes 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 fg 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 CFU 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    Wei Qi-Wei

    2012-09-01

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

  1. High sensitivity, loop-mediated isothermal amplification combined with colorimetric gold-nanoparticle probes for visual detection of high risk human papillomavirus genotypes 16 and 18.

    Science.gov (United States)

    Kumvongpin, Ratchanida; Jearanaikool, Patcharee; Wilailuckana, Chotechana; Sae-Ung, Nattaya; Prasongdee, Prinya; Daduang, Sakda; Wongsena, Metee; Boonsiri, Patcharee; Kiatpathomchai, Wansika; Swangvaree, Sukumarn Sanersak; Sandee, Alisa; Daduang, Jureerut

    2016-08-01

    High-risk human papillomavirus (HR-HPV) causes cervical cancer. HPV16 and HPV18 are the most prevalent strains of the virus reported in women worldwide. Loop-mediated isothermal amplification (LAMP) is an alternative method for DNA detection under isothermal conditions. However, it results in a turbid amplified product which is not easily detected by the naked eye. This study aimed to develop an improved technique by using gold nanoparticles (AuNPs) attached to a single-stranded DNA probe for the detection of HPV16 and HPV18. Detection of the LAMP product by AuNP color change was compared with detection by visual turbidity. The optimal conditions for this new LAMP-AuNP assay were an incubation time of 20min and a temperature of 65°C. After LAMP amplification was complete, its products were hybridized with the AuNP probe for 5min and then detected by the addition of magnesium salt. The color changed from red to blue as a result of aggregation of the AuNP probe under high ionic strength conditions produced by the addition of the salt. The sensitivity of the LAMP-AuNP assay was greater than the LAMP turbidity assay by up to 10-fold for both HPV genotypes. The LAMP-AuNP assay showed higher sensitivity and ease of visualization than did the LAMP turbidity for the detection of HPV16 and HPV18. Additionally, AuNP-HPV16 and AuNP-HPV18 probes were stable for over 1year. The combination of LAMP and the AuNP-probe colorimetric assay offers a simple, rapid and highly sensitive alternative diagnostic tool for the detection of HPV16 and HPV18 in district hospitals or field studies. PMID:27086727

  2. 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.

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

    International Nuclear Information System (INIS)

    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

  4. Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes

    Science.gov (United States)

    Arunrut, Narong; Kampeera, Jantana; Sirithammajak, Sarawut; Sanguanrut, Piyachat; Proespraiwong, Porranee; Suebsing, Rungkarn; Kiatpathomchai, Wansika

    2016-01-01

    Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non

  5. Progress in multiplex loop-mediated isothermal amplification tech-nology%多重环介导等温扩增技术研究进展

    Institute of Scientific and Technical Information of China (English)

    林文慧; 邹秉杰; 宋沁馨; 周国华

    2015-01-01

    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 im-proving 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.%环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP)因其扩增速度快、

  6. Loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia in HIV-uninfected immunocompromised patients with pulmonary infiltrates.

    Science.gov (United States)

    Nakashima, Kei; Aoshima, Masahiro; Ohkuni, Yoshihiro; Hoshino, Eri; Hashimoto, Kohei; Otsuka, Yoshihito

    2014-12-01

    Loop-mediated isothermal amplification (LAMP) is becoming an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. We retrospectively evaluated 78 consecutive HIV-uninfected patients who underwent LAMP method for diagnosing Pneumocystis pneumonia (PCP). Diagnosis of PCP was made by the detection of Pneumocystis jirovecii (P. jirovecii) with positive LAMP or conventional staining (CS) (Grocott methenamine silver staining or Diff-Quick™) on the basis of compatible clinical symptoms and radiologic findings. Additionally, we reviewed HIV-uninfected immunocompromised patients who underwent subcontract PCR as a historical control. LAMP was positive in 10 (90.9%) of 11 positive-CS patients. Among 13 negative-CS patients with positive LAMP, 11 (84.6%) had PCP, and the remaining 2 were categorized as having P. jirovecii colonization. LDH levels in negative-CS PCP were higher than in positive-CS PCP (p = 0.026). (1 → 3)-β-D-glucan levels in negative-CS PCP were lower than in positive-CS PCP (p = 0.011). The interval from symptom onset to diagnosis as PCP in LAMP group (3.45 ± 1.77 days; n = 22) was shorter than in subcontract PCR group (6.90 ± 2.28 days; n = 10; p cost-effective diagnostic method and is easy to administer in general hospitals. In-house LAMP method would realize early diagnosis of PCP, resulting in improving PCP prognosis and reducing unnecessary PCP-specific treatment. PMID:25187511

  7. Establishment of loop-mediated isothermal amplification (LAMP) for rapid detection of Brucella spp. and application to milk and blood samples.

    Science.gov (United States)

    Song, Liuyan; Li, Juntao; Hou, Shuiping; Li, Xunde; Chen, Shouyi

    2012-09-01

    Brucella spp. are facultative intracellular bacteria that infect humans and animals. In this study, the loop-mediated isothermal amplification (LAMP) was used to detect the Brucella-specific gene omp25. Reaction conditions were optimized as temperature 65°C, reaction time 60 min, Mg(2+) concentration 8.0 mmol/L, polymerase content Bst DNA, 0.5 μL, deoxyribonucleotide concentration 1.6 mmol/L, and inner/outer primer ratio 1:8. The LAMP method was evaluated with 4 Brucella species and 29 non-Brucella bacteria species. Positive reactions were observed on all the 4 Brucella species but not on any non-Brucella species. The limit of detection of the LAMP method was 3.81 CFU Brucella spp. Using the LAMP method, 7 of 110 raw milk samples and 5 of 59 sheep blood samples were detected positive of Brucella spp. Results indicated that LAMP is a fast, specific, sensitive, inexpensive, and suitable method for diagnosis of Brucella spp. infection.

  8. 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. PMID:25769803

  9. 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.

  10. 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.

  11. Development and application of a loop-mediated isothermal amplification assay for rapid identification of aflatoxigenic molds and their detection in food samples.

    Science.gov (United States)

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

    2012-10-15

    Aflatoxins are the most thoroughly studied mycotoxins. They are produced by several members of the genus Aspergillus in section Flavi with Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius being frequently isolated from contaminated food sources. In this work, we describe the development and evaluation of loop-mediated isothermal amplification (LAMP) assays for rapid detection of the three species in separate analyses. The acl1-gene of A. flavus and amy1-genes of A. nomius and A. parasiticus were used as target genes. The detection limits were 2.4, 7.6 and 20pg of pure DNA/reaction for A. flavus, A. nomius and A. parasiticus, respectively. For specificity testing, DNA extracted from mycelia of representative strains of 39 Aspergillus species, 23 Penicillium species, 75 Fusarium species and 37 other fungal species was used as a template for the specific LAMP primer sets developed for the three target species. The LAMP assay was combined with a DNA extraction method for the analysis of pure fungal cultures as well as artificially contaminated Brazil nuts, peanuts and green coffee beans. It is suggested that the developed LAMP assay is a promising tool in the prediction of a potential aflatoxin risk in food and food raw materials and may therefore be suitable for high throughput analysis in the food industry. PMID:23107500

  12. Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane.

    Science.gov (United States)

    Su, Yachun; Yang, Yuting; Peng, Qiong; Zhou, Dinggang; Chen, Yun; Wang, Zhuqing; Xu, Liping; Que, Youxiong

    2016-01-01

    Smut is a fungal disease with widespread prevalence in sugarcane planting areas. Early detection and proper identification of Sporisorium scitamineum are essential in smut management practices. In the present study, four specific primers targeting the core effector Pep1 gene of S. scitamineum were designed. Optimal concentrations of Mg(2+), primer and Bst DNA polymerase, the three important components of the loop-mediated isothermal amplification (LAMP) reaction system, were screened using a single factor experiment method and the L16(4(5)) orthogonal experimental design. Hence, a LAMP system suitable for detection of S. scitamineum was established. High specificity of the LAMP method was confirmed by the assay of S. scitamineum, Fusarium moniliforme, Pestalotia ginkgo, Helminthospcrium sacchari, Fusarium oxysporum and endophytes of Yacheng05-179 and ROC22. The sensitivity of the LAMP method was equal to that of the conventional PCR targeting Pep1 gene and was 100 times higher than that of the conventional PCR assay targeting bE gene in S. scitamineum. The results suggest that this novel LAMP system has strong specificity and high sensitivity. This method not only provides technological support for the epidemic monitoring of sugarcane smut, but also provides a good case for development of similar detection technology for other plant pathogens. PMID:27035751

  13. Development and evaluation of a loop-mediated isothermal amplification assay combined with enrichment culture for rapid detection of very low numbers of Vibrio parahaemolyticus in seafood samples.

    Science.gov (United States)

    Di, Huiling; Ye, Lei; Neogi, Sucharit Basu; Meng, Hecheng; Yan, He; Yamasaki, Shinji; Shi, Lei

    2015-01-01

    The aim of this study was to develop and evaluate a rapid and effective method to detect Vibrio parahaemolyticus, a leading pathogen causing seafood-borne gastroenteritis. A newly designed loop-mediated isothermal amplification (LAMP) assay including a short enrichment period was optimized. This assay correctly detected all the target strains (n=61) but none of the non-target strains (n=34). Very low numbers of V. parahaemolyticus (2 colony forming unit (CFU) per gram of seafood) could be detected within 3 h and the minimum time of the whole assay was only 5 h. Comparative screening of various seafood samples (n=70) indicated that the LAMP assay is superior to polymerase chain reaction (PCR) and conventional culture methods because it is more rapid and less complex. This highly sensitive LAMP assay can be applicable as the method of choice in large-scale and rapid screening of seafood and environmental samples to detect V. parahaemolyticus strains. PMID:25744462

  14. 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. PMID:24827529

  15. 环介导等温扩增技术的研究概况%The overview of loop-mediated isothermal amplification method research

    Institute of Scientific and Technical Information of China (English)

    崔学慧; 陈舜胜; 胡亚萍; 于翠; 印丽萍; 杨翠云

    2011-01-01

    A novel nucleic acid amplification method is termed loop - mediated isohermal a mplification ( LAMP) , Which is convenient, sensitive and specificitive. Through designing a pair of inner primers and a pair of outer primers by six specially sequences and employing a DNA polymerase that has activity of strand displacement DNA synthesis,The method can amplify target DNA fragment with high specificity isothermally. LAMP is applied to medicine field widely and develops rapidly in other fields. This article introduced LAMP from its principle, features and applications.%环介导等温扩增技术是一种操作简便、灵敏度高、特异性强的核酸扩增新方法,它通过针对靶序列的6个特异性区域分别设计两条内引物和两条外引物,利用Bst DNA聚合酶的链置换活性作为反应的动力,在恒温条件下实现对目标核酸的扩增.目前,在医学方面有较广泛的应用,同时这种方法在其他领域的研究也在不断发展.本文主要概述了环介导等温扩增技术(LAMP技术)的原理、特征和应用前景.

  16. Investigation of false positives associated with loop-mediated isothermal amplification assays for detection of Toxoplasma gondii in archived tissue samples of captive felids.

    Science.gov (United States)

    Suleman, Essa; Mtshali, Moses Sibusiso; Lane, Emily

    2016-09-01

    Toxoplasma gondii is a ubiquitous protozoan parasite that infects humans and many different animals, including felids. Many molecular and serologic tests have been developed for detection of T. gondii in a wide range of hosts. Loop-mediated isothermal amplification (LAMP) is a field-friendly technique that lacks the practical drawbacks of other molecular and serologic tests, and LAMP assays have been successfully developed for detection of T. gondii in fresh tissue samples. In the current study, both a previously published and a de-novo designed LAMP assay were compared to a quantitative real-time (q)PCR assay, for the detection of T. gondii in archived formalin-fixed, paraffin-embedded (FFPE) tissue samples from captive wildlife. The LAMP assays produced conflicting results, generating both false positives and false negatives. Furthermore, the LAMP assays were unable to positively identify samples with low levels of parasites as determined by qPCR and histopathology. Therefore, these LAMP assays may not be the most suitable assays for detection of T. gondii in archived FFPE and frozen tissue samples. PMID:27449130

  17. Development of a visual loop-mediated isothermal amplification method for rapid detection of the bacterial pathogen Pseudomonas putida of the large yellow croaker (Pseudosciaena crocea).

    Science.gov (United States)

    Mao, Zhijuan; Qiu, Yangyu; Zheng, Lei; Chen, Jigang; Yang, Jifang

    2012-06-01

    In recent years, the large yellow croaker (Pseudosciaena crocea), an important marine fish farmed in the coastal areas of Zhejiang province, east China, has become severely endangered as a result of the bacterial pathogen Pseudomonas putida. This paper reports the development of a visual loop-mediated isothermal amplification (LAMP) assay for rapid detection of the pathogen. Four primers, F3, B3, FIP and BIP, were designed on the basis of DNA sequence of the rpoN gene of P. putida. After optimization of the reaction conditions, the detection limit of LAMP assay was 4.8cfu per reaction, 10-fold higher than that of conventional PCR. The assay showed high specificity to discriminate all P. putida isolates from nine other Gram-negative bacteria. The assay also successfully detected the pathogen DNA in the tissues of infected fish. For visual LAMP without cross-contamination, SYBR Green I was embedded in a microcrystalline wax capsule and preset in the reaction tubes; after the reaction the wax was melted at 85°C to release the dye and allow intercalation with the amplicons. The simple, highly sensitive, highly specific and cost-effective characteristics of visual LAMP may encourage its application in the rapid diagnosis of this pathogen.

  18. Establishment of a novel one-step reverse transcription loop-mediated isothermal amplification assay for rapid identification of RNA from the severe fever with thrombocytopenia syndrome virus.

    Science.gov (United States)

    Xu, Haihong; Zhang, Lei; Shen, Guangqiang; Feng, Cen; Wang, Xinying; Yan, Jie; Zhang, Yanjun

    2013-12-01

    As an emerging infectious disease, severe fever with thrombocytopenia syndrome virus (SFTSV) infection has been found in many areas of China. Suitable laboratory diagnostic method is urgently needed in clinical detections and epidemiological investigations. In this study, a modified, low-cost and rapid visualized one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of RNA from the SFTSV has been established. In order to avoid the risk of aerosol contamination and facilitate the naked eye to observe, a microcrystalline wax-dye capsule wrapping the highly sensitive DNA fluorescence dye SYBR Green I was added to the RT-LAMP reaction tube before the initiation of the assay. The detection limit of the established RT-LAMP assay was 10 fg template RNA per reaction mixture. The RT-LAMP assay was confirmed to be high specific to SFTSV, and no cross-reaction was found with the detection of the Chikungunya fever virus, Hemorrhagic Fever with Renal Syndrome virus (HFRSV), and Dengue fever virus. The assay was then applied for the detection of SFTSV RNA in 32 clinical serum samples and showed 94.4% consistence with the detection results of the real-time RT-PCR. The whole process, from sample preparation to result reporting, can be completed within 2h. This adapted, cost efficient and quick visualized RT-LAMP method is feasible for SFTSV field diagnosis in resource-limited field settings.

  19. Rapid, simple, and sensitive detection of the ompB gene of spotted fever group rickettsiae by loop-mediated isothermal amplification

    Directory of Open Access Journals (Sweden)

    Pan Lei

    2012-10-01

    Full Text Available Abstract Background Spotted fever caused spotted fever group rickettsiae (SFGR is prevalent throughout China. In this study, we describe a rapid, simple, and sensitive loop-mediated isothermal amplification (LAMP assay targeting the ompB gene of spotted fever group rickettsiae ideal for application in China. The LAMP assay has the potential to detect spotted fever group rickettsiae early in infection and could therefore serve as an alternative to existing methods. Methods A set of universal primers which are specific 7 common species of spotted fever group rickettsiae in China were designed using PrimerExplorer V4 software based on conserved sequences of ompB gene. The sensitivity, specificity and reproducibility of the LAMP were evaluated. The LAMP assay for detecting SFGR was compared with conventional PCR assays for sensitivity and specificity in early phase blood samples obtained from 11 infected human subjects. Results The sensitivity of the LAMP assay was five copies per reaction (25 μL total volume, and the assay did not detect false-positive amplification across 42 strains of 27 members of the order Rickettsiales and 17 common clinical pathogens. The LAMP assay was negative to typhus group rickettsiae including R. prowazekii and R. typhi for no available conserved sequences of ompB was obtained for designing primers. To evaluate the clinical applicability of the LAMP assay, a total of 11 clinical samples, 10 samples confirmed serologically (3 cases, ecologically (1 case, by real-time polymerase chain reaction (PCR; 2 cases, ecologically and by real-time PCR (1 case, and serologically and by real-time PCR (3 cases were analyzed by the ompB LAMP assay. Data were validated using a previously established nested PCR protocol and real-time PCR. A positive LAMP result was obtained for 8 of the 10 confirmed cases (sensitivity, 73%; specificity, 100%, while none of these samples were positive by nested PCR (sensitivity, 0%; specificity, 100

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

    Directory of Open Access Journals (Sweden)

    Fenxia Fan

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

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

    Directory of Open Access Journals (Sweden)

    Khan Md Gulam Musawwir

    2012-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Feiwu Li

    2014-08-01

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

  3. Reverse transcription loop-mediated isothermal amplification assays for rapid identification of eastern and western strains of bluetongue virus in India.

    Science.gov (United States)

    Maan, S; Maan, N S; Batra, K; Kumar, A; Gupta, A; Rao, Panduranga P; Hemadri, Divakar; Reddy, Yella Narasimha; Guimera, M; Belaganahalli, M N; Mertens, P P C

    2016-08-01

    Bluetongue virus (BTV) infects all ruminants, including cattle, goats and camelids, causing bluetongue disease (BT) that is often severe in naïve deer and sheep. Reverse-transcription-loop-mediated-isothermal-amplification (RT-LAMP) assays were developed to detect eastern or western topotype of BTV strains circulating in India. Each assay uses four primers recognizing six distinct sequences of BTV genome-segment 1 (Seg-1). The eastern (e)RT-LAMP and western (w)RT-LAMP assay detected BTV RNA in all positive isolates that were tested (n=52, including Indian BTV-1, -2, -3, -5, -9, -10, -16, -21 -23, and -24 strains) with high specificity and efficiency. The analytical sensitivity of the RT-LAMP assays is comparable to real-time RT-PCR, but higher than conventional RT-PCR. The accelerated eRT-LAMP and wRT-LAMP assays generated detectable levels of amplified DNA, down to 0.216 fg of BTV RNA template or 108 fg of BTV RNA template within 60-90min respectively. The assays gave negative results with RNA from foot-and-mouth-disease virus (FMDV), peste des petits ruminants virus (PPRV), or DNA from Capripox viruses and Orf virus (n=10), all of which can cause clinical signs similar to BT. Both RT-LAMP assays did not show any cross-reaction among themselves. The assays are rapid, easy to perform, could be adapted as a 'penside' test making them suitable for 'front-line' diagnosis, helping to identify and contain field outbreaks of BTV. PMID:27054888

  4. Real-time loop-mediated isothermal amplification (LAMP) assay for group specific detection of important trichothecene producing Fusarium species in wheat.

    Science.gov (United States)

    Denschlag, Carla; Rieder, Johann; Vogel, Rudi F; Niessen, Ludwig

    2014-05-01

    Trichothecene mycotoxins such as deoxynivaneol (DON), nivalenol (NIV) and T2-Toxin are produced by a variety of Fusarium spp. on cereals in the field and may be ingested by consumption of commodities and products made thereof. The toxins inhibit eukaryotic protein biosynthesis and may thus impair human and animal health. Aimed at rapid and sensitive detection of the most important trichothecene producing Fusarium spp. in a single analysis, a real-time duplex loop-mediated isothermal amplification (LAMP) assay was set up. Two sets of LAMP primers were designed independently to amplify a partial sequence of the tri6 gene in Fusarium (F.) graminearum and of the tri5 gene in Fusarium sporotrichioides, respectively. Each of the two sets detected a limited number of the established trichothecene producing Fusarium-species. However, combination of the two sets in one duplex assay enabled detection of F. graminearum, Fusarium culmorum, Fusarium cerealis, F. sporotrichioides, Fusarium langsethiae and Fusarium poae in a group specific manner. No cross reactions were detected with purified DNA from 127 other fungal species or with cereal DNA. To demonstrate the usefulness of the assay, 100 wheat samples collected from all over the German state of Bavaria were analyzed for the trichothecene mycotoxin DON by HPLC and for the presence of trichothecene producers by the new real-time duplex LAMP assay in parallel analyses. The LAMP assay showed positive results for all samples with a DON concentration exceeding 163ppb. The major advantage of the duplex LAMP assay is that the presence of six of the major trichothecene producing Fusarium spp. can be detected in a rapid and user-friendly manner with only one single assay. To our knowledge this is the first report of the use of a multiplex LAMP assay for fungal organisms.

  5. Detection of Magnaporthe oryzae chrysovirus 1 in Japan and establishment of a rapid, sensitive and direct diagnostic method based on reverse transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Komatsu, Ken; Urayama, Syun-Ichi; Katoh, Yu; Fuji, Shin-Ichi; Hase, Shu; Fukuhara, Toshiyuki; Arie, Tsutomu; Teraoka, Tohru; Moriyama, Hiromitsu

    2016-02-01

    Magnaporthe oryzae chrysovirus 1 (MoCV1) is a mycovirus with a dsRNA genome that infects the rice blast fungus Magnaporthe oryzae and impairs its growth. To date, MoCV1 has only been found in Vietnamese isolates of M. oryzae, and the distribution of this virus in M. oryzae isolates from other parts of the world remains unknown. In this study, using a one-step reverse transcription PCR (RT-PCR) assay, we detected a MoCV1-related virus in M. oryzae in Japan (named MoCV1-AK) whose sequence shares considerable similarity with that of the MoCV1 Vietnamese isolate. To establish a system for a comprehensive survey of MoCV1 infection in the field, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for direct detection of the virus. The sensitivity of the RT-LAMP assay was at least as high as that of the one-step RT-PCR assay. In addition, we detected MoCV1-AK in M. oryzae-infected oatmeal agar plates and lesions on rice leaves using the RT-LAMP assay without dsRNA extraction, by simple sampling with a toothpick. Preliminary screening of MoCV1 in Japanese M. oryzae isolates indicated that MoCV1 is currently distributed in rice fields in Japan. Our results provide a first example of the application of RT-LAMP for the detection of mycoviruses, which will accelerate surveys for mycovirus infection. PMID:26547578

  6. A nationwide survey of pathogenic leptospires in urine of cattle and buffaloes by Loop-mediated isothermal amplification (LAMP) method in Thailand, 2011–2013

    Science.gov (United States)

    SUWANCHAROEN, Duangjai; LIMLERTVATEE, Supaluck; CHETIYAWAN, Philaiphon; TONGPAN, Phichet; SANGKAEW, Nongluck; SAWADDEE, Yaowarat; INTHAKAN, Kanya; WIRATSUDAKUL, Anuwat

    2016-01-01

    Leptospirosis is a worldwide distributed zoonosis which has long been endemic in Thailand. Cattle and buffaloes are important livestock species that live in close contact with humans, especially in rural areas. These animals may, therefore, act as long-term carriers of leptospirosis for humans and other livestock species. The present study employed loop-mediated isothermal amplification (LAMP) method to detect pathogenic leptospiral 16S rDNA in the urine of cattle and buffaloes for assessing associations between uroprevalence and species, sex, age and spatial distribution. A total of 3,657 urine samples were collected for laboratory diagnosis, and 312 of which turned positive to the test (true prevalence 5.90%; 95% CI 4.98–6.91). The highest true uroprevalence was found in lower northern region at 19.80% (95% CI 15.83–24.32) followed by upper and lower northeastern regions at 15.22% and 6.25%, respectively. However, the highest true uroprevalence in beef cattle, the majority of cattle in Thailand, was recorded in northeastern region which is the endemic area of human leptospirosis. The uroprevalence was not statistically different among species and types of examined animals. Male animals were over twice more likely to be infected compared to females. Excluding animals younger than one year of age due to small sample size, the uroprevalence upraised with increasing age. A collaborative investigation between veterinary and public health sectors is required to holistically explore the link between leptospirosis in humans and livestock, especially in high prevalent areas. PMID:27302016

  7. Detection of Plasmodium vivax and Plasmodium falciparum DNA in human saliva and urine: loop-mediated isothermal amplification for malaria diagnosis.

    Science.gov (United States)

    Ghayour Najafabadi, Zahra; Oormazdi, Hormozd; Akhlaghi, Lame; Meamar, Ahmad Reza; Nateghpour, Mehdi; Farivar, Leila; Razmjou, Elham

    2014-08-01

    This study investigated loop-mediated isothermal amplification (LAMP) detection of Plasmodium falciparum and Plasmodium vivax in urine and saliva of malaria patients. From May to November 2011, 108 febrile patients referred to health centers in Sistan and Baluchestan Province of south-eastern Iran participated in the study. Saliva, urine, and blood samples were analyzed with nested PCR and LAMP targeting the species-specific nucleotide sequence of small subunit ribosomal RNA gene (18S rRNA) of P. falciparum and P. vivax and evaluated for diagnostic accuracy by comparison to blood nested PCR assay. When nested PCR of blood is used as standard, microscopy and nested PCR of saliva and urine samples showed sensitivity of 97.2%, 89.4% and 71% and specificity of 100%, 97.3% and 100%, respectively. LAMP sensitivity of blood, saliva, and urine was 95.8%, 47% and 29%, respectively, whereas LAMP specificity of these samples was 100%. Microscopy and nested PCR of saliva and LAMP of blood were comparable to nested PCR of blood (к=0.95, 0.83, and 0.94, respectively), but agreement for nested PCR of urine was moderate (к=0.64) and poor to fair for saliva LAMP and urine LAMP (к=0.38 and 0.23, respectively). LAMP assay showed low sensitivity for detection of Plasmodium DNA in human saliva and urine compared to results with blood and to nested PCR of blood, saliva, and urine. However, considering the advantages of LAMP technology and of saliva and urine sampling, further research into the method is worthwhile. LAMP protocol and precise preparation protocols need to be defined and optimized for template DNA of saliva and urine.

  8. Evaluation of real-time loop-mediated isothermal amplification (RealAmp) for rapid detection of Mycobacterium tuberculosis from sputum samples.

    Science.gov (United States)

    Li, Yiming; Shi, Lei; Pan, Anqi; Cao, Weiwei; Chen, Xun; Meng, Hecheng; Yan, He; Miyoshi, Shin-ichi; Ye, Lei

    2014-09-01

    Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) leads to serious health problems as a chronic respiratory infectious disease. Here we established a real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) using a portable ESE Quant tube scanner as a convenient rapid detection method for MTB. The method efficacy from sputum samples was further investigated, and the reaction time was only 20min with the detection limit low to 10(2)CFU/ml concentration of MTB. We assessed a total of 1067 samples by the RealAmp assay, comparing the results with smear microscopy and conventional culture methods. To examine whether the failure to detect TB by culturing is due to low sensitivity or true absence, we examined the culture negative samples by commercial real time PCR MTB detection kit, and the results were compared with RealAmp. The data showed that RealAmp assay had a higher positive rate than that of sputum smear and culture methods. RealAmp had a sensitivity of 96.70% and a specificity of 91.55% when compared with culture. In addition, its sensitivity and specificity were 95.29% and 86.88% respectively compared with examination of smear samples using light microscopy. The sensitivity of RealAmp in comparison to real time PCR was 98.25% and specificity was 99.11% in validation of culture negative samples. The present study revealed the newly established RealAmp assay as a convenient, efficient, sensitive and specific method that could be an alternative for rapid detection of MTB and a tool to validate culture and smear negative samples. Furthermore, the portability of the ESE Quant tube scanner also contributed to the promising application for grassroots and field detection of MTB.

  9. An immunoassay-based reverse-transcription loop-mediated isothermal amplification assay for the rapid detection of avian influenza H5N1 virus viremia.

    Science.gov (United States)

    Tang, Yi; Yu, Xu; Chen, Hao; Diao, Youxiang

    2016-12-15

    Avian influenza virus (AIV) subtype H5N1 attracts particular consideration because it is a continuous threat to animals and public health systems. The viremia caused by AIV H5N1 infection may increase the risk of blood-borne transmission between humans. Therefore, there is a need to rapidly evaluate and implement screening measures for AIV H5N1 viremia that allows for rapid response to this potentially pandemic threat. The present report describes an immunoassay-based reverse-transcription loop-mediated isothermal amplification (immuno-RT-LAMP) assay for the rapid detection of AIV H5N1 in whole blood samples. Using PCR tubes coated with an H5 subtype monoclonal antibody, AIV H5N1 virions were specifically captured from blood samples. After a thermal lysis step, the released viral N1 gene was exponentially amplified using RT-LAMP on either a real-time PCR instrument for quantitative analysis, or in a water bath system for endpoint analysis. The detection limit of the newly developed immuno-RT-LAMP assay was as low as 1.62×10(1) 50% embryo infectious dose/mL of virus in both regular samples and simulated viremia samples. There were no cross-reactions with non-H5N1 influenza viruses or other avian viruses. The reproducibility of the assay was confirmed using intra- and inter-assay tests with variability ranging from 1.05% to 3.37%. Our results indicate that immuno-RT-LAMP is a novel, effective point-of-care virus identification solution for the rapid diagnosis and monitoring of AIV H5N1 in blood samples. PMID:27376196

  10. Performance testing of passive autocatalytic recombiners (PARs)

    Energy Technology Data Exchange (ETDEWEB)

    Blanchat, T. [Sandia National Laboratories, Albuquerque, NM (United States); Malliakos, A. [U.S. Nuclear Regulatory Commission, Washington, DC (United States)

    1997-03-01

    Passive autocatalytic recombiners (PARs) have been under consideration in the U.S. as a combustible gas control system in advanced light water reactor (ALWR) containments for design basis and severe accidents. PARs do not require a source of power. Instead they use palladium or platinum as a catalyst to recombine hydrogen and oxygen gases into water vapor upon contact with the catalyst. Energy from the recombination of hydrogen with oxygen is released at a relatively slow but continuous rate into the containment which prevents the pressure from becoming too high. The heat produced creates strong buoyancy effects which increases the influx of the surrounding gases to the recombiner. These natural convective flow currents promote mixing of combustible gases in the containment. PARs are self-starting and self-feeding under a very wide range of conditions. The recombination rate of the PAR system needs to be great enough to keep the concentration of hydrogen (or oxygen) below acceptable limits. There are several catalytic recombiner concepts under development worldwide. The USNRC is evaluating a specific design of a PAR which is in an advanced stage of engineering development and has been proposed for ALWR designs. Sandia National laboratories (SNL), under the sponsorship and the direction of the USNRC, is conducting an experimental program to evaluate the performance of PARs. The PAR will be tested at the SURTSEY facility at SNL. The test plan currently includes the following experiments: experiments will be conducted to define the startup characteristics of PARs (i.e., to define what is the lowest hydrogen concentration that the PAR starts recombining the hydrogen with oxygen); experiments will be used to define the hydrogen depletion rate of PARs as a function of hydrogen concentration; and experiments will be used to define the PAR performance in the presence of high concentrations of steam. (author)

  11. Autocatalytic silver-plating of aluminium radio frequency waveguides with autocatalytic nickel as the undercoat for space applications

    International Nuclear Information System (INIS)

    Autocatalytic plating, a technique used for evenly coating contoured items with deep cavities, such as microwave components, irrespective of shape and size of the item to be plated, was used in this work to coat a radio frequency waveguide. In this work, a process sequence was developed for autocatalytic silver plating on aluminum base material with autocatalytic nickel as the undercoat. The thickness of the deposited silver depends on variables like temperature, concentration of silver ions in the electrolyte, and the pH of the solution. The influence of these variables was studied under different process conditions. Silver-coated rectangular plates were subjected to various tests, including a bend test, a heat resistance test, a thermal cycling test, a thermo vacuum test, a solderability test, and a humidity resistance test. Autocatalytic silver-coated RF waveguide WR28 was tested for insertion loss and return loss. Autocatalytic silver-plated rectangular plates and waveguides were found to withstand a simulated space environment. (paper)

  12. 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.

  13. The Sensitivity and Specificity of Loop-Mediated Isothermal Amplification (LAMP Assay for Tuberculosis Diagnosis in Adults with Chronic Cough in Malawi.

    Directory of Open Access Journals (Sweden)

    Marriott Nliwasa

    Full Text Available Current tuberculosis diagnostics lack sensitivity, and are expensive. Highly accurate, rapid and cheaper diagnostic tests are required for point of care use in low resource settings with high HIV prevalence.To investigate the sensitivity and specificity, and cost of loop-mediated isothermal amplification (LAMP assay for tuberculosis diagnosis in adults with chronic cough compared to Xpert® MTB/RIF, fluorescence smear microscopy.Between October 2013 and March 2014, consecutive adults at a primary care clinic were screened for cough, offered HIV testing and assessed for tuberculosis using LAMP, Xpert® MTB/RIF and fluorescence smear microscopy. Sensitivity and specificity (with culture as reference standard, and costs were estimated.Of 273 adults recruited, 44.3% (121/273 were HIV-positive and 19.4% (53/273 had bacteriogically confirmed tuberculosis. The sensitivity of LAMP compared to culture was 65.0% (95% CI: 48.3% to 79.4% with 100% (95% CI: 98.0% to 100% specificity. The sensitivity of Xpert® MTB/RIF (77.5%, 95% CI: 61.5% to 89.2% was similar to that of LAMP, p = 0.132. The sensitivity of concentrated fluorescence smear microscopy with routine double reading (87.5%, 95% CI: 73.2% to 95.8% was higher than that of LAMP, p = 0.020. All three tests had high specificity. The lowest cost per test of LAMP was at batch size of 14 samples (US$ 9.98; this was lower than Xpert® MTB/RIF (US$ 13.38 but higher than fluorescence smear microscopy (US$ 0.65.The sensitivity of LAMP was similar to Xpert® MTB/RIF but lower than fluorescence smear microscopy; all three tests had high specificity. These findings support the Malawi policy that recommends a combination of fluorescence smear microscopy and Xpert® MTB/RIF prioritised for people living with HIV, already found to be smear-negative, or being considered for retreatment of tuberculosis.

  14. The Sensitivity and Specificity of Loop-Mediated Isothermal Amplification (LAMP) Assay for Tuberculosis Diagnosis in Adults with Chronic Cough in Malawi

    Science.gov (United States)

    Nliwasa, Marriott; MacPherson, Peter; Chisala, Palesa; Kamdolozi, Mercy; Khundi, McEwen; Kaswaswa, Kruger; Mwapasa, Mphatso; Msefula, Chisomo; Sohn, Hojoon; Flach, Clare; Corbett, Elizabeth L.

    2016-01-01

    Background Current tuberculosis diagnostics lack sensitivity, and are expensive. Highly accurate, rapid and cheaper diagnostic tests are required for point of care use in low resource settings with high HIV prevalence. Objective To investigate the sensitivity and specificity, and cost of loop-mediated isothermal amplification (LAMP) assay for tuberculosis diagnosis in adults with chronic cough compared to Xpert® MTB/RIF, fluorescence smear microscopy. Methods Between October 2013 and March 2014, consecutive adults at a primary care clinic were screened for cough, offered HIV testing and assessed for tuberculosis using LAMP, Xpert® MTB/RIF and fluorescence smear microscopy. Sensitivity and specificity (with culture as reference standard), and costs were estimated. Results Of 273 adults recruited, 44.3% (121/273) were HIV-positive and 19.4% (53/273) had bacteriogically confirmed tuberculosis. The sensitivity of LAMP compared to culture was 65.0% (95% CI: 48.3% to 79.4%) with 100% (95% CI: 98.0% to 100%) specificity. The sensitivity of Xpert® MTB/RIF (77.5%, 95% CI: 61.5% to 89.2%) was similar to that of LAMP, p = 0.132. The sensitivity of concentrated fluorescence smear microscopy with routine double reading (87.5%, 95% CI: 73.2% to 95.8%) was higher than that of LAMP, p = 0.020. All three tests had high specificity. The lowest cost per test of LAMP was at batch size of 14 samples (US$ 9.98); this was lower than Xpert® MTB/RIF (US$ 13.38) but higher than fluorescence smear microscopy (US$ 0.65). Conclusion The sensitivity of LAMP was similar to Xpert® MTB/RIF but lower than fluorescence smear microscopy; all three tests had high specificity. These findings support the Malawi policy that recommends a combination of fluorescence smear microscopy and Xpert® MTB/RIF prioritised for people living with HIV, already found to be smear-negative, or being considered for retreatment of tuberculosis. PMID:27171380

  15. 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

    amplification (LAMP) for rapid and quantitative detection of Salmonella spp. in food samples. The whole diagnostic procedures including DNA isolation, isothermal amplification, and real-time detection were accomplished in a single chamber. Up to eight samples could be handled simultaneously and the system...... 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...

  16. Use of loop-mediated isothermal amplification in parasitology research%环介导等温扩增技术及其在寄生虫学研究中的应用

    Institute of Scientific and Technical Information of China (English)

    刘航; 李彦; 杨毅梅

    2011-01-01

    A novel method of nucleic acid amplification, termed loop-mediated isothermal amplification (LAMP), employs two pairs of specially designed primers and DNA polymerase with strand displacement activity.LAMP can efficiently amplify DNA with high specificity under isothermal conditions.The final products are stem-loop DNA with several inverted repeats of the target sequence and cauliflower-like structures with multiple loops.DNA synthesis is accompanied by a byproduct in the form of a white precipitate of magnesium pyrophosphate, providing a visible result.As a new technique for nucleic acid amplification, LAMP has been used in the study of parasitic diseases such as malaria and schistosomiasis.Research has shown that LAMP is sensitive, specific, and easy to perform.LAMP has a range of potential applications in parasitology research.%环介导等温扩增(LAMP)技术是利用2对特殊设计的引物,一种具有链置换活性的DNA聚合酶,在恒温条件下特异、高效地扩增DNA的新技术,扩增产物为一系列反向重复的靶序列构成的茎环结构和多环花椰菜样结构的DNA片段混合物,电泳图谱呈阶梯式带状分布,反应伴有白色的副产物焦磷酸镁沉淀产生,可通过肉眼定性判断反应结果:.作为一种新的核酸扩增技术,已被应用于疟疾、血吸虫等寄生虫的研究中.LAMP方法:敏感、特异,简便,在寄生虫学研究中有广阔的应用前景.

  17. Loop-mediated isothermal amplification and its application in virus detection%环媒恒温扩增技术在病毒感染检测中的应用

    Institute of Scientific and Technical Information of China (English)

    拜晓勃; 韩洪起; 许丰雯; 乔文涛

    2009-01-01

    Loop-mediated isothermal amplification ( LAMP ) , a novel isothermal nucleic acid amplification method, employs a DNA polymerase and four specially designed primers that recognize a total of six distinct sequences on the target DNA to amplify the target via stem-loop DNA structure. LAMP allows the synthesis of large amounts of DNA in a short period of time with high specificity under isothermal conditions. As the LAMP reaction progresses, the reaction by-product pyrophosphate ions bind to magnesium ions and form a white precipitate of magnesium pyrophosphate. Real-time turbidity measurements of the LAMP reaction make it to be a very useful molecular method for quantitutire analysis of nucleic acid.%环媒恒温扩增法(loop mediated isothermal amplification,LAMP)是一种新型的恒温核酸扩增技术,它利用了2对特异引物和一种有链取代活性的DNA聚合酶,通过使扩增产物形成单链环结构实现核酸的恒温扩增.LAMP自身的一系列特性使其可以达到对特定核酸片段高效、特异的扩增.由于反应结果可以通过测定反应副产物--焦磷酸镁沉淀引起的浊度来判断,因此使其成为一种十分有效的分子检测手段.

  18. Stability analysis of an autocatalytic protein model

    Science.gov (United States)

    Lee, Julian

    2016-05-01

    A self-regulatory genetic circuit, where a protein acts as a positive regulator of its own production, is known to be the simplest biological network with a positive feedback loop. Although at least three components—DNA, RNA, and the protein—are required to form such a circuit, stability analysis of the fixed points of this self-regulatory circuit has been performed only after reducing the system to a two-component system, either by assuming a fast equilibration of the DNA component or by removing the RNA component. Here, stability of the fixed points of the three-component positive feedback loop is analyzed by obtaining eigenvalues of the full three-dimensional Hessian matrix. In addition to rigorously identifying the stable fixed points and saddle points, detailed information about the system can be obtained, such as the existence of complex eigenvalues near a fixed point.

  19. LAMP检测无乳链球菌方法的建立及应用%Development and Application of Loop-Mediated Isothermal Amplification for Detection of Streptococcus agalactiae

    Institute of Scientific and Technical Information of China (English)

    王永; 赵新; 景海春; 兰青阔; 朱珠; 程奕

    2009-01-01

    以无乳链球菌纤连蛋白fbs基因为主要研究对象,采取环介导等温扩增技术(Loop-Mediated Isothermal Amplification,LAMP),针对fbs基因的6个区域设计4条特异性引物,利用一种链置换DNA聚合酶(Bst DNA polymerase)在63℃保温1 h,通过荧光显色即可完成对无乳链球菌的检测工作.结果显示,LAMP方法能够特异性检测fbs基因,其检测灵敏度是常规PCR方法的100倍,并与实时荧光定量PCR方法相当.所建立的针对无乳链球菌fbs基因的LAMP检测方法具有高度的特异性及稳定性,结果可靠,非常适合无乳链球菌的快速检测.%To develop a technique for detecting the fbsB gene of Streptococcus agalactiae using a novel DNA amplification method designated Loop-Mediated Isothermal Amplification (LAMP).The detection assay is based on the loop-mediated isothermal amplification (LAMP) reaction.The fbsB gene was amplified by a set of four specially primers that recognize six distinct sequences of the target.The amplification can be obtained in 1 h by incubating all of the reagents in a single tube with Bst DNA polymerase at 63℃.Results showed that the developed LAMP assay demonstrated an exceptionally higher than the conventional PCR.This assay results was found to be 100 times more sensitive than the PCR assay,and consistent with the results of real-time PCR.Our results clearly demonstrate that the LAMP-based assay is a sensitive and reliable means for the detection of Streptococcus agalactiae.

  20. CFD analysis of passive autocatalytic recombiner interaction with atmosphere

    Energy Technology Data Exchange (ETDEWEB)

    Gera, B.; Sharma, Pavan K.; Singh, R.K.; Vaze, K.K. [Bhabha Atomic Research Centre, Trombay, Mumbai (India). Reactor Safety Div.

    2011-05-15

    In water cooled power reactors, significant quantities of hydrogen could be produced following a severe accident (loss-of coolant-accident along with non availability of Emergency Core Cooling System) from the reaction between steam and zirconium at high fuel clad temperature. In order to prevent the containment and other safety relevant components from incurring serious damage caused by a detonation of the hydrogen/air-mixture generated during a severe accident in water cooled power reactors, passive autocatalytic recombiners (PAR) are used for hydrogen removal in an increasing number of French, German and Russian plants. These devices make use of the fact that hydrogen and oxygen react exothermally on catalytic surfaces generating steam and heat. Numerous tests and simulations have been conducted in the past to investigate passive autocatalytic recombiners behaviour in situations representative of severe accidents. Numerical models were developed from the experimental data for codes like COCOSYS or ASTEC in order to optimise the passive autocatalytic recombiners location and to assess the efficiency of passive autocatalytic recombiners implementation in different scenarios. However, these models are usually simple (black-box type) and based on the manufacturer's correlation to calculate the hydrogen depletion rate. Recently, uses of enhanced CFD models have shown significant improvements towards modeling such phenomenon in complex geometry. The work presents CFD analysis of interaction of a representative nuclear power plant containment atmosphere with passive autocatalytic recombiners simulated using the commercial Computational Fluid Dynamics code for PAR Interaction Studies (PARIS benchmarks) exercise. A two-dimensional geometrical model of the simulation domain was used. The containment was represented by an adiabatic rectangular box with two PAR located at intermediate elevations near opposite walls. The flow in the simulation domain was modelled as

  1. Application of loop-mediated isothermal amplification for malaria diagnosis during a follow-up study in São Tomé

    Directory of Open Access Journals (Sweden)

    Lee Pei-Wen

    2012-12-01

    Full Text Available Abstract Background A reliable and simple test for the detection of malaria parasite is crucial in providing effective treatment and therapeutic follow-up, especially in malaria elimination programmes. A comparison of four methods, including nested polymerase chain reaction (PCR and loop-mediated isothermal amplification (LAMP were used for the malaria diagnosis and treatment follow-up in São Tomé and Príncipe, during a successful pre-elimination campaign. Method During the period September to November 2009, blood samples from 128 children (five to 14 years old with temperature ≥38°C (tympanic in the District of Agua Grande were examined using four different methods, i.e., histidine-rich protein 2 (HRP-2 based rapid diagnostic tests (HRP-2-RDTs, optical microscopy, nested PCR, and LAMP. First-line treatment with artesunate-amodiaquine was given for uncomplicated malaria and intravenous quinine was given for complicated malaria. Children with persistent positivity for malaria by microscopy, or either by nested PCR, or by LAMP on day 7 were given second-line treatment with artemether-lumefantrine. Treatment follow-up was made weekly, for up to four weeks. Results On day 0, positive results for HRP-2-RDTs, microscopy, nested PCR, and LAMP, were 68(53%, 47(37%, 64(50%, and 65(51%, respectively. When nested PCR was used as a reference standard, only LAMP was comparable; both HRP-2-RDTs and microscopy had moderate sensitivity; HRP-2-RDTs had poor positive predictive value (PPV and a moderate negative predictive value (NPV for the treatment follow-up. Seventy-one children with uncomplicated malaria and eight children with complicated falciparum malaria were diagnosed based on at least one positive result from the four tests as well as clinical criteria. Twelve of the 79 children receiving first-line treatment had positive results by nested PCR on day 7 (nested PCR-corrected day 7 cure rate was 85%. After the second-line treatment, nested PCR

  2. 环介导等温扩增技术快速检测奶粉中的单增李斯特菌%Rapid Detection of Listeria monocytogenes in Milk Powder by Loop-mediated Isothermal Amplification

    Institute of Scientific and Technical Information of China (English)

    路彦霞; 孟兆祥; 马晓燕; 王羽; 张先舟; 张伟

    2012-01-01

    Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification technology with high specificity, efficiency and rapidity under isothermal conditions. In this research, a set of specific LAMP primers were designed to amplify the specific hlyA gene of Listeria monocytogenes by LAMP technology. The results showed that the detection sensitivity and detection limit of the LAMP method for purely cultured Listeria monocytogenes were 2.45×101 cfu/mL and 7.32× 101cfu/mL, respectively. For other food pathogens, the target bands were not found. This method was suitable for rapid detection of Listeria monocytogenes in contaminated foods.%环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)是一种在等温务件下特异、灵敏、快速的新型基因扩增技术.试验以单增李斯特菌为研究对象,根据其特有的hlyA基因设计了一套特异性引物,进行LAMP扩增.结果表明,LAMP检测单增李斯特菌的灵敏度为2.45×101 cfu/mL,其对人工感染奶粉样品的检出限为7.32×101 cfu/mL.对其它食品病原菌进行检测,结果均未出现目的条带,特异性强.表明LAMP法适合于食品中污染单增李斯特菌的快速检测.

  3. Computing autocatalytic sets to unravel inconsistencies in metabolic network reconstructions

    DEFF Research Database (Denmark)

    Schmidt, R.; Waschina, S.; Boettger-Schmidt, D.;

    2015-01-01

    MOTIVATION: Genome-scale metabolic network reconstructions have been established as a powerful tool for the prediction of cellular phenotypes and metabolic capabilities of organisms. In recent years, the number of network reconstructions has been constantly increasing, mostly because...... of the availability of novel (semi-)automated procedures, which enabled the reconstruction of metabolic models based on individual genomes and their annotation. The resulting models are widely used in numerous applications. However, the accuracy and predictive power of network reconstructions are commonly limited...... by inherent inconsistencies and gaps. RESULTS: Here we present a novel method to validate metabolic network reconstructions based on the concept of autocatalytic sets. Autocatalytic sets correspond to collections of metabolites that, besides enzymes and a growth medium, are required to produce all biomass...

  4. Autocatalytic fission-fusion microexplosions for nuclear pulse propulsion

    Science.gov (United States)

    Winterberg, F.

    2000-12-01

    Autocatalytic fission-fusion microexplosions, mutually amplifying fission and fusion reactions, are proposed for propulsion. Autocatalytic fission-fusion microexplosions can be realized by imploding a shell of uranium 235 (or plutonium) onto a magnetized deuterium-tritium (DT) plasma. After having reached a high temperature, the DT plasma releases fusion neutrons making fission reactions in the fissile shell increasing the implosion velocity which in turn increases the fusion reaction rate until full ignition of the DT plasma. To implode the fissile shell a small amount of high explosive and to magnetize the DT plasma a small auxiliary electric discharge are required. In comparison to nuclear bomb pulse propulsion, the energy released per pulse is much smaller and the efficiency higher. And in comparison to laser- or particle-beam-ignited fusion microexplosions, there is no need for a massive fusion ignition driver.

  5. On the magnetic properties of iron nanostructures fabricated via focused electron beam induced deposition and autocatalytic growth processes.

    Science.gov (United States)

    Tu, F; Drost, M; Vollnhals, F; Späth, A; Carrasco, E; Fink, R H; Marbach, H

    2016-09-01

    We employ Electron beam induced deposition (EBID) in combination with autocatalytic growth (AG) processes to fabricate magnetic nanostructures with controllable shapes and thicknesses. Following this route, different Fe deposits were prepared on silicon nitride membranes under ultra-high vacuum conditions and studied by scanning electron microscopy (SEM) and scanning transmission x-ray microspectroscopy (STXM). The originally deposited Fe nanostructures are composed of pure iron, especially when fabricated via autocatalytic growth processes. Quantitative near-edge x-ray absorption fine structure (NEXAFS) spectroscopy was employed to derive information on the thickness dependent composition. X-ray magnetic circular dichroism (XMCD) in STXM was used to derive the magnetic properties of the EBID prepared structures. STXM and XMCD analysis evinces the existence of a thin iron oxide layer at the deposit-vacuum interface, which is formed during exposure to ambient conditions. We were able to extract magnetic hysteresis loops for individual deposits from XMCD micrographs with varying external magnetic field. Within the investigated thickness range (2-16 nm), the magnetic coercivity, as evaluated from the width of the hysteresis loops, increases with deposit thickness and reaches a maximum value of ∼160 Oe at around 10 nm. In summary, we present a viable technique to fabricate ferromagnetic nanostructures in a controllable way and gain detailed insight into their chemical and magnetic properties. PMID:27454990

  6. On the magnetic properties of iron nanostructures fabricated via focused electron beam induced deposition and autocatalytic growth processes

    Science.gov (United States)

    Tu, F.; Drost, M.; Vollnhals, F.; Späth, A.; Carrasco, E.; Fink, R. H.; Marbach, H.

    2016-09-01

    We employ Electron beam induced deposition (EBID) in combination with autocatalytic growth (AG) processes to fabricate magnetic nanostructures with controllable shapes and thicknesses. Following this route, different Fe deposits were prepared on silicon nitride membranes under ultra-high vacuum conditions and studied by scanning electron microscopy (SEM) and scanning transmission x-ray microspectroscopy (STXM). The originally deposited Fe nanostructures are composed of pure iron, especially when fabricated via autocatalytic growth processes. Quantitative near-edge x-ray absorption fine structure (NEXAFS) spectroscopy was employed to derive information on the thickness dependent composition. X-ray magnetic circular dichroism (XMCD) in STXM was used to derive the magnetic properties of the EBID prepared structures. STXM and XMCD analysis evinces the existence of a thin iron oxide layer at the deposit-vacuum interface, which is formed during exposure to ambient conditions. We were able to extract magnetic hysteresis loops for individual deposits from XMCD micrographs with varying external magnetic field. Within the investigated thickness range (2-16 nm), the magnetic coercivity, as evaluated from the width of the hysteresis loops, increases with deposit thickness and reaches a maximum value of ˜160 Oe at around 10 nm. In summary, we present a viable technique to fabricate ferromagnetic nanostructures in a controllable way and gain detailed insight into their chemical and magnetic properties.

  7. 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

  8. 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.

  9. 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.

  10. Establishment and application of loop-mediated isothermal amplification method for rapid detection of Campylobacter jejuni%空肠弯曲杆菌LAMP检测方法的建立及初步应用

    Institute of Scientific and Technical Information of China (English)

    唐梦君; 周生; 张小燕; 唐修君; 顾荣; 高玉时

    2012-01-01

    In order to establish a method of loop-mediated isothermal amplification (LAMP) for detection of Campylobacter jejuni , specific loop-mediated isothermal amplification primers were designed, and a novel and highly specific loop-mediated isothermal amplification assay for the sensitive and rapid detection of Campylobacter jejuni were developed. According to Campylobacter jejuni gyrA gene sequences published on GenBank, the natural infection rate of Campylobacter jejuni was tested in ten chicken breeds. The results show that the products of LAMP demonstrated the typical ladder patterns and digested fragments were found with the predicted sizes. Sensitivity of the LAMP assay for direct detection of Campylobacter jejuni in pure cultures was 20 cfu/mL. It was 10-fold more sensitive than that of the conventional PCR assay. The assay identified Campylobacter jejuni correctly, but did not detect 7 non-Campylobacter jejuni strains. The natural infection rate of Campylobacter jejuni was 6%-90% in the ten chicken breeds. The LAMP assay is a sensitive, rapid and simple tool for the detection of Campylobacter jejuni and will play a role in clinical detection.%目的 建立一种灵敏的环介导等温扩增方法(Loop-mediated Isothermal Amplification,LAMP)用于空肠弯曲杆菌(Campylobacter jejuni)的快速检测.方法 根据已公布的空肠弯曲杆菌旋转酶基因(gyrA)设计引物,建立并优化LAMP反应体系,对检测方法的特异性和灵敏度进行验证,并运用该方法对我国不同地方鸡种空肠弯曲杆菌的携带率进行调查.结果 该方法能扩增出典型的梯形条带,且扩增产物的酶切鉴定结果与理论值相符;对单增李斯特菌等8株实验菌株进行检测,仅空肠弯曲杆菌的LAMP结果为阳性;该方法检测空肠弯曲杆菌纯培养物的灵敏度为20 cfu/mL,高于常规PCR;不同地方鸡种空肠弯曲杆菌携带率介于6%~90%之间,差异显著.结论 本试验建立的空肠弯曲杆菌LAMP检测

  11. Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in Guinea

    Science.gov (United States)

    Kurosaki, Yohei; Magassouba, N’Faly; Oloniniyi, Olamide K.; Cherif, Mahamoud S.; Sakabe, Saori; Takada, Ayato; Hirayama, Kenji; Yasuda, Jiro

    2016-01-01

    Given the current absence of specific drugs or vaccines for Ebola virus disease (EVD), rapid, sensitive, and reliable diagnostic methods are required to stem the transmission chain of the disease. We have developed a rapid detection assay for Zaire ebolavirus based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and coupled with a novel portable isothermal amplification and detection platform. The RT-LAMP assay is based on primer sets that target the untranscribed trailer region or nucleoprotein coding region of the viral RNA. The test could specifically detect viral RNAs of Central and West African Ebola virus strains within 15 minutes with no cross-reactivity to other hemorrhagic fever viruses and arboviruses, which cause febrile disease. The assay was evaluated using a total of 100 clinical specimens (serum, n = 44; oral swab, n = 56) collected from suspected EVD cases in Guinea. The specificity of this diagnostic test was 100% for both primer sets, while the sensitivity was 100% and 97.9% for the trailer and nucleoprotein primer sets, respectively, compared with a reference standard RT-PCR test. These observations suggest that our diagnostic assay is useful for identifying EVD cases, especially in the field or in settings with insufficient infrastructure. PMID:26900929

  12. Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in Guinea.

    Science.gov (United States)

    Kurosaki, Yohei; Magassouba, N'Faly; Oloniniyi, Olamide K; Cherif, Mahamoud S; Sakabe, Saori; Takada, Ayato; Hirayama, Kenji; Yasuda, Jiro

    2016-02-01

    Given the current absence of specific drugs or vaccines for Ebola virus disease (EVD), rapid, sensitive, and reliable diagnostic methods are required to stem the transmission chain of the disease. We have developed a rapid detection assay for Zaire ebolavirus based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and coupled with a novel portable isothermal amplification and detection platform. The RT-LAMP assay is based on primer sets that target the untranscribed trailer region or nucleoprotein coding region of the viral RNA. The test could specifically detect viral RNAs of Central and West African Ebola virus strains within 15 minutes with no cross-reactivity to other hemorrhagic fever viruses and arboviruses, which cause febrile disease. The assay was evaluated using a total of 100 clinical specimens (serum, n = 44; oral swab, n = 56) collected from suspected EVD cases in Guinea. The specificity of this diagnostic test was 100% for both primer sets, while the sensitivity was 100% and 97.9% for the trailer and nucleoprotein primer sets, respectively, compared with a reference standard RT-PCR test. These observations suggest that our diagnostic assay is useful for identifying EVD cases, especially in the field or in settings with insufficient infrastructure.

  13. An ultrasensitive chemiluminescence biosensor for carcinoembryonic antigen based on autocatalytic enlargement of immunogold nanoprobes.

    Science.gov (United States)

    Hao, Minjia; Ma, Zhanfang

    2012-01-01

    A sensitive flow injection chemiluminescence assay for carcinoembryonic antigen (CEA) detection based on signal amplification with gold nanoparticles (NPs) is reported in the present work. The sandwich system of CEA/anti-CEA/goat-anti-mouse IgG functionalized Au nanoparticles was used as the sensing platform. In order to improve detection sensitivity, a further gold enlargement step was developed based on the autocatalytic Au deposition of gold nanoprobes via the reduction of AuCl(4)- to Au0 on their surface in the presence of NH(2)OH·HCl. AuCl(4)-, which is a soluble product of gold nanoprobes, served as an analyte in the CL reaction for the indirect measurement of CEA. Under optimized conditions, the CL intensity of the system was linearly related to the logarithm of CEA concentration in the range of 100 pg∙mL-1 to 1,000 ng∙mL-1, with a detection limit of 20 pg∙mL-1. PMID:23443399

  14. An Ultrasensitive Chemiluminescence Biosensor for Carcinoembryonic Antigen Based on Autocatalytic Enlargement of Immunogold Nanoprobes

    Directory of Open Access Journals (Sweden)

    Minjia Hao

    2012-12-01

    Full Text Available A sensitive flow injection chemiluminescence assay for carcinoembryonic antigen (CEA detection based on signal amplification with gold nanoparticles (NPs is reported in the present work. The sandwich system of CEA/anti-CEA/goat-anti-mouse IgG functionalized Au nanoparticles was used as the sensing platform. In order to improve detection sensitivity, a further gold enlargement step was developed based on the autocatalytic Au deposition of gold nanoprobes via the reduction of AuCl4− to Au0 on their surface in the presence of NH2OH·HCl. AuCl4−, which is a soluble product of gold nanoprobes, served as an analyte in the CL reaction for the indirect measurement of CEA. Under optimized conditions, the CL intensity of the system was linearly related to the logarithm of CEA concentration in the range of 100 pg∙mL−1 to 1,000 ng∙mL−1, with a detection limit of 20 pg∙mL−1.

  15. Structure and selection in an autocatalytic binary polymer model

    DEFF Research Database (Denmark)

    Tanaka, Shinpei; Fellermann, Harold; Rasmussen, Steen

    2014-01-01

    An autocatalytic binary polymer system is studied as an abstract model for a chemical reaction network capable to evolve. Due to autocatalysis, long polymers appear spontaneously and their concentration is shown to be maintained at the same level as that of monomers. When the reaction starts from....... Stability, fluctuations, and dynamic selection mechanisms are investigated for the involved self-organizing processes. Copyright (C) EPLA, 2014...... a pool of monomers, highly ordered populations with particular sequence patterns are dynamically selected out of a vast number of possible states. The interplay between the selected microscopic sequence patterns and the macroscopic cooperative structures is examined both analytically and in simulation...

  16. Development of a Real-time Turbidimeter-based Loop-mediated Isothermal Amplification Assay for Detection of Transgenic Soybean%应用LAMP实时浊度法检测转基因大豆

    Institute of Scientific and Technical Information of China (English)

    袁瑛娜; 单潇潇; 王宗德; 石磊; 袁秀金; 谭贵良

    2011-01-01

    Loop-mediated isothermal amplification method (LAMP) is a novel nucleic acid ampli- fication technology. The LAMP method amplifies DNA with rapidity, high specificity and sensitivity under isothermal conditions. Since turbidity of the reaction mixture would increase in correlation with the DNA yield, real-time monitoring of the LAMP reaction was achieved by real-time turbidimeter. Enolpyruvl Shimimate phosphate syntheses gene was amplified by a set of four specially primers that recognize six distinct sequences of the target. The amplification can be obtained in 1 h by incubating all of the reagents in a single tube by real-time turbidimeter at 63 ℃. Results from this study showed that the LAMP method was an effective method for the rapid detection of Transgenic Soybean and their test results were consistent with the results of conventional PCR methods. LAMP assay results were found to be 10 times more sensitive than the conventional PCR. The LAMP detection method was specific, stable and reliable, and will be an effective tool for rapid detection of Transgenic Soybean.%DNA环介导等温扩增(Loop Mediated Isothermal Amplification,LAMP)方法是一种新型的核酸扩增检测方法,该方法操作简便、所需时间短、灵敏度高、特异性强.实时浊度法可以实时检测反应过程中所产生的白色沉淀,从而实现对LAMP整个反应过程的实时监控.本研究以抗草甘膦转基因大豆为研究对象,针对外源基因cp4-epsps的保守区域设计特异性引物,通过实时浊度法在63℃恒温条件下完成转基因大豆的检测工作.结果显示,LAMP实时浊度法能够特异性检测cp4-epsps基因,其检测灵敏度是常规定性PCR方法的10倍.本研究建立了针对转基因大豆cp4-epsps基因的LAMP实时浊度检测方法,该方法具有高度的稳定性与特异性,结果准确,适合于转基因抗草甘膦大豆的快速检测.

  17. Rapid detection of Typhoid Bacilus by loop-mediated isothemal amplification assay%利用环介导恒温扩增法快速检测伤寒杆菌

    Institute of Scientific and Technical Information of China (English)

    刘琳琳; 蒋栋能; 蒲晓允

    2013-01-01

    Objective Use loop mediated isothermal amplification (LAMP) technology to design method for rapid Salmonella typhi detection,and study the reaction's characteristics in order to apply to the clinical inspection. Methods (l)By gene alignment and primer design,build LAMP detection method for Salmonella typhi. (2) According to the amplification by LAMP method for the detection of Salmonella typhi and other interference bacteria,its specific assessment was made. (3)Detection of the LAMP method for different concentrations of Salmonella typhi amplification,observe the minimum detection limit,and sensitivity assessment was made. Results The LAMP method only worked for Salmonella typhi, other interference bacteria were not amplificated, showing good specificity. The minimum detection limit of the method of Salmonella typhi was 1 × 101 CFU/mL,the sensitivity was good. Conclusion The research designed a LAMP method for Salmonella typhi detection, the LAMP method has good specificity and high sensitivity,and can be used for Salmonella typhi's clinical inspection.%目的 利用环介导恒温扩增技术(LAMP),设计快速检测伤寒杆菌的方法,研究其反应特性,以期能应用于临床现场检验.方法 (1)通过基因比对与引物设计,建立针对伤寒杆菌的LAMP检测方法.(2)根据该方法对伤寒杆菌及其他干扰菌进行检测时的扩增情况,对其特异性进行评价.(3)根据该LAMP方法对不同浓度伤寒杆菌的扩增情况,得出其最低检测限,对其灵敏度进行评价.结果 该LAMP检测方法只针对伤寒杆菌有扩增,对其他干扰菌不扩增,显示出良好的特异性.该方法对伤寒杆菌的最低检测限为1×101 CFU/mL,其灵敏度较高.结论 本试验设计出了针对伤寒杆菌的LAMP检测方法,该LAMP检测方法具有良好的特异性和较高的灵敏度,能够用于伤寒杆菌的临床快速检测.

  18. Preliminary application and evaluation of loop-mediated isothermal amplification (LAMP for detection of bovine theileriosis and trypanosomosis in Tanzania : research communication

    Directory of Open Access Journals (Sweden)

    O.M.M. Thekisoe

    2007-09-01

    Full Text Available The sensitivity of LAMP, PCR and microscopy to detect Theileria spp. and Trypanosoma congolense in field-derived bovine blood samples from Tanzania was evaluated and compared. No parasites were detected by microscopy. Furthermore, no bovine Theileria spp. were detected by LAMP and PCR from all the 24 samples collected from Arusha. Four and one out of 24 samples were positive for Theileria congolense infection by LAMP and PCR respectively while, 18 and nine out of 40 samples from Dar es Salaam were positive by LAMP and PCR for Theileria spp. Infection, respectively. Although all samples from Dar es Salaam were negative for Trypanosoma congolense infections by PCR, 12 out of 40 samples were LAMP positive. Whilst PCR is an established gene amplification method for the detection of Theileria and trypanosome parasites, this study introduces LAMP as an alternative molecular diagnostic tool that could be used in large-scale epidemiological surveys.

  19. Detection of Haemophilus parasuis isolates from South China by loop-mediated isothermal amplification and isolate characterisation

    OpenAIRE

    Jian-min Zhang; Hai-yan Shen; Ming Liao; Tao Ren; Li-li Guo; Cheng-gang Xu; Sai-xiang Feng; Hui-ying Fan; Jing-yi Li; Ji-dang Chen; Bin Zhang,

    2012-01-01

    Haemophilus parasuis is the etiological agent of Glässer’s disease, which is characterised by fibrinous polyserositis, meningitis and polyarthritis, causing severe economic losses to the swine industry. In this study, a loop-mediated isothermal amplification (LAMP) test was developed to improve the specificity, facility and speed of diagnosis of H. parasuis isolates. The LAMP assay rapidly amplified the target gene within 50 min incubation at 63 °C in a laboratory water bath. The LAMP amplicon...

  20. 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.

  1. Development of a Reverse Transcription Loop-mediated Isothermal Amplification Assay for Visual Detection of Chicken Infectious Anemia%鸡传染性贫血病毒病LAMP快速可视化检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    邓显文; 谢芝勋; 谢志勤; 刘加波; 庞耀珊; 谢丽基; 彭宜; 范晴

    2011-01-01

    为建立一种能快速检测鸡传染性贫血病毒病(CIAV)的检测方法,根据基因库中鸡传染性贫血病毒病的保守序列,设计一套特异性环介导等温扩增(LAMP)引物,建立了CIAV的LAMP可视化检测方法.该法敏感性可达10 fg,高于常规PCR方法10倍;全部反应可在1h内完成;可通过肉眼观察颜色直接判定结果;对其它鸡常见病原体的检测结果均为阴性.结果表明建立的LAMP方法简便、快速、灵敏、特异,可用于CIAV感染的快速检测.%A loop-mediated isothermal amplification (LAMP) assay was developed for detection of chicken infectious anemia(CIAV). According to the sequences of CIAV in GenBank, six primers were designed, and the reaction conditions were optimized. The results showed that the detection limit of this LAMP method was 10 fg, which was higher than the routine PCR. The amplification could be finished within 1h, and the presence of CIAV could be detected by naked eyes. There was no cross-reactivity with the other pathogen of chiken. These results suggest that this LAMP assay is a simple and specific method for rapid detection of CIAV in clinical samples.

  2. 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.

  3. Loop-Mediated Isothermal Amplification Method and Its Application in Pathogen Inspection%环介导等温扩增技术在病原菌检测中的应用

    Institute of Scientific and Technical Information of China (English)

    刘芳; 黄静敏; 何永盛; 李传礼

    2013-01-01

    Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering a rapid, accurate, and cost-effective diagnosis of infectious diseases. This technology has been developed for a variety of pathogens, including bacteria and viruses. The current focus on LAMP methodology is to explore LAMP as a diagnostic system in resource-limited laboratories in developing countries, where many fatal tropical diseases are endemic. The combination of LAMP and novel microfluidic technologies may facilitate the realization of genetic point-of-care testing systems to be used in the near future. This review described the historical, current, and future developments of such technologies.%环介导等温扩增是一种新的核酸扩增技术,具有简单、快速、特异性强的特点。针对多种食源性致病菌和病毒的环介导等温扩增检测方法已建立了起来,现在主要是发展成为发展中国家一些高发传染病的快速诊断方法,今后环介导等温扩增和微流体等技术的联用有望推动基因床边诊断的实现。本文针对环介导等温扩增技术过去、现在的应用和未来的发展方向展开了论述。

  4. 罗非鱼无乳链球菌LAMP快速检测方法的建立%Development of loop-mediated isothermal amplification for detection of Streptococcus agalactiae from Tilapia

    Institute of Scientific and Technical Information of China (English)

    郑磊; 樊海平; 吴斌; 曾占壮; 钟全福; 张新艳

    2015-01-01

    针对无乳链球菌高度保守的表面免疫相关蛋白基因,采用环介导等温扩增技术( LAMP),设计4条特异性引物,优化反应体系,建立罗非鱼无乳链球菌快速诊断方法。研究结果表明, LAMP方法检测灵敏度达到30 CFU· mL-1,且对2株罗非鱼源无乳链球菌均能扩增出特异性片段,而对14株非无乳链球菌均未扩增出相应片段。建立的LAMP检测方法具有高度的特异性和灵敏性,反应在1 h内完成,为罗非鱼无乳链球菌病的快速诊断提供了一种重要的技术手段。%To develop a technique for rapid detection of Streptococcus agalactiae from Tilapia, the loop-mediated isothermal amplification( LAMP) was introduced to amplify the surface immunogenic protein ( SIP) gene.The detection method was established by using optimum reaction system with four specif-ic primers of the target gene.The results showed that the method had perfect specificity, and the sen-sitivity was 30 CFU · mL-1 , as the DNA of 2 strains of Streptococcus agalactiae were able to be amplified, while the other 14 strains of non -Streptococcus agalactiae were not.The amplification process can be finished in 1 hour.The method provides a better choice for rapid detection of Strepto-coccus agalactiae.

  5. 检测鸡蛋中沙门氏菌的LAMP方法的建立及初步应用%Loop-mediated isothermal amplification assay for rapid detection of Salmonella spp.

    Institute of Scientific and Technical Information of China (English)

    唐梦君; 周生; 张小燕; 顾荣; 蒲俊华; 葛庆联; 高玉时

    2011-01-01

    Salmonella spp. is an important kind of food-borne pathogenic bacteria which can cause human and animal disease. Salmonella invA gene is known as a major virulence gene. According to published Salmonella invA gene sequence in GenBank, we designed one sets of specific loop-mediated isothermal amplification primers and developed a novel and highly specific loop-mediated isothermal amplification assay for the sensitive and rapid detection of Salmonella spp. The assay correctly identified Salmonella spp., but did not detect 7 non-Salmonella spp. strains. Sensitivity of the LAMP asssay for direct detection of Salmonella spp. in pure cultures was 1.04×102 cfu·mL-1. The results of LAMP detection of 100 eggs were consistent with the traditional method. The LAMP assay is a sensitive, rapid and simple tool for the detection of Salmonella spp. and will facilitate the surveillance for control of contamination of Salmonella spp. in egg.%根据GenBank上公布的沙门氏菌invA基因序列中的保守区域,设计一套环介导等温扩增(LAMP)引物,将其用于沙门氏菌的检测,结果成功地扩增出特异性的梯形条带.LAMP方法检测沙门氏菌纯培养的灵敏度为1.04×102 cfu·mL-1,对11株不同细菌进行LAMP检测,仅沙门氏菌获得阳性结果.应用该方法对扬州市场上销售的鸡蛋进行沙门氏菌检测,检测结果与传统培养方法相符合.因此,LAMP方法检测沙门氏菌具有灵敏度高,特异性强,耗时短,方法简便等特点,有望发展成为快速检测鸡蛋中沙门氏菌的有效手段.

  6. Development of Loop-Mediate Isothermal Amplification Assay for Salmonella Detection in Food%食品中沙门氏菌LAMP快速检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    黄金海; 孙跃辉; 陈瑞; 庄世文; 刘莹; 王静思

    2012-01-01

    It is important to detect Salmonella in food or food materials in order to assure the food safety. A rapid sensitive loop-mediated isothermal amplification (LAMP) method assay for the food-borne pathogen Salmonella detection was developed. A set of primers were designed according to the nucleotide sequence of the target invA gene in Salmonella, 7 strains of Salmonella and 4 non--Salmonella bacteria were detected by LAMP, and meanwhile the mode of artificially contaminated food was constructed to evaluate the sensitivity of LAMP assay and bacterial cell counting method. The results showed that all the 7 Salmonella bacterial strains had specific amplification, but the 4 non-Salmonella bacterial strains submitted negative reactions. Sensitivity of LAMP assay for pure cell culture of Salmonella was 336 mL-1. However, an 8.25 g-1 detection limit was obtained for Salmonella artificially contaminated food following a bacterial culture enrichment process, while there was no amplification from the negative control. In conclusion, the LAMP assay developed in the present study is a specific, sensitive, simple and convenient method for the rapid detection of Salmonella in contaminated foods.%食品及其原料中沙门氏茵的快速、现场检测对食品安全控制具有重要意义.根据沙门氏茵invA基因核苷酸序列设计一组引物,应用环介导等温扩增技术(LAMP),分别对7种不同血清型沙门氏茵和4种非沙门氏茵进行扩增,同时建立沙门氏茵人工污染的食品模型,比较了LAMP法与活茵计数检测的敏感性.结果表明,该方法仅对沙门氏菌产生特异性扩增,灵敏度高达336mL-1,食品样品经细菌富集培养后,检测灵敏度高达8.25g-1.所建立的检测沙门氏茵方法具有较高的特异性与灵敏性,操作简单、快速,可用于沙门氏茵污染食品的快速检测.

  7. Loop-mediated isothermal amplification as a good tool to study changing Leptosphaeria populations in oilseed rape plants and air samples

    Directory of Open Access Journals (Sweden)

    Małgorzata Jędryczka

    2014-01-01

    Full Text Available LAMP is an innovative, simple, rapid, specific and cost-effective nucleic acid amplification method. Due to the use of a special enzyme – GspSSD polymerase, the reaction takes a short time and can be performed at isothermal conditions. The sensitivity and specificity of LAMP technique is significantly higher, than standard PCR techniques, as two or three specific primer pairs are used. The technique is regarded as a useful tool for the detection and identification of plant pathogens. In this work, LAMP was used to study the composition of the population of fungi of the genus Leptosphaeria, causing a damaging disease of oilseed rape, called blackleg or stem canker. The detection concerned DNA present in fungal spores contained in air samples obtained using Hirst-type volumetric trap, in Pomerania (north Poland in 2010. The results achieved using the LAMP technique were similar to these obtained with previously used, highly specific method of Real-time PCR. Conducting LAMP reaction was much easier and less time-and cost-consuming, due to a simplified method of DNA isolation of pathogens from plant tissues. Then, the LAMP technique was used to assess the composition of the population of Leptosphaeria spp. in plants of oil- seed rape collected from the field in the Opole region (south-we- stern part of Poland in 2013. In contrast to studies conducted in 2002–2003, the analysis of leaf symptoms showed a higher pro- portion of L. maculans compared to L. biglobosa, what reflects changes in the composition of pathogen population of fungi causing blackleg on oilseed rape in this part of Poland.

  8. 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.

  9. The origin of large molecules in primordial autocatalytic reaction networks

    CERN Document Server

    Giri, Varun

    2011-01-01

    Large molecules such as proteins and nucleic acids are crucial for life, yet their primordial origin remains a major puzzle. The production of large molecules, as we know it today, requires good catalysts, and the only good catalysts we know that can accomplish this task consist of large molecules. Thus the origin of large molecules is a chicken and egg problem in chemistry. Here we present a mechanism, based on autocatalytic sets (ACSs), that is a possible solution to this problem. We discuss a mathematical model describing the population dynamics of molecules in a stylized but prebiotically plausible chemistry. Large molecules can be produced in this chemistry by the coalescing of smaller ones, with the smallest molecules, the `food set', being buffered. Some of the reactions can be catalyzed by molecules within the chemistry with varying catalytic strengths. Normally the concentrations of large molecules in such a scenario are very small, diminishing exponentially with their size. ACSs, if present in the c...

  10. Weber's law for biological responses in autocatalytic networks of chemical reactions.

    Science.gov (United States)

    Inoue, Masayo; Kaneko, Kunihiko

    2011-07-22

    Biological responses often obey Weber's law, according to which the magnitude of the response depends only on the fold change in the external input. In this study, we demonstrate that a system involving a simple autocatalytic reaction shows such a response when a chemical is slowly synthesized by the reaction from a faster influx process. We also show that an autocatalytic reaction process occurring in series or in parallel can obey Weber's law with an oscillatory adaptive response. Considering the simplicity and ubiquity of the autocatalytic process, our proposed mechanism is thought to be commonly observed in biological reactions. PMID:21867048

  11. Establishment of a rapid detection method for Brucella spp by loop-mediated isothermal amplification%布鲁杆菌环介导等温扩增快速检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    李秀梅; 郭恋; 郭立力; 李颖; 石瑜; 杨爱华; 王健春; 王红军

    2013-01-01

    A loop-mediated isothermal amplification(LAMP) method for rapidly detecting Brucella spp was established using two sets of Brucella-specific primers according to the species-specific OMP25 gene and optimizing the reaction condition. The results of specificity and sensitivity tests showed that the method was able to detect Brucella spp specifically and the limit of detection was as low as 5 copies/μL. The amplified products were judged by agarose gel electrophoresis and chro-mogenic reaction. The LAMP method is suitable for field detection.%研究针对布鲁杆菌保守的OMP25基因设计了4条LAMP的特异性引物,通过优化反应条件,建立了布鲁杆菌的LAMP快速检测方法.特异性和敏感性试验结果显示,该方法能够特异地检测布鲁杆菌,其最低检测限为5 copies/μL.扩增产物可通过琼脂糖凝胶电泳、显色反应进行判定.LAMP可以快速、直观、准确地检测布鲁杆菌,是一种适合基层现场应用的检测方法.

  12. 环介导等温扩增技术检测肉及肉制品中的沙门氏菌%Loop-mediated Isothermal Amplification Assay for the Detection of Salmonella in Meat and Meat Products

    Institute of Scientific and Technical Information of China (English)

    王羽; 王贞强; 张伟

    2010-01-01

    建立用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)检测肉及肉制品中沙门氏菌的方法.利用靶基因的6个区段设计4条引物,应用具有链置换活性的Bst DNA聚合酶,能够在恒温(65℃左右)条件下特异、高效、快速地扩增待测物的DNA.针对沙门氏菌的特异基因invA基因设计两对LAMP引物,建立LAMP检测沙门氏菌的新方法,并对肉及肉制品中的沙门氏菌进行检测.结果表明:建立LAMP技术检测肉及肉制品中沙门氏菌的方法,该方法能够特异检测沙门氏菌,且灵敏性可达10-9CFU/mL,对48份样品检测,该方法检出率为91.6%、敏感性100%、特异性70.0%、符合率为93.8%、检出时间1.5h.

  13. Development of an allele-specific, loop-mediated, isothermal amplification method (AS-LAMP to detect the L1014F kdr-w mutation in Anopheles gambiae s. l.

    Directory of Open Access Journals (Sweden)

    Badolo Athanase

    2012-07-01

    Full Text Available Abstract Background Malaria control relies heavily on treated bed nets and indoor residual spraying with pyrethroid insecticides. Unfortunately, the resistance to pyrethroid insecticides, mainly due to the kdr mutation, is spreading in the main malaria vector Anopheles gambiae s.l., decreasing the insecticides’ efficacy. To manage the insecticide resistance rapidly and flexibly, simple and effective tools for the early detection of resistant mosquitoes are needed. This study aimed to develop an allele-specific, loop-mediated, isothermal amplification (AS-LAMP method to detect the West African-type kdr mutation (kdr-w; L1014F in field-collected mosquitoes. Methods DNA fragments of the wild-type and the mutated kdr gene were used to select the primers and develop the method. The primers were designed with the mutation at the 5’ end of the backward inner primer (BIP. The AS-LAMP method was compared to the AS-PCR method using the genomic DNA of 120 field-collected mosquitoes. Results The AS-LAMP method could discriminate between the wild-type homozygote, the heterozygote, and the kdr-w homozygote within 75 min. The AS-LAMP method has the advantage of being faster and at least as sensitive and specific as the AS-PCR method. Conclusions The AS-LAMP method can be used to detect the kdr mutation for quick decision-making, even in less well-equipped laboratories.

  14. Meat species authenticity identification by loop-mediated isothermal amplification assay in beef and mutton%环介导恒温扩增法鉴定牛羊肉中的搀杂肉

    Institute of Scientific and Technical Information of China (English)

    侯东军; 杨红菊; 于雷; 李颖; 王海; 姜艳彬

    2012-01-01

    A loop-mediated isothermal amplification(Lamp) method was developed for meat ingredients authenticity identification in beef and mutton. A set of universal primer used for Lamp at 63℃ was chosen to differentiate animal gene encoding cytochrome b with high sensitivity. 0.01%~2% pork could be detected in beef and mutton by the method. Besides,the method could be used to detect animal meats processed or unprocessed.%建立了一个鉴定牛羊肉中搀杂杂动物肉的环介导恒温扩增检测方法。确定了一套可在牛羊肉中特异并灵敏地检测出搀杂肉成分的引物对,以动物细胞色素b基因组为模板可在恒温63℃恒温特异性扩增出猪等基因片段而无其他扩增片段影响。可检测牛肉中0.01%~2%的猪肉成分。经与PCR方法比对,结果表明,该方法可有效用于实际生鲜肉或加工肉制品样本的鉴定。

  15. 快速检测单核细胞增生李斯特菌LAMP方法的建立%To develop a loop-mediated isothermal amplification assay forquick detection of Listeria monocytogenes

    Institute of Scientific and Technical Information of China (English)

    姚栋; 张如胜; 欧新华; 宋克云

    2012-01-01

    Objective: To establish a loop-mediated isothermal amplification( LAMP ) assay for the rapid and specific detection of Listeria monocy to genes. Methods: Four primers regions on the hlyA gene of Listeria monocytogenes were designed and used for LAMP assay. Listeria monocytogenes DNA was amplified under i-sothermal conditions! 65°C )for 60 min in water bath, then the amplified product was judged by naked eye, SYBR Green 1 staining, and electrophoresis analysis. To evaluate the specificity of the assay, 1 strain of Listeria monocytogenes and 10 strains of none -Listeria monocytogenes were tested by LAMP and conventional PCR assay. In addition, the detection limit of LAMP was compared with that of PCR by using the Listeria monocytogenes strain,that were serially diluted and were amplified by LAMP and PCR. Results: With one strain of Listeria monocytogenes, observation with naked eyes, SYBR Green I staining and electrophoretic a-nalysis were able to detect the products in the LAMP assay, and amplification were not observed when 10 strains of non-Listeria monocytogenes were tested. The sensitivity of LAMP was higher than that of PCR assay. The detection limit of LAMP assay for Listeria monocytogenes was 3 cfu/ml and that of PCR was > 300 cfu/ml. LAMP method was superior to conventional PCR for its rapid detection of Listeria monocytogenes, which can complete in 60 min. Conclusion: LAMP for rapid and specific detection of Listeria monocytogenes was established in this study.%目的:建立一种检测单核细胞增生李斯特菌核酸的快速、特异的环介导等温扩增(Loop-mediated isothermal amplification,LAMP)方法.方法:针对单核细胞增生李斯特菌的李斯特菌溶血素hlyA基因设计4条LAMP引物,在水浴箱内65 ℃保温约60 min,完成对单核细胞增生李斯特菌的扩增,扩增产物经肉眼、SYBR Green I染色和电泳鉴定.利用LAMP和PCR方法同时检测1株单核细胞增生李斯特菌和10株非单核细胞增生李斯特

  16. Rapid detection of Orientia tsutsugamushi causing scrub typhus using a loop-mediated isothermal amplification assay%恙虫病东方体环介导恒温扩增可视化快检方法的建立

    Institute of Scientific and Technical Information of China (English)

    耿美玲; 操敏; 张锦海; 吕恒; 胡丹; 郑峰; 朱进; 潘秀珍; 王长军

    2013-01-01

    Objective A loop-mediated isothermal amplification (LAMP) assay was established for rapid detection of Orientia tsutsugamushi in rural areas and/or endemic foci of scrub typhus.Methods Two sets of LAMP primers targeting the 56 kd outer membrane protein gene of O.tsutsugamushi were designed.The more effective set was selected and reaction conditions were optimized.Evaluation included testing the standard O.tsutsugamushi strains from chick embryo cultures and O.tsutsugamushi isolates from animal organs and patient blood samples.Specificity was evaluated using five members of the genus Rickettsia,which are closely related to O.tsutsugamushi.The detection limit for LAMP was assessed via serial dilution using the same target gene and results were compared to those of conventional PCR.Results LAMP rapidly detected (within an hour) the 56 kd gene specific to O.tsutsugamushi in various types of samples,providing results that could be observed visually or based on turbidity.No corresponding amplification was noted when using DNA of the five rickettsiae as a template,indicating that LAMP was highly specific.The detection limit for LAMP was 5.3 × 101 copies while it was 5.3 × 104 copies for conventional PCR,indicating a 3-fold increase.Conclusion LAMP proved to be a simple,rapid,sensitive,and specific way to detect O.tsutsugamushi and may be a useful way to diagnose scrub typhus in poor rural areas.%目的 建立快速检测恙虫病东方体(Orientia tsutsugamushi,Ot)的环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP),以期用于基层医院及疫区恙虫病快速筛查. 方法 以恙虫病东方体56 ku外膜蛋白基因为检测靶序列,设计2组恒温扩增引物,筛选最佳引物,优化反应条件,对多种培养物中的Ot标准株及地方株进行验证检测,以经典PCR检测为对照,评估LAMP检测方法的敏感性和特异性. 结果 建立的LAMP法可快速检测鸡胚培养物、动物脏器及病人血样中的Ot DNA,经63

  17. Sliding wear performance of electroplated hard chromium and autocatalytic nickel-phosphorus coatings at elevated temperatures

    OpenAIRE

    Eriksson, Mats

    2014-01-01

    This thesis was written for a Swedish valve manufacturer to find out in what temperature regimes it was possible to replace electroplated hard chromium with autocatalytic electroless nickel-phosphorus. In this work the dry sliding wear properties of electroplated hard chromium and autocatalytic electroless nickel-phosphorus(10% P) were compared. All tests and investigations were done by using available equipment at Karlstads University. The tests were made to find out how the wear of these co...

  18. Development of Visual Loop-Mediate Isothermal Amplification Assay for CpTI gene%抗虫转CpTI基因成分现场可视化检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    王永; 兰青阔; 朱珠; 赵新; 陈锐; 刘娜; 李欧静; 郭永泽; 程奕

    2014-01-01

    It is important to detect Cowpea Trypsin Inhibitor (CpTI) gene in food or feed materials in order to prevent the illegal diffusion. A rapid visual loop-mediated isothermal amplification (LAMP) method assay for CpTI gene detection was developed. A set of primers were designed according to the nucleotide sequence of the target CpTI gene, seven genetically modified organisms (GMO) were detected by LAMP for method specificity, and meanwhile the mode of artificially contaminated food was constructed to evaluate the sensitivity of LAMP assay and qualitative PCR method. The results showed that the CpTI gene had specific amplification, but the non- CpTI GMO submitted negative reactions. Sensitivity of LAMP assay for Kefeng No.6 genetically modified rice was 0.005%. In conclusion, the LAMP assay developed in the present study is a specific, sensitive, simple and convenient method for the rapid screening of CpTI gene in contaminated foods.%豇豆胰蛋白酶抑制剂(CpTI)基因是目前仅次于Bt基因的广谱性抗虫基因,常用于转基因水稻和转基因棉花的基因工程研究中。根据CpTI基因特异性碱基序列设计引物,应用可视化环介导等温扩增技术,对转基因水稻科丰6号进行扩增,同时建立转基因成分人工污染的食品模型,比较了 LAMP 法与定性PCR方法检测的敏感性。结果表明:该方法仅对CpTI基因产生特异性扩增,灵敏度达0.005%;所建立的CpTI基因现场可视化筛查方法具有较高的特异性与灵敏性,操作简单、快速,可用于含有CpTI基因转基因成分污染的快速检测。

  19. 羊丝状支原体簇环介导等温扩增检测方法的建立%Detection of Mycoplasma mycoides Cluster by Loop-mediated Isothermal Amplification

    Institute of Scientific and Technical Information of China (English)

    谢秀兰; 康晓冬; 储岳峰; 马小明; 岳彩娟

    2012-01-01

    In this study, a sensitive loop-mediated isothermal amplification (LAMP) was developed for rapidly detecting Mycoplasma mycoides cluster. A set of four primers.two outer and two inner primers, were designed from genomic sequences according to the conserved region of the 16S rRNA gene of Mycoplasma mycoides cluster. The reaction was performed in one step in a single tube at 65 ℃ for 60 min, with hydroxynaphthol blue (HNB) dye added prior to amplification. Blue-positive results were significantly different from the purple-negative. The assay could amplify from Mycoplasma mycoides cluster members, Mmc, Mmm LC and Mccp.but no cross-reaction from other bacteria. The sensitivity test showed that it could detect 10 pg/μL DNA from Mmc strain PG3. The study had developed a diagnostic procedure which was rapid and sensitive for Mycoplasma mycoides cluster.%本试验利用环介导等温扩增技术(LAMP)建立了羊丝状支原体簇的快速检测方法,该方法以羊丝状支原体簇成员的保守性基因16S rRNA为靶序列,设计了4条特异性引物,在65℃等温条件下,60 min一步完成反应.在反应管中预先添加羟基萘酚蓝(HNB),阳性呈蓝色,阴性呈紫色.丝状支原体簇成员——丝状支原体山羊亚种(Mmc)、丝状支原体丝状亚种LC型(Mmm LC)、山羊支原体山羊肺炎亚种(Mccp)的LAMP检测为阳性;对其他病原菌没有交叉反应.以Mmc的核酸为模板进行灵敏度检测,LAMP的最低检出限为10 pg/μL.结果表明,本试验建立了一种特异、敏感、快速、简便的羊丝状支原体簇的LAMP方法.

  20. 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.

  1. Development of Loop-mediated Isothermal Amplification for Rapid Detection of Mycoplasma bovis%牛支原体的环介导等温扩增快速检测技术研究

    Institute of Scientific and Technical Information of China (English)

    侯轩; 付萍; 张海燕; 张跃伟; 吴文学

    2012-01-01

    牛支原体(Mycoplasma bovis是感染牛的一种重要的致病性病原体.本研究利用环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术建立了牛支原体的快速检测方法.该方法以牛支原体保守性基因uvrC为靶序列设计了5条特异引物,在58℃等温条件下,60 min即可完成反应.LAMP方法能检测出20 pg牛支原体DNA,较PCR方法高100倍,用牛鼻支原体(M.bovirhinis)、无乳支原体(M.agalactiae)、精氨酸支原体(M.ariginini)、牛副流感病毒(BPIV)、牛腺病毒(BADV)、牛传染性鼻气管炎病毒(IBRV)作特异性检测,两种方法均有很高的特异性.本研究还将荧光显色剂钙黄绿素(calcein)和Mn2+用于LAMP反应结果的可视化检测.临床鼻拭子样品煮沸后,可直接进行等温扩增,省去了DNA提取步骤.在对167个临床鼻拭子样本的检测中,LAMP方法检测结果阳性率(26.95%)高于PCR方法检测出阳性率(19.16%),这证明本研究所建立的方法,与PCR法比较更适于临床样品的检测.研究结果表明,LAMP方法具有操作简便、快速、高效、敏感、特异、经济等特点,适于基层实验室监测的广泛使用.%Mycoplasma bovis is an important pathogen in cattle. In this research we developed a new and rapid test method for Mycoplasma bovis with loop-mediated isothermal amplification(LAMP). With the 5 specific primers targeting to a total of six distinct sequences on the conservative gene wvrC, the new method could be completed in 60 min under the isothermal condition of 58 ℃. With the LAMP method, we could detect 20 pg DNA of M. Bovis, which was 100 times lower than using the method of PCR. In addition, using M. Bovirhinis, M. Agalactiae, M. Arginini, Bovine parainfluenza virus (BPFV), Bovine adenoviruses (BADV) and Infectious Bovine rhinotracheitis virus (IBRV) to determine the specificity of LAMP and PCR methods, both the methods of LAMP and PCR displayed the high specificity results. The research had

  2. Simple diagnosis of Pine Wilt Disease using loop-mediated isothermal amplification%利用环介导恒温扩增法简易诊断松萎蔫病

    Institute of Scientific and Technical Information of China (English)

    Takuya Aikawa; Natsumi Kanzaki; Taisei Kikuchi

    2011-01-01

    诊断松萎蔫病的传统方法是从木质组织中分离松材线虫,然后用显微镜进行形态学鉴定.基于DNA分子检测的方法尽管很灵敏,不需要固定发育阶段的松材线虫,但是仍然需要用Baermann漏斗法分离线虫,并且需要昂贵的仪器.笔者研发了利用环介导恒温扩增法检测松材线虫的存在,该方法包括:(1)从木块中提取DNA;(2) DNA扩增;(3)通过反应溶液的颜色做出诊断.该方法仅使用培养箱且整个过程只需要90 min,比以往方法更加简便和快速.%Pine wilt disease (PWD) , a serious threat to pine forests in East Asia and Europe, is caused by the pinewood nematode, Bursaphelenchus xylophilus. Because the nematodes are transmitted by insect vectors from dead to healthy trees, PWD spreads extremely rapid. Accurate and rapid diagnosis and then complete removal of the nematode-infected trees from pine forests are thus very important for control of PWD. To diagnose PWD, B. Xylophilus must be detected in infected trees. Generally, the nematode is extracted from wood tissues by u-sing the Baermann funnel technique and is then identified under stereo and light microscopes. However, this method requires technical knowledge of nematode morphology and expensive equipment such as microscopes. In addition, nematode extraction takes a long time (usually 1 or 2 days) , and identification of the nematode is possible only when adults ( both male and female) are extracted. Several DNA-based protocols for the identification of B. Xylophilus have recently been developed. Although these molecular techniques are sensitive and independent of the life stage of the nematode, they require both the use of a Baermann funnel to collect the nematodes from the wood tissues and expensive equipment such as a thermocycler or real-time PCR apparatus to amplify the DNA. We developed a simple and low-cost method of determining the presence of PWD by using loop-mediated isothermal amplification ( LAMP

  3. Highly sensitive and visible detection of virus by loop-mediated isothermal amplification%高灵敏度等温扩增法可视化检测血液中的病毒核酸

    Institute of Scientific and Technical Information of China (English)

    梁超; 成思佳; 武海萍; 周国华

    2011-01-01

    目的 探讨便携式、可灵敏、快速检测血液标本中病毒核酸的方法.方法 以HBV病毒为例,针对其基因组保守序列设计特异性引物,采用环介导等温扩增法(LAMP)对待测HBV标本做核酸目标序列扩增,以高灵敏双链DNA嵌合荧光染料为指示剂,根据荧光信号指示扩增反应产物的有无;将本法与实时荧光PCR法对临床标本做HBV检测的对照分析.结果 建立了血液病毒核酸的可视化检测方法,研制出扩增检测一体化仪器;灵敏度达到可在<1h完成10 cp/管的HBV核酸扩增反应和产物检测.62例实时荧光PCR检测为HBV阳性的临床标本,本法亦都检测为阳性;而从42例实时荧光PCR检测为HBV阴性的临床标本中,本法检出了6例HBV阳性.结论 所建立的方法和装置可实现高灵敏度单管快速检测血液病毒核酸,为特殊现场环境中的血液安全性筛查提供了新的手段.%Objective To develop a sensitive,rapid,and convenient method for detection of viral nucleic acid in blood samples,such as HBV. Method The primers were designed according to the conservative regions of HBV genome,and the loop-mediated isothermal amplification ( LAMP) was carried out to amplify the target sequences of HBV. SYBR Green I was used as the indicator for the PCR products. The assay would be scored as positive if a fluorescent signal was detected. Results We have successfully developed a method for visible detection of blood virus and invented a portable device that combined the amplification and detection in one platform. The sensitivity of our proposed method is 10 copies of HBV per tube. The amplification and detection process could be done within 1 hour. In the testing of 62 clinical specimens, which have been determined as HBV positive by quantitative PCR,all specimens were positive and an accuracy of 100 % was a-chieved. We also detected 6 positive specimens from 42 clinical samples which have been determined as HBV negative

  4. 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.

  5. 利用环介导恒温扩增法快速检测空肠弯曲菌%Rapid Detection of Campylobacter Jejuni Basing on Loop-mediated Isothermal Amplification

    Institute of Scientific and Technical Information of China (English)

    刘孝波; 蒋栋能; 蒲晓允

    2013-01-01

    目的 利用环介导恒温扩增技术,以空肠弯曲菌的VS1基因设计引物,建立快速检测空肠弯曲菌的方法,以期能应用于急性腹泻和肠炎等疾病的快速检验.方法 ①通过基因比对与引物设计,设计针对空肠弯曲菌的环介导恒温扩增检测(LAMP)方法;②用钙黄绿素、亚甲基蓝、结晶紫、溴化乙锭4种DNA染料对扩增产物进行染色,评价各染料染色效果;③检测该LAMP方法对空肠弯曲菌及其他干扰菌的扩增情况,评价其特异性;④将该LAMP方法与培养法比较,进行统计学分析.结果 ①设计出针对空肠弯曲菌的LAMP检测方法;②钙黄绿素与扩增产物结合后颜色变化明显,是该LAMP方法中较好的DNA染料物质;③本LAMP检测方法只针对空肠弯曲菌有扩增,对其他干扰菌不扩增,显示出良好的特异性;④试验共检测了60株临床和标准菌株,对于菌株的培养物,LAMP检测结果与培养鉴定法比较差异无统计学意义(x2 =0.3333,P>0.05).结论 本试验设计出了针对空肠弯曲菌LAMP检测方法,该LAMP法较好的DNA染料物质是钙黄绿素,该方法具有良好的特异性,与培养鉴定法比较具有较高的阳性、阴性及总体符合率,能够用于空肠弯曲菌的快速检验.%Objective Basing on the loop-mediated isothermal amplification (LAMP) technology,to design a rapid detection method of Campylobacter jejuni,so as to be applied in clinical inspection of C.jejuni in acute diarrhea and gastroenteritis.Methods By gene alignment and primer design,a set of loop mediated isothermal amplification (LAMP) method for detection of C.jejuni was designed.Four kinds of DNA dye,calcein,methylene blue,crystal violet and EB,were used to stain amplification products,then the dyeing effect could be evaluated.The LAMP method of detecting C.jejuni and other interference bacteria was tested on their specific evaluation.LAMP was Compared with the culture method of identification

  6. 环介导等温扩增快速检测临床假丝酵母菌属方法建立与应用%Establishment and application of loop-mediated isothermal amplification for rapid detection of clinical isolates of Candida spp

    Institute of Scientific and Technical Information of China (English)

    鲁勇; 汪一萍; 应建飞; 俞燕红; 贺明阳

    2015-01-01

    目的:建立一种环介导等温扩增(LAMP)反应,根据假丝酵母菌属 D1~ D226S rDNA 保守区域设计通用引物,直接对临床标本进行假丝酵母菌属检测。方法根据假丝酵母属的特异 D1~ D226S rDNA 基因序列比对后的相对保守区设计特异引物,分别提取5种假丝酵母菌、临床对照菌株以及临床标本 DNA ,然后以 LAMP 技术扩增,并对白色假丝酵母菌孢子悬液进行系列梯度稀释检测,确定最低检测限。结果5种假丝酵母菌直接提取 DNA 经 LAMP 和 PCR 方法检测均为阳性;30份经培养证实假丝酵母菌属感染标本,经 LAMP 检测有27份标本阳性,阳性检出率为90.0%;另10份临床对照标本和11种非假丝酵母菌属来源 DNA 样本经 LAMP 方法检测后结果均为阴性,无非特异性反应发生;LAMP 最低检测限达到103 CFU /ml 。结论结果证明 LAMP 是一种简便、快速、特异和敏感的检测方法,可以检测临床和环境标本中的常见假丝酵母菌属。%OBJECTIVE To establish the loop-mediated isothermal amplification for direct detection of Candida spp in clinical samples by designing universal primers on the basis of conserved region of D ~ D2 26S rDNA of Candida spp .METHODS The specific primers were designed from the relative conservation region of the published sequence of D1-D2 26S rDNA genes of the Candida sp p ,then the genomic DNA was extracted from five species of Candida spp ,clinical control strains ,and clinical specimens and was amplified by using loop-mediated isothermal amplifica-tion ,and the serial dilutions were carried out for the spore suspensions of Candida albicans so as to determine the minimum detection limit .RESULTS The loop-mediated isothermal amplification and PCR assays obtained positive results for the DNA that was directly extracted from the five species of Candida sp p .Of 30 specimens that were proved infected with Candida sp p through culture

  7. 环介导等温扩增技术检测小肠结肠炎耶尔森氏菌的研究%Development of a loop-mediated isothermal amplification forthe detection of Yersinia enterocolitica

    Institute of Scientific and Technical Information of China (English)

    梁磊; 李英军; 孟兆祥; 马晓燕; 张伟

    2011-01-01

    采用环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)快速检测小肠结肠炎耶尔森氏菌(Yersinia enterocolitica)。以小肠结肠炎耶尔森氏菌(AY004311.1)Ail基因序列作为靶序列,设计内、外引物,通过肉眼观察沉淀判断检测结果。结果表明:LAMP检测小肠结肠炎耶尔森氏菌的灵敏度为6.3cfu/mL,人工污染鸡肉的检出限为340cfu/g。PCR检测小肠结肠炎耶尔森氏菌的灵敏度为630cfu/mL,人工污染鸡肉的检出限为3.4×104cfu/g。采用试剂盒法提取DNA,从样品处理到报告结果,LAMP方法耗时2h,PCR方法耗时3h。因此,LAMP检测小肠结肠炎耶尔森氏菌的灵敏度高,耗时短,特异性好,操作简便,无需特殊的仪器设备,适合在我国广大基层实验室开展应用,为快速检测食源性致病菌构建了一个新的技术平台。%A loop-mediated isothermal amplification(LAMP) technology with two loop primers was developed for rapidly detecting Yersinia enterocolitica in chicken. Sequences of Ail gene of Y.e (AY004311.1) were used as target sequences, to design outer primers, inner primers. We judged the results of detection, through visible to the naked eye of the white precipitate. The result indicates: we extracted DNA using the method of Chemical reagent. The detection limit of pure bacterial culture was 6.3 cfu/mL and the detection limit of artificially contaminated chicken was 340 cfu/g with improved LAMP detection for two hours. In contrast, the detection limit of pure bacterial culture was 630 cfu/mL and the detection limit of artificially contaminated chicken was 3.4×104 cfu/g with PCR detection for three hours. Therefore, LAMP has the potential to replace PCR because of its simplicity, rapidity, specificity, no special equipment, and cost-effectiveness. It is very suitable for the majority of the our local laboratory on the application. For the rapid detection of foodborne pathogens build a technology

  8. Modelling of the operational behaviour of passive autocatalytic recombiners

    International Nuclear Information System (INIS)

    Due to severe accidents in nuclear power plants, a significant amount of hydrogen can be produced. In pressurized water reactors, a possible and wide-spread measurement is the use of auto-catalytic recombiners. There are numerous numerical models describing the operational behaviour of recombiners for containment codes. The numerical model REKO-DIREKT was developed at the Forschungszentrum Juelich. This model describes the chemical reaction on the catalytic sheets by a physical model, as opposed to the usual codes based on empirical correlations. Additionally, there have been experimental studies concerning the catalytic recombination of hydrogen since the 1990s. The aim of this work is the further development of the program REKO-DIREKT to an independent recombiner model for severe accident and containment codes. Therefore, the catalyst model already existed has been submitted by a parameter optimization with an experimental database expanded during this work. In addition, a chimney model has been implemented which allows the calculation of the free convection flow through the recombiner housing due to the exothermal reaction. This model has been tested by experimental data gained by a recently built test facility. The complete recombiner model REKO-DIREKT has been validated by data from literature. Another aim of this work is the derivation of the reaction kinetics for recombiner designs regarding future reactor concepts. Therefore, experimental studies both on single catalytic coated meshes as well as on two meshes installed in a row have been performed in laboratory scale. By means of the measured data, a theoretical approach for the determination of the reaction rate has been derived.

  9. Sensitive and rapid detection of Vibrio cholerae O139 by loop-mediated isothermal amplification%应用环介导等温扩增法快速检测O139群霍乱弧菌

    Institute of Scientific and Technical Information of China (English)

    朱水荣; 陈寅; 王志刚; 张政; 朱敏; 卢亦愚

    2009-01-01

    Objective To develop a loop-mediated isothermal amplification method for rapid diag-nosing Vibrio cholerae O139 in inspection department and small-scale laboratory. Methods Four primers (2 inner primer and 2 outer primer) for the LAMP test were designed by targeting the wbfR gene of Vibrio chol-erae O139, and the reaction condition and reaction system of LAMP were optimized. Thirty Vibrio cholerae O139, 13 Vibrio cholerae reference strains, 10 O1 biotype Vibrio cholera* and 32 other enterobacterias were analyzed and the LAMP results were determined by visual inspection or electrophoretie analysis . Results All of the Vibrio cholerae O139's amplification products were observed as green by visual inspection and had a ladder-like pattern on the gel, but O1 biotype Vibrio cholera* and other enterobacteria's products were dis-played as orange by visual examination and had no ladder-like pattern on the gel. In addition, the reaction time of the LAMP method was only 1.5 h and the detection limit of this assay was 63 CFU/reaction. Con-clnsion LAMP assay targeting the wbfR gene of Vibrio cholera* O139 is rapid, specific, and sensitive for the detection of Vibrio cholerae O139. This method not only reduced the dependence of complicated equip-ment but also had a potential for wider use in inspection department, small-scale laboratory, emergency mo-tor vehicles and field survey.%目的 建立一种适合基层检验部门及小型实验室使用的快速检测O139群霍乱弧菌的方法.方法 应用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP),针对O139霍乱弧菌wbfR基因设计4条引物(2条内引物、2条外引物);优化LAMP反应条件和反应体系,对反应体系中的引物、dNTP、Mg2+/Mn2+离子及Calcium等浓度进行优化;并对13株种系背景明确的霍乱弧菌不同实验对照株、30株O139群霍乱弧菌地方分离株、10株O1群霍乱弧菌地方分离株、32株其他肠道菌进行检测,验证该方法的特异

  10. Loop-mediated isothermal amplification for visual detection of hepatitis B virus%应用环介导等温扩增技术可视化检测乙型肝炎病毒

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    目的 建立利用煮沸血清可视化检测乙型肝炎病毒(HBV)的环介导等温扩增技术(LAMP)方法. 方法 根据GenBank上提交的HBV的S基因序列比对后的相对保守区设计特异LAMP引物,分别用试剂盒法和煮沸法提取样本DNA.对反应条件进行优化,通过检测HBV标准毒株和临床样本来评价LAMP的特异性、灵敏度和抗干扰性,将实验结果和PCR进行比较.同时,对LAMP实验结果进行可视化检测.应用SPSS17.0软件进行一致性检验. 结果 建立了最适LAMP反应条件,LAMP特异性高,没有产生非特异性扩增.无论使用哪种核酸提取方法,LAMP灵敏度均为10拷贝/管.染料羟基萘芬兰(HNB)的灵敏度和电泳检测、SYBR Green Ⅰ效果相当,而不似染料SYBR Green Ⅰ容易造成气溶胶污染.另外,以荧光定量PCR (FQ-PCR)为金标准,煮沸法的LAMP和FQ-PCR具有的一致性较好(Kappa=0.762,P>0.05).然而,煮沸法的PCR和FQ-PCR的一致性较差(Kappa=0.186,P<0.05). 结论 LAMP在检测HBV感染中有优于PCR的特点,利用LAMP技术有利于在现场或基层医院检测HBV.%Objective To investigate the method of loop-mediated isothermal amplification (LAMP) using boiled serum for visual detection of hepatitis B virus (HBV).Methods Specific LAMP primers were designed according to the conservative region compared with the sequence of S genes in GenBank,and DNA of the samples was extracted by kit and boiling methods.The reaction conditions of LAMP were optimized,and HBV standard strains and clinical samples were examined to evaluate the specificity,sensitivity,and antiinterference ability of LAMP.Visual detection was performed for the results of LAMP,and SPSS 17.0 was used for consistency test.Results The optimal reaction conditions of LAMP were established.LAMP had a high specificity,and there was no nonspecific amplification.The sensitivity of LAMP was 10 copies/tube,regardless of the method for nucleic acid extraction.Hydroxynaphthol blue (HNB

  11. 鱼类致病鰤鱼诺卡氏菌(Nocardia seriolae)的LAMP检测技术建立与应用%DEVELOPMENT AND APPLICATION OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR DETECTION OF FISH PATHOGENIC NOCARDIA SERIOLAE

    Institute of Scientific and Technical Information of China (English)

    王国良; 刘璐; 徐益军

    2011-01-01

    针对鲫鱼诺卡氏菌16S-23S rRNA基因内转录间隔序列,设计了四条LAMP引物,在外内引物浓度比1:8、dNTP浓度0.8-1.2mmol/L、Mg浓度10-14mmol/L、反应温度60-65℃、反应时间60min的优化条件下,扩增产物经电泳后呈现特异性的LAMP梯形条带,建立的LAMP法具有高灵敏度,达到10μg/μl DNA浓度,比常规PCR方法高100倍.将该方法应用于取自养殖场的乌鳢、大黄鱼、黄姑鱼组织样品检测,结果在16份组织样品中有7份检出自然感染的鰤鱼诺卡氏菌,与同步取样品鱼组织的细菌分离、培养检查结果相一致,并显示既能检测发病鱼,又能检出未发病且已感染的病鱼.分析表明,这是一种能快速、简易、特异、敏感的检测鱼类致病鰤鱼诺卡氏菌的诊断方法.%To develop a loop-mediated isothermal amplification (LAMP) assay for detection of fish pathogenic Nocardia seriolae. Four primers were designed based on the sequence of the 16S-23S ribosomal RNA internal transcribed spacer region of N. seriolae for the use of LAMP assay. The assay was optimized to the following conditions: 1:8 outer primer concentration ratio, 0.8-l.2mmol/L of dNTP, 10- 14mmol/L of Mg2+, at 60-65 ℃ for 60min. LAMP amplification products had ladder-like bands when electrophoresed on an agarose gel. The assay could amplify up to 10-7μg/μl of DNA. Hence, the sensitivity of LAMP assay was 100-fold higher than the standard PCR protocol. The assay was applied to detect the presence of Nocardia seriolae in different tissue samples from farm Ophiocephalus argus, Pseudnosciaena crocea, Niber albifiora. Seven naturally infected N. seriolae were detected in 16 tissue samples, and the results from the bacterial culture test were consistent with fish tissue samples taken simultaneously. This study showed LAMP assay could be used not only to diagnose the infected fishes, but also to recognize the carrier ofN. seriolae as well. LAMP assay is a rapid,simple, specific and

  12. 利用环介导等温核酸扩增技术(LAMP)快速检测霍乱弧菌%Rapid detection of Vibrio cholerae by Loop-Mediated Isothermal Amplification

    Institute of Scientific and Technical Information of China (English)

    燕勇; 曹家穗; 高雯洁; 高蕾; 王恒辉; 陈黎霞

    2011-01-01

    Objective: The aims of this study were to research Loop -Mediated Isothermal Amplification (LAMP), a novel technology of nucleic acid amplification, so to establish a rapid, reliable and importantly easy - to - use detection method of V. cholerae, providing references and sevices to the early diagnosis of pathogen and disease survey of Cholera. Methods: The target sequences of the V. cholerae ctxA genes( available from Genbank) were analyzed and selected for LAMP usage by some softwares,BLAST, GENtle, DNAMan and so on. The LAMP primers were designed by special software, PrimerExplorer version 4. The LAMP assay for V. cholerae was developed on the realtime PCR cycler using fluorescent SYBR Green I for detection, improved through a series of optimization tests including temperature, time and component concentration of reaction, and compared with a ordinary PCR assays aiming to familiar target genes, ace, rtxA and ctxA of V. cholerae. Results: The LAMP assay correctly identiffed all 5 strains of CT - producing V. cholerae with ctxA primers, from 20 tested bacteria strains comprising 8 genera. The detection limits of the assay in pure cultrue were determined to be 5 cfu/μl( 10 cfn per reaction) of V. cholerae cells. Conclusion: The LAMP assay is a more rapid, reliable, simple detection method of V. cholerae, worthy of popularization, with important significance to disease survey and emergency detection of Cholera.%目的:建立基于环介导等温核酸扩增技术(LAMP)的霍乱弧菌快速检测方法,为霍乱疫情监测与预防提供及时的应急检测服务和参考结果.方法:通过Genbank检索、下载霍乱弧菌ctxA基因序列,使用BLAST、GENtle、DNAMan等相关核酸序列分析软件比对分析确定目标序列,使用PrimerExplorer v4软件设计霍乱弧菌LAMP引物.以SYBR Green I作为荧光剂在荧光定量PCR仪上实时检测LAMP反应结果,作为本研究的主要研究方式.通过优化反应温度、时间、组成浓度等实验

  13. Rapid detection of vibrio parahaemolyticus with loop-mediated isothermal amplification%环介导等温扩增技术快速检测副溶血性弧菌

    Institute of Scientific and Technical Information of China (English)

    张如胜; 宋克云; 苏良; 魏泉德

    2009-01-01

    Objective To develop a loop-mediated isothermal amplification(LAMP) assay for the rapid and specific detection of vibrio parahaemolyticus(Vp). Methods Four primers which recog-nized 6 distinct regions on the thermolabite hemolysin(tlh) gene of Vp were designed and used for LAMP assay. Vp DNA was amplified under isothermal conditions(65 ℃) for 60 rain, then the ampli-fied product was judged by naked eye, SYBR Green I staining, electrophoretie analysis and restriction digestion. To evaluate the specificity of the assay, 1 strain of Vp and 12 strains of non-Vp were tested by LAMP and conventional PCR simultaneously. In addition, the detection limit of LAMP was com-pared with that of PCR by using the Vp strain, that were 10-fold serially diluted and was amplified by LAMP and PCR. Results With 1 strain of Vp, the naked eye, SYBR Green I staining and electro-phoretic analysis could detect the products in the LAMP assay. The specificity of LAMP products was confirmed by digestion of LAMP products using restriction enzymes. Amplification of LAMP was not observed when 12 strains of non-Vp were tested. The specificity and sensitivity of LAMP were similar to those of conventional PCR assay and the detection limit of LAMP assay was 15 cfu/mL. Conclusion These results indicate that the LAMP assay is a rapid, specific and sensitive detection method for Vp. The method is suitable fo: daily monitoring and rapid diagnosis of Vp.%目的 建立一种检测副溶血性弧菌(Vp)快速且特异的环介导等温扩增(LAMP)检测方法.方法 针对副溶血性弧菌小耐热溶血素(tlh)基因特异性序列的6个位点设计4条LAMP引物,65℃保温约60 min,完成对副溶血性弧菌的扩增,扩增产物经肉眼、SYBR Green I染色、电泳和酶切鉴定.利用I.AMP和普通PCR方法同时检测1株副溶血性弧菌和12株非副溶血性弧菌来验证LAMP方法的特异性;将副溶血性弧菌菌液作一系列lO倍稀释后用LAMP和PCR方

  14. Establishment and application of a loop-mediated isothermal amplification method for rapid diagnosis of Vibrio cholerae%霍乱弧菌LAMP快速检测方法的建立及应用

    Institute of Scientific and Technical Information of China (English)

    柯雪梅; 陈胤瑜; 高璐璐; 杜正平; 冯雪梅; 廖如燕; 陈志永; 曹以诚; 陈清

    2009-01-01

    目的 建立环介导等温扩增技术(LAMP)快速检测霍乱弧菌.方法 针对霍乱弧菌外膜蛋白的ompW基因设计特异引物,优化反应条件,建立霍乱弧菌的LAMP检测方法.通过对47株细菌及模拟污染现场,检测该方法的灵敏度、特异度和实用性.结果 活菌计数方法表明建立的LAMP方法检测霍乱弧菌的灵敏度为1.6×10~2 cfu/ml.在人工污染霍乱弧菌的粪便及海水标本中检测的敏感度为1.6×10~3 cfu/ml,在牛奶标本中敏感度为1.6×10~4 cfu/ml.检测21株霍乱弧菌均为阳性,26株非霍乱菌则全部阴性,特异度为100%.结论 建立的LAMP方法检测霍乱弧菌灵敏度、特异度高,可用于霍乱现场监测和流行病学调查.%Objective To establish a loop-mediated isothermal amplification (LAMP) method for rapid diagnosis of Vibrio cholerae. Method Based on the ompW nucleic sequence of Vibrio cholerae, a pair of primers was designed for LAMP. The reaction conditions were optimized, and the specificity, sensitivity, and practicability of LAMP were tested using 47 baterial strains and simulated contaminated sites. Results The results of viable bacterium count showed that LAMP was capable of detecting Vibrio cholerae at a level as low as 1.6×10~2 cfu/ml. The minimal detectable concentration was 1.6×10~3 cfu/ml for simulated contaminated samples such as feces and seawater, and 1.6×10~4 cfu/ml for contaminated milk. All the 21 strains of Vibrio cholerae yielded positive results in LAMP, and the 26 strains of other bacteria all showed negative results, with a detection specificity of 100%. Conclusion The established LAMP method has high specificity and sensitivity for detecting Vibrio cholerae and is applicable in field monitoring and epidemiological study of Vibrio cholerae.

  15. The application of loop mediated isothermal amplification in surveillance for leptospira interrogans%环介导等温扩增技术在钩端螺旋体病监测中的应用

    Institute of Scientific and Technical Information of China (English)

    张晶; 周勇; 陈守义

    2012-01-01

    目的:评价LAMP技术在钩端螺旋体检测与监测中的可行性.方法:采用LAMP技术对感染病人血清及钩体标准菌株感染鼠肾进行检测,同时与卫生行业标准推荐的引物G1/G2 PCR方法进行对比.结果:LAMP快速检测方法60min内即可完成检测,具有较高的检测灵敏度,对纯培养的钩体检测灵敏度可达1.5 CFU/ml,比PCR法高出两个数量级,结果肉眼直接可观.同时对21种共42株细菌进行LAMP扩增,仅钩体结果为阳性,显示该LAMP方法具有较强特异性.结论:LAMP方法操作简便、特异性强、灵敏度高且成本低廉,在钩体的快速检测和疫情监控中具有良好的应用前景.%Objective:To discuss the feasibility of LAMP for detecting and monitoring Leptospira interrogans. Methods: The specificity and sensitivity of detection for leptospiral DNA in human serum and rat kidney based on Loop mediated isothermal amplification ( LAMP) were compared with those of the recommended standard G1/G2 PCR. Results: 42 bacterial strains were selected to test specificity of the LAMP method and only Leptospira interrogans showed positive result by LAMP, indicating the high specificity of the LAMP method. The LAMP method could be completed in 60 min and showed a good sensitivity of 1. 5 CFU/ml for cultured Leptospira interrogans. Conclusion: The LAMP assay is a sensitive, rapid and simple tool for the detection of Leptospira interrogans, performing a bright future for the detection and monitoring of Leptospira interrogans.

  16. Loop-mediated isothermal amplification technology in the rapid detection of Bacillus anthracis%环介导等温扩增技术在快速检测炭疽芽胞杆菌中的应用

    Institute of Scientific and Technical Information of China (English)

    段圣亮; 陆晔; 田桢干; 王桂江

    2012-01-01

    The present paper aims to establish a rapid detection method for Bacillus anthracis. The primes were designed according to Bacillus anthracis strain-specific gene fragment, and loop-mediated isothermal amplification (LAMP) was used to establish the detection method . The results showed that LAMP can effectively identify the specific target bacteria with sensitivity of 102 -103 CFU/ml. It is suggested that LAMP is simple and fast in detection of bioterrorism bacteria such as Bacillus anthracis in acidic , alkaline and viscous media. High-salt environment influences LAMP results , so it is necessary to effectively remove salt out of nucleic acid before application of LAMP .%本文旨在建立适合国境口岸现场应用的生物恐怖防控快速检测方法,从而保障口岸安全.针对生物恐怖炭疽芽胞杆菌,选择目标菌种特异性基因片段,设计引物,运用环介导等温扩增(LAMP)技术建立一套简便、高效的检测方法,并模拟生物恐怖炭疽芽胞杆菌可能存在的基质条件,评价LAMP技术在快速筛查中的适用性.结果显示,LAMP技术排查生物恐怖炭疽芽胞杆菌简便、快速、特异,检测灵敏度为102~103 CFU/ml;且能有效检出在偏酸、偏碱及黏稠基质中的炭疽芽胞杆菌.而高盐环境对该反应影响较大,有必要采用能有效去除盐分的核酸抽提方法.

  17. Rapid detection for Mycobacterium tuberculosis by loop-mediated isothermal amplification%环介导等温扩增技术快速检测结核分枝杆菌核酸

    Institute of Scientific and Technical Information of China (English)

    沈会平; 张耀祺; 杨坚; 石磊; 陈涛; 李国周; 赵红波; 莫自耀

    2011-01-01

    目的 建立一种快速准确的检测结核分枝杆菌(MTB)核酸的环介导等温扩增(LAMP)方法.方法 以重复插入序列IS6110为目的基因,设计LAMP引物,特异检测MTB核酸.用本法与痰涂片抗酸染色镜检法、实时荧光PCR法对100例可疑患者痰标本进行对比检查.结果 LAMP法特异性强,仅扩增MTB复合群核酸;灵敏度高,检测限达100 fg;而实时荧光PCR检测限为1 pg.对100例疑似结核病患者痰液标本检测,涂片抗酸染色法、LAMP法、实时荧光PCR法的阳性率分别为28%、39%和38%.结论 本研究建立的LAMP方法检测MTB核酸特异性强、灵敏度高、时间短且操作简便,有望成为临床快速检测MTB的新方法.%Objective To develop a rapid loop-mediated isothermal amplification (LAMP) method for detecting the nuclear acid of Mycobacterium tuberculosis ( MTB). Methods The repetitive insertion sequence IS6110 was used as target gene to develop an efficient LAMP method by which MTB was specifically detected. MTB in 100 clinical sputum samples were detected simultaneously by direct smear, LAMP and real-time fluorescent PCR. Results The developed LAMP method showed high specificity for detecting pathogenic strains of MTB complex. The detection limit of LAMP assay was as low as 100 fg of DNA for each tube, while the detection limit of conventional PCR was 10 pg for each tube. In the 100 clinical sputum samples the positive rate of MTB detected by direct smear, LAMP and real-time PCR was 28% , 39% and 38% , respectively. Conclusion The developed LAMP in this study was effective method with high specificity and sensitivity for detection of MTB, so it is expected to become a valuable tool for rapid detection and I dentification of MTB in clinical laboratory.

  18. 环介导等温扩增法在结核病诊断中的应用%Application of loop-mediated isothermal amplification in the diagnosis of tuberculosis

    Institute of Scientific and Technical Information of China (English)

    杨秉芬; 王心静; 蒋静; 翟斐; 曹志红; 程小星

    2015-01-01

    Obejective To study the diagnostic value of loop-mediated isothermal amplification( LAMP) in different tuberculosis clinical samples. Methods LAMP was used to detect mycobacterium tuberculosis complex-specific gene IS1081 in bronchoalveolar lavage fluid ( BALF ) , puncture fluid, ascites, and cerebrospinal fluid ( CSF) . And the result was compared with real-time PCR ( RT-PCR) . Results The sensitivity of LAMP was lower than RT-PCR (80% vs 92%) in BALF and puncture fluid, in which there were more bacteria. The sensitivity of LAMP was higher than RT-PCR (34. 6% vs 15. 4%) in ascites and CSF, in which the mount of bacteria was ex-tremely low, but the specificity of LAMP decreased slightly (80% vs 93. 3%). The sensitivities of LAMP and RT-PCR calculated in all samples showed no significant difference ( 56. 9% vs 52. 9%) . Conclusion LAMP has high sensitivity and specificity in detecting mycobacterium tuberculosis complex.%目的:研究环介导等温扩增法在不同临床样本中的结核病诊断价值。方法使用环介导等温扩增法检测肺泡灌洗液、局部穿刺液、腹水和脑脊液中的结核分枝杆菌复合群的特异性基因IS1081,并与实时荧光定量PCR进行比较。结果在荷菌量较高的肺泡灌洗液和局部穿刺液中,环介导等温扩增法敏感性比实时荧光定量PCR低(80% vs 92%);在荷菌量极低的腹水和脑脊液中,环介导等温扩增法敏感性比实时荧光定量PCR高(34.6% vs 15.4%),但特异性略有下降(80% vs 93.3%);综合计算所有样本的敏感性,环介导等温扩增法与实时荧光定量PCR无显著差别(56.9% vs 52.9%)。结论环介导等温扩增法具有较高敏感性和特异性,可以用于结核病诊断。

  19. Detecting My cobacterium Tuberculosis in Sputum Specimens by Technique of Loop-mediated Isothermal Amplification%环介导等温扩增技术检测痰标本中结核分枝杆菌的研究

    Institute of Scientific and Technical Information of China (English)

    易海华; 丁永健; 钱志娟; 房超; 吴萍兰; 房婷

    2008-01-01

    [目的]寻找1种快速的,且敏感度高,特异性强的结核分枝杆菌的检测方法.[方法]环介导等温扩增基因检测(Loop-mediated isothermal amplification LAMP)是一种便捷、灵敏度高而又特异性强的核酸基因扩增检测技术.本课题组根据结核分枝杆菌gyrB特征性序列设计3对特征性引物对痰液中和培养基中的结核分枝杆菌进行检测.其中1对环状引物结合于特征性序列中环状结构部分,可极大地加快LAMP反应速度,且不干扰内引物在LAMP反应中作用.[结果]实验结果表明:使用环状引物的LAMP反应可在0.5h内完成,总共分析时间不超过1h.LAMP检测技术的灵敏度比经典PCR技术高100倍左右,LAMP检测技术具有与实时PCR(Real-time PCR)技术相同的特异度.另外,由于LAMP检测技术是在恒温的情况下进行,不需要特别昂贵的仪器设备,检测方法简单,结果判定直接.[结论]该方法的特点和优势可以使之在基层实验室及现场监测方面广泛使用,在快速检测方面具有一定的开发潜力.

  20. Rapid detection of Enterobacter sakazakii in milk by loop-mediated isothermal amplification%环介导等温扩增法快速检测乳中阪崎肠杆菌

    Institute of Scientific and Technical Information of China (English)

    董鑫悦; 满朝新; 卢雁; 郭颖; 王今雨; 杨相宜; 郎友; 庞心怡; 姜毓君

    2013-01-01

    利用环介导等温扩增方法的快速、简便等优点,建立一种检测乳中阪崎肠杆菌的方法.以阪崎肠杆菌的OmpA序列为靶基因,设计特异性引物.优化并建立LAMP检测乳中阪崎肠杆菌的方法.结果表明,LAMP检测阪崎肠杆菌纯培养物的灵敏度为3.7×101cfu/mL,其灵敏度是PCR方法的10倍.人工污染阪崎肠杆菌灭菌乳的检测限为4.3×101 cfu/mL.对23株致病菌进行特异性实验,特异性良好.该方法具有特异性强、灵敏度高、设备简便、耗时短等优点,在食品检测中具有良好的应用前景.%In order to rapidly and conveniently detect Enterobacter sakazakii in milk,a new method was developed by loop-mediated isothermal amplification (LAMP) .The LAMP primers were designed on the basis of outer membrane protein A gene ( OmpA) in Enterobacter sakazakii.The LAMP reaction mixture was optimized.The determination limit of LAMP when testing E. sakazakii templates in pure culture was 3.7×101 cfu/mL, and its sensitive was better than PCR.For dairy samples,LAMP could specifically detect E.sakazakii cells as low as 4.3× 101 cfu/mL.Only four Enterobacter sakazakii strains were amplified using LAMP,and no amplicon was observed in other 19 bacteria strains.Therefore LAMP method was an effective technology with high specificity and sensitivity,to conveniently detect E.sakazakii in milk.

  1. Kinetics of the First Order Autocatalytic Decomposition Reaction of Nitrocellulose (13.86% N)

    Institute of Scientific and Technical Information of China (English)

    GUO,Peng-Jiang(郭鹏江); HU,Rong-Zu(胡荣祖); NING,Bin-Ke(宁斌科); YANG,Zheng-Quan(杨正权); SONG,Ji-Rong(宋纪蓉); SHI,Qi-Zhen(史启祯); LU,Gui-E(路桂娥); JIANG,Jin-You(江劲勇)

    2004-01-01

    The kinetics of the first order autocatalytic decomposition reaction of nitrocellulose (NC, 13.86% N) was studied by using DSC. The results show that the DSC curve for the initial 50% of conversion degree of NC can be described by the first order autocatalytic equation dy/dt=-1016.3exp(-181860/RT)y-1016.7exp(-173050)y(1-y) and that for the latter 50% conversion degree of NC described by the reaction equations dy/dt=-1016.4exp(-154820/RT)y(n=1) and dy/dt=-1016.9exp(-155270/RT)y2.80(n≠1).

  2. Detection of Brucella by loop-mediated isothermal amplification%布鲁杆菌环介导等温扩增快速检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    刘锴

    2013-01-01

    Objective A loop-mediated isothermal amplification (LAMP)assay was developed for rapid detection of Brucella.Methods Using Primer 4.0,we designed four specific primers at brookings coli outer membrane protein (OMP31) gene conservative area,under the action of Bst polymerase larger pieces to realize the step-like isothermal amplification of DNA.In this method,on the basis of optimal amplification conditions,the specificity and sensitivity of this method was compared with conventional PCR,and LAMP visualization experiment was conducted.Results The detection results of Brucella abortus (B.abortus) strain 544,B.abortus strain 104M,Brucella melitensis (B.melitensis) strain Rev-1,B.melitensis strain 16M,Brucella suis (B.suis) strain S2,B.suis strainand 1330S,Brucella canis (B.canis) strain RM6/66,Brucellá ovis (B.ovis) strain 63/290,and Brucella neotomae.(B.neotomae) strain 5K33 were positive,and Yersinia enterocolitica (Y.enterocolitica) O ∶ 9,Escherichia coli (E.coli) O157 ∶H7,and (Salmonella typhimurium,S.typhimurium) 47729 were negative.The minimum detection limit was 8.5 × 10-8 mg/L.LAMP was more sensitive than PCR.The results can be determined by electrophoresis or through visual judgment.Conclusions LAMP is a simple,specific and sensitive method.LAMP assay is a useful tool for rapid detection of Brucella.%目的 建立一种环介导等温扩增(LAMP)快速检测动物布鲁杆菌的方法.方法 使用Primer 4.0,针对布鲁杆菌外膜蛋白(OMP31)基因保守区设计4条特异引物,在Bst大片段聚合酶的作用下,实现DNA的梯状等温扩增,并在此方法最适扩增条件优化的基础上,对其特异性、灵敏度进行实验,同时与普通PCR灵敏度进行比较,以及进行LAMP可视化实验.结果 本研究建立的检测方法对牛种布鲁杆菌(Brucella abortus,B.abortus)544A和104M、羊种布鲁杆菌(Brucella melitensis,B.melitensis)Rev-1和16M、猪种布鲁杆菌(Brucella suis,B.suis)S2和1330S

  3. 环介导恒温扩增技术快速检测短凯伦藻%Rapid Detection of Karenia brevis (Dinophyceae) by Loop-mediated Isothermal Amplification (LAMP)

    Institute of Scientific and Technical Information of China (English)

    张凤英; 石彦红; 马凌波; 徐兆礼

    2012-01-01

    短凯伦藻(Karenia brevis)是一种有毒赤潮微藻,所产生的短藻毒素对海洋生物乃至人类都有毒害作用,为加强对短凯伦藻赤潮的监控,建立了稳定的短凯伦藻环介导恒温扩增(LAMP)鉴定体系,在此基础上进行了特异性和灵敏性验证.特异性实验结果显示只有短凯伦藻或者含短凯伦藻的模板呈现阳性反应,其他微藻为阴性反应,从而验证了该LAMP方法的特异性;同时,对短凯伦藻的基因组DNA进行一系列10倍稀释作为敏感度实验的模板,并与常规PCR做了对比,结果表明:短凯伦藻的LAMP方法最低检测限度为50 pg,敏感度比常规PCR高10倍.LAMP产物鉴定不需要常规的胶电泳过程,直接采用肉眼观察的方法,在含有短凯伦藻的阳性反应管中会出现白色混浊,加入syBRò Green I染料呈现绿色,而未含有短凯伦藻的阴性管为澄清,染色后仍为原来的橙色.因此,该方法操作简便、特异性强、灵敏度高而成本低,在赤潮原因种检测监控方面具有良好的应用前景.%Karenia brevis (Gymnodinium breve) is an unarmored, non-peridinin-containing dinoflagellate. It produces lipophilic brevetoxin that can harm many marine animals, eg. fish, birds and shellfish. As for human, brevetoxin can cause respiratory distress by inhalation and food poisoning by consumption of contaminated shellfish. Previous methods for the detection of A', brevis depend on microscopy analysis, which is time-consuming and requires a considerable amount of expertise and skill. Molecular methods have also been applied to detect K. brevis, e.g. real-time PCR and DNA hybridization assay. However, there are some inevitable disadvantages for the two methods, such as expensive reagents and equipment, or fussy approaches. Loop-mediated isothermal amplification (LAMP) is a specific nucleic acid amplification method that is easy to perform. The LAMP method can amplify nucleic acids under isothermal conditions at

  4. Electron-beam induced deposition and autocatalytic decomposition of Co(CO)3NO

    Science.gov (United States)

    Vollnhals, Florian; Drost, Martin; Tu, Fan; Carrasco, Esther; Späth, Andreas; Fink, Rainer H; Steinrück, Hans-Peter

    2014-01-01

    Summary The autocatalytic growth of arbitrarily shaped nanostructures fabricated by electron beam-induced deposition (EBID) and electron beam-induced surface activation (EBISA) is studied for two precursors: iron pentacarbonyl, Fe(CO)5, and cobalt tricarbonyl nitrosyl, Co(CO)3NO. Different deposits are prepared on silicon nitride membranes and silicon wafers under ultrahigh vacuum conditions, and are studied by scanning electron microscopy (SEM) and scanning transmission X-ray microscopy (STXM), including near edge X-ray absorption fine structure (NEXAFS) spectroscopy. It has previously been shown that Fe(CO)5 decomposes autocatalytically on Fe seed layers (EBID) and on certain electron beam-activated surfaces, yielding high purity, polycrystalline Fe nanostructures. In this contribution, we investigate the growth of structures from Co(CO)3NO and compare it to results obtained from Fe(CO)5. Co(CO)3NO exhibits autocatalytic growth on Co-containing seed layers prepared by EBID using the same precursor. The growth yields granular, oxygen-, carbon- and nitrogen-containing deposits. In contrast to Fe(CO)5 no decomposition on electron beam-activated surfaces is observed. In addition, we show that the autocatalytic growth of nanostructures from Co(CO)3NO can also be initiated by an Fe seed layer, which presents a novel approach to the fabrication of layered nanostructures. PMID:25161851

  5. Electron-beam induced deposition and autocatalytic decomposition of Co(CO)3NO.

    Science.gov (United States)

    Vollnhals, Florian; Drost, Martin; Tu, Fan; Carrasco, Esther; Späth, Andreas; Fink, Rainer H; Steinrück, Hans-Peter; Marbach, Hubertus

    2014-01-01

    The autocatalytic growth of arbitrarily shaped nanostructures fabricated by electron beam-induced deposition (EBID) and electron beam-induced surface activation (EBISA) is studied for two precursors: iron pentacarbonyl, Fe(CO)5, and cobalt tricarbonyl nitrosyl, Co(CO)3NO. Different deposits are prepared on silicon nitride membranes and silicon wafers under ultrahigh vacuum conditions, and are studied by scanning electron microscopy (SEM) and scanning transmission X-ray microscopy (STXM), including near edge X-ray absorption fine structure (NEXAFS) spectroscopy. It has previously been shown that Fe(CO)5 decomposes autocatalytically on Fe seed layers (EBID) and on certain electron beam-activated surfaces, yielding high purity, polycrystalline Fe nanostructures. In this contribution, we investigate the growth of structures from Co(CO)3NO and compare it to results obtained from Fe(CO)5. Co(CO)3NO exhibits autocatalytic growth on Co-containing seed layers prepared by EBID using the same precursor. The growth yields granular, oxygen-, carbon- and nitrogen-containing deposits. In contrast to Fe(CO)5 no decomposition on electron beam-activated surfaces is observed. In addition, we show that the autocatalytic growth of nanostructures from Co(CO)3NO can also be initiated by an Fe seed layer, which presents a novel approach to the fabrication of layered nanostructures.

  6. Electron-beam induced deposition and autocatalytic decomposition of Co(CO3NO

    Directory of Open Access Journals (Sweden)

    Florian Vollnhals

    2014-07-01

    Full Text Available The autocatalytic growth of arbitrarily shaped nanostructures fabricated by electron beam-induced deposition (EBID and electron beam-induced surface activation (EBISA is studied for two precursors: iron pentacarbonyl, Fe(CO5, and cobalt tricarbonyl nitrosyl, Co(CO3NO. Different deposits are prepared on silicon nitride membranes and silicon wafers under ultrahigh vacuum conditions, and are studied by scanning electron microscopy (SEM and scanning transmission X-ray microscopy (STXM, including near edge X-ray absorption fine structure (NEXAFS spectroscopy. It has previously been shown that Fe(CO5 decomposes autocatalytically on Fe seed layers (EBID and on certain electron beam-activated surfaces, yielding high purity, polycrystalline Fe nanostructures. In this contribution, we investigate the growth of structures from Co(CO3NO and compare it to results obtained from Fe(CO5. Co(CO3NO exhibits autocatalytic growth on Co-containing seed layers prepared by EBID using the same precursor. The growth yields granular, oxygen-, carbon- and nitrogen-containing deposits. In contrast to Fe(CO5 no decomposition on electron beam-activated surfaces is observed. In addition, we show that the autocatalytic growth of nanostructures from Co(CO3NO can also be initiated by an Fe seed layer, which presents a novel approach to the fabrication of layered nanostructures.

  7. 环介导等温扩增技术检测卡氏肺孢子虫的研究%Detection of Pneumocystis carinii DNA by loop-mediated isothermal amplification

    Institute of Scientific and Technical Information of China (English)

    杨秋林; 张如胜; 伍和平; 王可耕; 张愉快

    2008-01-01

    Objective To detect Pneumocystis carinii (Pc) DNA by loop-mediated isothermal amplification (LAMP). Methods After injected with hydrocortisone acetate for 8 weeks, the bronchoalveolar lavage fluid (BALF) of Wistar rats were collected and a portion of BALF were examined for identifying Pc organisms using microscope. Then Pc DNA was extracted by phenol-chloroform extraction. Four primers which recognized 6 distinct regions on the mtrRNA gene of Pc were designed and used for LAMP assay. To evaluate the specificity of the assay, M. tuberculosis, M. pneumoniae, C. pneumoniae, P. gondii and rat leucocyte were used as negative controls. To compare the sensitivity of the LAMP to that of conventional PCR, Pc DNA were 10-fold serially diluted and was amplified by LAMP and PCR. LAMP results were judged by naked eye, electrophoretic analysis and restriction digestion. Results Pc organisms were detected from BALF of rats injected with hydrocortisone acetate. After LAMP reaction, positive signal was observef rats injected with hydrocortisone acetate. By contrast, no positive signal was observed for the negative controls in the study. The amplified product digested by restriction enzyme demonstrated 3 bands (82, 135, 189 lip) upon agarose gel electrophoresis, in good agreement with the predicted sizes. The detection limit of LAMP assay was 1 pg/μl of Pc DNA per reaction and that of PCR was 10 pg/μ1 of Pc DNA per reaction. Conclusion LAMP assay has usefulness for rapid detection of Pc.%目的 环介导等温扩增(LAMP)技术检测卡氏肺孢子虫(Pc).方法 醋酸可的松经皮下注射Wistar大鼠诱导Pc,收集支气管肺泡灌洗液(BALF)提取Pc基因组DNA.设计4条扩增Pc线粒体核糖体大亚基(mtrRNA)基因的LAMP引物,以结核杆菌、肺炎支原体、肺炎衣原体、弓形虫、大鼠白细胞为对照,进行LAMP反应.LAMP产物经显色、电泳及酶切鉴定.将Pc DNA 10倍稀释后同时进行LAMP和PCR,比较其敏感性.结果 Pc检测管经显

  8. 空肠弯曲菌环介导等温扩增检测方法的建立和优化%Development and Optimization of a Loop-mediated Isothermal Amplification Assay for Detection of Campylobacter jejuni

    Institute of Scientific and Technical Information of China (English)

    许紫建; 杨兵; 苏霞; 周宏专; 史爱华; 徐福洲

    2013-01-01

      首先根据空肠弯曲菌 mapA 基因序列设计 LAMP 引物,建立检测空肠弯曲菌的 LAMP 检测方法。继而对LAMP 方法中不同反应组分的浓度分别进行筛选,结果显示,当内外引物浓度分别为1.6,0.2μmol/L、Mg 2+浓度为8 mmol/L、dNTP 浓度为1.4 mmol/L、Betaine 浓度为0.8 mol/L 时获得最佳的反应结果。最后对 LAMP 反应的特异性和敏感性进行检测,特异性检测结果显示,对其他13种革兰氏阴性和革兰氏阳性细菌扩增结果均为阴性;敏感性检测结果显示,对空肠弯曲菌基因组 DNA 的检测限为每个反应管100 fg,对空肠弯曲菌培养菌液检测限为每个反应管7.5 cfu。试验建立的 LAMP 方法为快速简便地自动物源性产品中检测空肠弯曲菌奠定了基础。%  In this study,the specific mapA gene was used to design a set of primers to develop a loop -mediated isothermal amplification (LAMP) assay for detection of C.jejuni.To optimize the LAMP assay,the reaction compo-nents in the assay were screened to determine the optimal concentration .The final LAMP assay comprised 1.6μmol/L each of inner primers FIP and BIP,0.2 μmol/L each of outer primers F3 and B3,8 mmol /L Mg mmol/L dNTP,0.8 mol/L Betaine.The specificity test showed that the assay correctly identified all C.jejuni strains but not 13 other bacterial species.The sensitivity of the assay was 100 fg per test tube for C.jejuni genomic DNA and 7.5 cfu per test tube for C.jejuni bacterial culture.This LAMP assay is a rapid and simple tool for detection of C.jejuni and will provide an essential role in facilitating early diagnosis of this organism from animal products .

  9. Preliminary study on detection of Echinococcus granulosus DNA by loop-mediated isothermal amplification%环介导等温扩增技术检测细粒棘球绦虫DNA的初步研究

    Institute of Scientific and Technical Information of China (English)

    徐祥珍; 金小林; 李健; 江文才; 蒋岗

    2011-01-01

    Objective To evaluate the sensitivity of loop-mediated isothermal amplification (LAMP) on the detection of Echi-nococcus granulosus. Methods The DNAs were extracted from Echinococcus granulosus eggs and adults. According to Echinococcus mitochondrial 12S rRNA sequences and the mechanism of LAMP, 4 Echinococcus specific primers were designed and used for LAMP assay, and Bubble taenia and the blank were used as the negative control for evaluation of the specificity. The LAMP products were stained by SYBR Green I and analyzed by electrophoresis, and 1000, 100, 10, 1 eggs of Echinococcus granulosus per 200 μl were amplified by LAMP for evaluating the sensitivity. Results The LAMP products of Echinococcus granulosus adult DNA became turbid and green after staining while the products of control DNA kept clarify and brown after staining. Electrophoresis analysis showed that the LAMP products of Echinococcus granulosus eggs presented characteristic ladders, but the products of control did not. The detection limit of LAMP assay was 1 egg of Echinococcus granulosus per reaction. Conclusions LAMP assay is a simple, sensitive and specific method for detection of hydatid disease pathogens and could be used for the disease surveillance.%目的 评价环介导等温扩增技术(LAMP)检测细粒棘球绦虫的敏感性.方法 提取细粒棘球绦虫虫卵及成虫DNA,根据棘球绦虫线粒体12SrRNA基因序列及LAMP法原理,设计4条细粒棘球绦虫特异性引物,进行LAMP反应,反应产物经SYBR Green Ⅰ显色及1.5%琼脂糖电泳鉴定,同时设置泡状带绦虫及空白对照.用1000、100、10、1个虫卵/200μl细粒棘球绦虫虫卵DNA进行LAMP反应,评价其敏感性.结果 细粒棘球绦虫检测管反应液呈混浊沉淀,显色后为绿色;对照组澄清,显色后为棕色.虫卵产物电泳后呈LAMP特征性梯状条带,对照组无扩增产物.LAMP可检测到的虫卵最低数量为1个.结论 LAMP法敏感、特异、简便,可用于棘

  10. 玉米细菌性枯萎病菌的环介导恒温扩增(LAMP)检测方法%Loop-mediated isothermal amplification (LAMP) for detection of Pantoea stewartii subsp.stewartii

    Institute of Scientific and Technical Information of China (English)

    封立平; 倪新; 吴兴海; 伦才智; 吴翠萍; 栾晶

    2015-01-01

    为了提高口岸和基层实验室检疫和监测玉米细菌性枯萎病菌的准确性和工作效率,利用环介导恒温扩增技术(LAMP),根据内切葡聚糖酶(EGase)基因前导序列,设计2个内引物和2个外引物,对玉米细菌性枯萎病菌进行快速检测.结果表明,使用玉米细菌性枯萎病菌的近缘种或引致相似症状的病原菌菊欧文氏菌玉米致病变种Erwinia chrysanthemi pv.zeae、玉米内州萎蔫病菌Clavibacter michiganensis subsp.nebraskensis、燕麦假单胞菌Pseudomonas avenae、杓兰欧文氏菌Erwinia cypripedii检测其特异性,仅玉米细菌性枯萎病菌有扩增.LAMP检测灵敏度达到2 pgDNA,为普通PCR的100倍;与其它检测方法相比,LAMP方法检测时间短,效率高,不仅降低了设备投入,易于操作,而且具有较高的灵敏度和特异性,适合玉米细菌性枯萎病菌的现场检疫和大规模检测.%In order to improve the accuracy and efficiency of quarantine and monitoring of Pantoea stewartii subsp.stewartii for the port and the basic-level test labs,and establish a rapid detection method,the loop mediated isothermal amplification (LAMP) technology was applied in the laboratory.Based on the endoglucanase (EGase) gene leading sequence,two inner primers and two outer primers were designed through a simple LAMP to detect P.stewartii subsp.stewartii.The results showed that the LAMP primers did not react with suspensions of Erwinia chrysanthemi pv.zeae,Clavibacter michiganensis subsp.nebraskensis,Pseudomonas avenae,and Erwinia cypripedii.The LAMP detection system of P.stewartii subsp.stewartii was designed successfully with the lowest detection limit of 2 pg DNA,100 times higher than ordinary PCR technology.Compared with other detection methods,the LAMP method for detection of P.stewartii subsp.stewartii in this study was rapid,with higher efficiency and lower equipment investment.The results indicated that LAMP method is easy to conduct,and has high sensitivity and

  11. 环介导恒温核酸扩增技术快速检测志贺菌研究%Rapid and sensitive detection of Shigella by Loop -Mediated Isothermal Amplification

    Institute of Scientific and Technical Information of China (English)

    梅玲玲; 朱敏; 占利; 龚璞; 张俊彦

    2011-01-01

    目的:建立一种快速检测志贺菌的环介导恒温核酸扩增(LAMP)技术,为志贺菌食源性疾病监测、诊断提供技术支撑.方法:在对志贺菌基因序列进行分析、对比基础上,根据志贺菌ipaH基因序列保守区域设计6条特异性引物;通过对LAMP技术反应条件优化,实现对志贺菌的快速检测;用不同来源志贺菌及沙门菌、大肠杆菌、副溶血性弧菌、柠檬酸杆菌、金黄色葡萄球菌、乙型溶血性链球菌等菌株验证方法的敏感性和特异性.结果:当25μl反应体系中含1.6 μmol/L内引物(FIP、BIP)、0.2 μmol/L外引物(F3、B3)、0.2μmol/L环引物(LF、LB)、1 mmol/L dNTP、6 mmol/L MgSO4,0.8 mol/L甜菜碱、1×反应缓冲液、DNA模版,8 U Bst DNA聚合酶,扩增条件为60℃,60 min时,LAMP技术对志贺菌最低检测浓度为200 CFU/ml,经8 h增菌,最低检测浓度可达到20 CFU/ml.对32株不同来源的志贺菌检测结果,所有反应管内肉眼观察有混浊,为阳性,而13种139株其它非志贺菌株反应结果均呈透明为阴性.对50件食品检测结果与常规培养方法及实时荧光PCR方法相比无显著性差异(P>0.05,x2=0.27).结论:本研究建立的志贺菌LAMP检测方法具有特异性强、灵敏度高、方便快捷、成本低等特点,适合在基层、小型实验室以及无条件购买荧光定量PCR仪的病原微生物检验检测机构推广使用.%Objective:To develop a technique for rapid detection of Shigella using a novel DNA amplification procedure designated Loop - mediated isothermal amplification (LAMP). To provide technical support for foodborne disease surveillance and diagnose. Methods: Six Shigella - specific primers were designed from the published ipaH gene sequences of Shigella by using the LAMP primer supported software program. The specificity and sensitivity of LAMP were examined using Salmonella, Escherichia coli, Vibrio Parahemolyticus, Staphylococcus aureus and other species. Results: It

  12. Laser Induced Selective Activation For Subsequent Autocatalytic Electroless Plating

    DEFF Research Database (Denmark)

    Zhang, Yang

    that chemical bonds exist. However, it is still not excluded that chemical bonding is part of the mechanism. The second hypothesis is that the laser track has a stronger attraction work to the activation solution. This is proved by a calculation using van Oss et al., theory based on contact angle measurement...... of an antenna. Two simple antenna shapes, planar dipole, and planar loop were studied, as well as a co-planar stripline. The antennas made by LISA exhibited impedance characteristics expected from the respective antenna shapes and comparable to antennas made by PCB technology....

  13. Hybrid Chirped Pulse Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Jovanovic, I; Barty, C P J

    2002-05-07

    We present a novel chirped pulse amplification method which combines optical parametric amplification and laser amplification. We have demonstrated this hybrid CPA concept with a combination of beta-barium borate and Ti:sapphire. High-efficiency, multi-terawatt compatible amplification is achieved without gain narrowing and without electro-optic modulators using a simple commercial pump laser.

  14. Estimation of Kinetic Parameters for Autocatalytic Oxidation of Cyclohexane Based on a Modified Adaptive Genetic Algorithm

    Institute of Scientific and Technical Information of China (English)

    刘平乐; 邹丽珊; 罗和安; 王良芥; 郑金华

    2004-01-01

    A modified genetic algorithm of multiple selection strategies, crossover strategies and adaptive operator is constructed, and it is used to estimate the kinetic parameters in autocatalytic oxidation of cyclohexane. The influences of selection strategy, crossover strategy and mutation strategy on algorithm performance are discussed. This algorithm with a specially designed adaptive operator avoids the problem of local optimum usually associated with using standard genetic algorithm and simplex method. The kinetic parameters obtained from the modified genetic algorithm are credible and the calculation results using these parameters agree well with experimental data. Furthermore, a new kinetic model of cyclohexane autocatalytic oxidation is established and the kinetic parameters are estimated by using the modified genetic algorithm.

  15. 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)

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

    International Nuclear Information System (INIS)

    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)

  17. Compositional and stable carbon isotopic fractionation during non-autocatalytic thermochemical sulfate reduction by gaseous hydrocarbons

    Science.gov (United States)

    Xia, Xinyu; Ellis, Geoffrey S.; Ma, Qisheng; Tang, Yongchun

    2014-01-01

    The possibility of autocatalysis during thermochemical sulfate reduction (TSR) by gaseous hydrocarbons was investigated by examination of previously reported laboratory and field data. This reaction was found to be a kinetically controlled non-autocatalytic process, and the apparent lack of autocatalysis is thought to be due to the absence of the required intermediate species. Kinetic parameters for chemical and carbon isotopic fractionations of gaseous hydrocarbons affected by TSR were calculated and found to be consistent with experimentally derived values for TSR involving long-chain hydrocarbons. Model predictions based on these kinetic values indicate that TSR by gaseous hydrocarbon requires high-temperature conditions. The oxidation of C2–5 hydrocarbons by sulfate reduction is accompanied by carbon isotopic fractionation with the residual C2–5 hydrocarbons becoming more enriched in 13C. Kinetic parameters were calculated for the stable carbon isotopic fractionation of gaseous hydrocarbons that have experienced TSR. Model predictions based on these kinetics indicate that it may be difficult to distinguish the effects of TSR from those of thermal maturation at lower levels of hydrocarbon oxidation; however, unusually heavy δ13C2+ values (>−10‰) can be diagnostic of high levels of conversion (>50%). Stoichiometric and stable carbon isotopic data show that methane is stable under the investigated reaction conditions and is likely a product of TSR by other gaseous hydrocarbons rather than a significant reactant. These results indicate that the overall TSR reaction mechanism for oxidation of organic substrates containing long-chain hydrocarbons involves three distinct phases as follows: (1) an initial slow and non-autocatalytic stage characterized by the reduction of reactive sulfate by long-chain saturated hydrocarbons; (2) a second autocatalytic reaction phase dominated by reactions involving reduced sulfur species and partially oxidized hydrocarbons; (3

  18. Permanganate oxidation of α-amino acids: kinetic correlations for the nonautocatalytic and autocatalytic reaction pathways.

    Science.gov (United States)

    Perez-Benito, Joaquin F

    2011-09-01

    The reactions of permanganate ion with seven α-amino acids in aqueous KH(2)PO(4)/K(2)HPO(4) buffers have been followed spectrophotometrically at two different wavelengths: 526 nm (decay of MnO(4)(-)) and 418 nm (formation of colloidal MnO(2)). All of the reactions studied were autocatalyzed by colloidal MnO(2), with the contribution of the autocatalytic reaction pathway decreasing in the order glycine > l-threonine > l-alanine > l-glutamic acid > l-leucine > l-isoleucine > l-valine. The rate constants corresponding to the nonautocatalytic and autocatalytic pathways were obtained by means of either a differential rate law or an integrated one, the latter requiring the use of an iterative method for its implementation. The activation parameters for the two pathways were determined and analyzed to obtain statistically significant correlations for the series of reactions studied. The activation enthalpy of the nonautocatalytic pathway showed a strong, positive dependence on the standard Gibbs energy for the dissociation of the protonated amino group of the α-amino acid. Linear enthalpy-entropy correlations were found for both pathways, leading to isokinetic temperatures of 370 ± 21 K (nonautocatalytic) and 364 ± 28 K (autocatalytic). Mechanisms in agreement with the experimental data are proposed for the two reaction pathways.

  19. 环介导等温扩增(LAMP)技术检测蜜蜂球囊菌%Diagnosis of the Ascosphaera apis by the Loop-Mediated Isothermal Amplification

    Institute of Scientific and Technical Information of China (English)

    席伟军; 李江红; 陈大福; 粱勤

    2016-01-01

    [目的]建立一种快速、灵敏的检测蜜蜂球囊菌(Ascosphaera apis)的环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法,为监测和防治蜜蜂白垩病提供技术支撑.[方法]根据蜜蜂球囊菌特异性序列ITS区,用在线引物设计软件PrimerExplorer V4.0设计并合成4条特异性引物A.apis-F3(5'-ACATTGCGCCCTCTGGTA-3')、A.apis-B3(5’-TGGTTAGACCGGACAGTCG-3’)、A.apis-FIP(5’-TAAGACGGGACGATCGCCC AACCTGTCCGAGCGTCATTG-3')和A.apis-BIP(5'-GAAAGGCAGTGACGGCGTCGGGCCACTAGAGCGAAAGAC-3’),进行LAMP扩增试验,分别设置Mg2+终浓度为0、2、4、6、8、1 0、12、14 mmol·L-1,dNPTs终浓度为0、0.2、0.4、0.6、0.8、1.0、1.2、1.4、1.6、1.8 mmol·L-1,内引物FIB/BIF终浓度为0.2、0.4、0.6、0.8、1.0、1.2、1.4、1.6、1.8mmol·L-1,甜菜碱终浓度分别为0、0.2、0.4、0.6、0.8、1.0、1.2 mol·L-1,反应温度为58、60、63、65℃,反应时间分别为30、40、50、60 min,并利用实时浊度仪测定浊度值和扩增产物进行琼脂糖凝胶电泳来检测LAMP反应的结果,确定优化的LAMP检测体系.以东方蜜蜂微孢子虫(Nosema ceranae)、蜜蜂微孢子虫(Nosema apis)、蜜蜂残翅病毒(Deformed wing virus,DWV)、蜜蜂囊状幼虫病毒(Sacbrood virus,SBV)、黑蜂王台病毒(Black queen cell virus,BQCV)和以色列急性麻痹病毒(Israel acute paralysis virus,IAPV)基因组为模板进行特异性验证.并用PvuI酶切验证产物,最终确定LAMP反应体系的准确性;进而通过将蜜蜂球囊菌基因组DNA进行10倍梯度稀释,分别获得DNA浓度0.2231、0.2231×10-1、0.2231×10-2、0.2231×10-3、0.2231×10-4、02231×10-3、0.2231×10-5、0.2231×10-1、0.2231×10-8 μg·μL-1作为模板,比较LAMP和普通PCR检测灵敏度,并检测验证该技术在临床应用上的可行性.[结果]建立了一种特异检测蜜蜂球囊菌的方法,反应条件优化后的检测体系为4mmol·L-1Mg2+、1.2 mmol·L-1 dNTPs、1.6 mmol·L-1FIP/BIP、0.4 mol·L-1

  20. 食品中单核细胞增生李斯特菌DNA环介导恒温扩增快速检测方法的建立%Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Listeria monocytogenes in Foods

    Institute of Scientific and Technical Information of China (English)

    徐义刚; 崔丽春; 李丹丹; 刘忠梅; 李苏龙; 张子群; 张国财

    2012-01-01

    According to the iap gene of Listeria monocytogenes, two pairs of specific primers were designed, and then a rapid loop-mediated isothermal amplification (LAMP) assay for detecting Listeria monocytogenes in foods was developed using real- time turbidity of the amplification byproduct magnesium pyrophosphate as the positive criterion. Twenty-nine bacterial strains were used to evaluate the specificity of the LAMP method. Our results showed that all tested Listeria monocytogenes were positive, while other strains were negative to LAMP detection, suggesting that this LAMP method was highly specific to the target bacteria. The sensitivity for cultivated Listeria monocytogenes and its contaminated foods were was 8 CFU and 12 CFU per test tube, respectively. The LAMP method developed in this study provides a sensitive, rapid and simple approach for the detection of Listeria monocytogenes.%基于单核细胞增生李斯特菌胞壁质水解酶iap基因,设计两对特异性引物,利用DNA环介导恒温扩增(loop-mediated isothermal amplification,LAMP)技术,以扩增副产物焦磷酸镁实时浊度为判定标准,建立食品中单核细胞增生李斯特菌LAMP快速检测方法。结果显示,本LAMP方法特异性强,经过对29株细菌进行检测,所试单核细胞增生李斯特菌均为LAMP阳性,其他菌株为阴性;本LAMP方法对单核细胞增生李斯特菌纯培养菌的检测灵敏度为8CFU/管,对污染食品中单核细胞增生李斯特菌的检测灵敏度为12CFU/管。本研究建立的LAMP检测方法简便快速、结果判断直观。

  1. 环介导等温扩增技术检测含有CaMV35S的转基因玉米%Loop-mediated Isothermal Amplification for the Detection of CaMV35S Promoter in Genetically Modified Maize

    Institute of Scientific and Technical Information of China (English)

    陈金松; 黄丛林; 张秀海; 吴忠义

    2011-01-01

    The objective is to develop a loop-mediated isothermal amplification method for detection of CaMV-35S promoter in Genetically Modified maize. Loop-mediated isothermal amplification (LAMP) , a novel nucleic acid amplification method, was developed for the rapid detection of Genetically Modified maize. Cauliflower mosaic virus 35S (CaMV35S) promoter gene was amplified by a set of four primers that recognize six distinct sequences of the target. The optimized conditions for LAMP were studied using different Mg2+ , dNTPs, Betaine and primers concentrations. Furthermore, the sensitivity of LAMP was tested comparing with PCR. The LAMP method can detect CaMV35S promoter from genetically modified maize and their test results were consistent with the results of conventional PCR method. LAMP assay results were found to be more sensitive than the conventional PCR. The LAMP assay is an extremely rapid, highly sensitive, specific method, and will be an effective tool for rapid detection of Genetically Modified maize.%DNA环介导等温扩增技术是一种特异、灵敏、快速的新型基因检测技术.针对玉米表达载体的花椰菜花叶病毒35 S启动子(CaMV35S)的6个区域设计4种特异引物,对LAMP反应的MgSO4、dNTPs、Betaine、内引物、外引物各个成分进行了优化,此外还对LAMP和PCR两种不同方法的特异性进行了比较.建立转基因玉米花椰菜花叶病毒35 S启动子环介导等温扩增技术检测方法,LAMP与PCR相比具有更高的灵敏度.环介导等温扩增技术检测方法具有时间少,成本低,特异性高,检测方法多样等优势,为检测转基因玉米花椰菜花叶病毒35 S启动子提供了一种更加简便快速的方法.

  2. Autocatalytic growth of Co on pure Co surfaces using Co{sub 2}(CO){sub 8} precursor

    Energy Technology Data Exchange (ETDEWEB)

    Cordoba, R.; Sese, J.; Ibarra, M.R. [Laboratorio de Microscopias Avanzadas (LMA), Instituto de Nanociencia de Aragon (INA), Universidad de Zaragoza, E-50018 Zaragoza (Spain); Departamento de Fisica de la Materia Condensada, Universidad de Zaragoza, E-50009 Zaragoza (Spain); De Teresa, J.M., E-mail: deteresa@unizar.es [Laboratorio de Microscopias Avanzadas (LMA), Instituto de Nanociencia de Aragon (INA), Universidad de Zaragoza, E-50018 Zaragoza (Spain); Departamento de Fisica de la Materia Condensada, Universidad de Zaragoza, E-50009 Zaragoza (Spain); Instituto de Ciencia de Materiales de Aragon (ICMA), Facultad de Ciencias, Universidad de Zaragoza-CSIC, E-50009 Zaragoza (Spain)

    2012-12-15

    Highlights: Black-Right-Pointing-Pointer We investigate the autocatalytic growth of Co using Co{sub 2}(CO){sub 8} precursor. Black-Right-Pointing-Pointer On Si wafers and Co grown by FEBID, no role is played by autocatalytic growth. Black-Right-Pointing-Pointer On Co films grown by sputtering, Co grows autocatalytically. Black-Right-Pointing-Pointer Implications of the results on Co by FEBID are discussed. - Abstract: The autocatalytic growth of Co on different surfaces using the Co{sub 2}(CO){sub 8} precursor is investigated. It is observed that Co{sub 2}(CO){sub 8} molecules dissociate spontaneously on pure Co surfaces grown by sputtering, forming a pure Co film. The microstructure of this film consists of Co nanocrystals with size below 100 nm. However, when the same type of experiment is done on a Co surface grown by focused-electron-beam induced deposition there is no autocatalytic growth of Co. On other surfaces such as Si substrates and Al films grown by sputtering, the spontaneous dissociation of the Co{sub 2}(CO){sub 8} molecules does not occur. The origin and implications of these results are discussed.

  3. 环介导恒温扩增技术快速检测对虾白斑综合征病毒方法的建立%Establishment of Loop-mediated Isothermal Amplification for Detection of White spot syndrome virus

    Institute of Scientific and Technical Information of China (English)

    李明云; 丁文超; 陈炯; 史雨红

    2011-01-01

    DNA环介导恒温扩增(loop-mediated isothermal amplification,LAMP)技术是一种特异、灵敏、快速的新型基因检测技术,其检测结果可以通过琼脂糖凝胶电泳观察,也可以通过加入核酸染料SYBR Green I进行肉眼观察.本研究根据对虾白斑综合征病毒(White spot syndrome virus,WSSV)的结构蛋白VP26基因序列,设计一套引物,成功建立了针对WSSV的LAMP检测方法.应用该方法,在65℃温育1h的条件下快速扩增病毒基因组DNA,琼脂糖凝胶电泳得到特异性梯度条带;在反应体系中添加SYBR Green I染料后,可通过肉眼观察有无荧光直接判定结果.该研究建立的LAMP法能特异性检出WSSV,其检测下限比PCR法低一个数量级,相当于0.563 pg/μL的质粒印MD19-T-VP26)浓度.表明所建立的WSSV LAMP检测法特异性好、灵敏度高、操作简单方便,有望作为WSSV快速诊断的常规方法.%Loop-mediated isothermal amplification (LAMP) method is a novel gene detection technique with specificity, sensitive and rapidity, the products of which can be electrophoresed in 1.5% agarose gels, or visually inspected by adding nucleic acid dye (SYBR Green Ⅰ ). In this paper, a one step loop-mediated isothermal amplification (LAMP) assay was developed for detection of White spot syndrome virus (WSSV). A set of primers were designed from the VP26 gene sequence of WSSV. The assay was established to amplify WSSV genomic DNA by incubation at 65℃ for only 1 h, and required only a simple water bath or heating block to provide a constant temperature of 65℃. LAMP amplification products had a ladder-like appearance when electrophoresed on an agarose gel. With the addition of SYBR Green Ⅰ, the presence of the target DNA could be detected by naked eyes. The detection limit of plasmid DNA (pMD19-T-VP26) by LAMP was 0.563 pg/μL which was found to be lower than that of the commonly used PCR method. The results indicate the suitability and simplicity of the test as

  4. The Transition Towards Immortality: Non-linear Autocatalytic Growth of Citations to Scientific Papers

    Science.gov (United States)

    Golosovsky, Michael; Solomon, Sorin

    2013-04-01

    We discuss microscopic mechanisms of complex network growth, with the special emphasis of how these mechanisms can be evaluated from the measurements on real networks. As an example we consider the network of citations to scientific papers. Contrary to common belief that its growth is determined by the linear preferential attachment, our microscopic measurements show that it is driven by the nonlinear autocatalytic growth. This invalidates the scale-free hypothesis for the citation network. The nonlinearity is responsible for a dramatic dynamical phase transition: while the citation lifetime of majority of papers is 6-10 years, the highly-cited papers have practically infinite lifetime.

  5. The transition towards immortality: non-linear autocatalytic growth of citations to scientific papers

    CERN Document Server

    Golosovsky, Michael

    2013-01-01

    We discuss microscopic mechanisms of complex network growth, with the special emphasis of how these mechanisms can be evaluated from the measurements on real networks. As an example we consider the network of citations to scientific papers. Contrary to common belief that its growth is determined by the linear preferential attachment, our microscopic measurements show that it is driven by the nonlinear autocatalytic growth. This invalidates the scale-free hypothesis for the citation network. The nonlinearity is responsible for a dramatic dynamical phase transition: while the citation lifetime of majority of papers is 6-10 years, the highly-cited papers have practically infinite lifetime.

  6. Development of new type passive autocatalytic recombiner. Part 2. Proposal of conceptual structure

    International Nuclear Information System (INIS)

    A new type passive autocatalytic recombiner to mitigate released hydrogen gas at the accident of nuclear power plants and related facilities has been developed. In order to realize easy and anywhere installation, this new recombiner has advanced automotive catalysts with features such as light and small, high durability, and high performance. Downsizing and light-weighting of recombiner has been conducted with thermal hydraulic and structural analysis incorporating an automotive catalyst model and its characterization results obtained through experiments. Thermal hydraulic and structural analysis show PAR concept proposed is feasible for hydrogen mitigation in nuclear power plants and related facilities. (author)

  7. Estimation of Critical Rate of Temperature Rise for Thermal Explosion of First Order Autocatalytic Decomposition Reaction Systems by Using Non-isothermal DSC

    Institute of Scientific and Technical Information of China (English)

    GUO Peng-jiang; LU Gui-e; JIANG Ji-you; HU Rong-zu; ZHANG Hai; XIA Zhi-ming; SONG Ji-rong; GAO Sheng-li; NING Bin-ke; SHI Qi-zhen; LIU Rong

    2004-01-01

    A method of estimating the critical rate of temperature rise for the thermal explosion of first order autocatalytic decomposition reaction systems by using non-isothermal DSC is presented. The information was obtained on the increasing rate of temperature for the first order autocatalytic decomposition of nitrocellulose containing 13.86% nitrogen converting into the thermal explosion.

  8. Demonstrative testing of honeycomb passive autocatalytic recombiner for nuclear power plant

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jae-Won, E-mail: jpark@kepco-enc.com [KEPCO E and C, 257 Yonggudaero, Giheung-gu, Yongin-si, Gyeonggi-do (Korea, Republic of); Koh, Byung-Ryung [Korea Nuclear Technology, 1687-2, Shinil-dong, Daeduk-gu, Daejeon (Korea, Republic of); Suh, Kune Y. [Department of Nuclear Engineering, Seoul National University, San 56-1, Sillim-dong, Kwanak-gu, Seoul (Korea, Republic of)

    2011-10-15

    Highlights: > A new PAR (Passive autocatalytic recombiner) for hydrogen control. > It uses honeycomb type catalyst. > It has an improved performance of hydrogen removal. > Performance tests in specially manufactured test facilities. > Development of correlation of hydrogen depletion rate from the test results. - Abstract: Passive autocatalytic recombiners (PAR) are widely being used as hydrogen control device in the current and advanced light water reactors (ALWRs). The PARs lend themselves to very effective means of circumventing buildup of combustible or detonable hydrogen gas mixtures in the reactor containment. Korea Nuclear Technology Inc. has recently developed a new PAR system with high porous catalyst material in the shape of honeycomb. The honeycomb PAR catalyst has a design characteristic of improved hydrogen removal performance by increasing the surface area and enhancing the flow rate through the catalyst at the same time, without increasing PAR size compared to the conventional PARs. The experimental study was focused on the development of the hydrogen depletion rate correlation of the honeycomb PAR. Two different sizes of PARs, KPAR-40 and KPAR-T2, have been employed in the tailor-made Integral Test Facility and Performance Test Facility. Multiple tests were conducted in various conditions of pressure, temperature, and hydrogen concentration. The hydrogen depletion rate correlation and the PAR performance constant were determined from the experimental results, which can be applied to the honeycomb PAR system. Also determined was the scale effect due to the PAR size, i.e., the number of catalysts in a PAR.

  9. Prebiotic Synthesis of Autocatalytic Products From Formaldehyde-Derived Sugars as the Carbon and Energy Source

    Science.gov (United States)

    Weber, Arthur L.

    2003-01-01

    Our research objective is to understand and model the chemical processes on the primitive Earth that generated the first autocatalytic molecules and microstructures involved in the origin of life. Our approach involves: (a) investigation of a model origin-of-life process named the Sugar Model that is based on the reaction of formaldehyde- derived sugars (trioses and tetroses) with ammonia, and (b) elucidation of the constraints imposed on the chemistry of the origin of life by the fixed energies and rates of C,H,O-organic reactions under mild aqueous conditions. Recently, we demonstrated that under mild aqueous conditions the Sugar Model process yields autocatalytic products, and generates organic micropherules (2-20 micron dia.) that exhibit budding, size uniformity, and chain formation. We also discovered that the sugar substrates of the Sugar Model are capable of reducing nitrite to ammonia under mild aqueous conditions. In addition studies done in collaboration with Sandra Pizzarrello (Arizona State University) revealed that chiral amino acids (including meteoritic isovaline) catalyze both the synthesis and specific handedness of chiral sugars. Our systematic survey of the energies and rates of reactions of C,H,O-organic substrates under mild aqueous conditions revealed several general principles (rules) that govern the direction and rate of organic reactions. These reactivity principles constrain the structure of chemical pathways used in the origin of life, and in modern and primitive metabolism.

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

    KAUST Repository

    Bukhryakov, Konstantin V.

    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.

  11. 人细小病毒 B19的环介导等温扩增快速检测方法的建立%Establishment of A rapid method for detection of the human parvovirus B19 by a loop-mediated isothermal amplification

    Institute of Scientific and Technical Information of China (English)

    张彦东; 邬巍; 马宁; 刘涛; 王凯; 姜振宁

    2015-01-01

    目的:建立一种简单、快速的核酸检测新方法,即环介导等温扩增方法(LAMP)来检测人细小病毒 B19的核酸。方法从 GenBank 中获得 B19病毒 VP2基因序列,利用 PrimerExplorer V3软件在序列保守区域设计 LAMP引物,同时对试验中的几个重要参教进行优化,并评价其敏感性和特异性。结果 LAMP 检测方法的敏感性高于PCR 方法,并且对 B19病毒的检测具有特异性。结论建立了环介导等温扩增快速检测方法,此方法灵敏性高、特异性强,不仅适用于实验室检测,更能够广泛用于临床检测。%Objective To establish a simple and rapid new method for nucleic acid detection,loop-mediated isother-mal amplification (LAMP)to detect human parvovirus B19 nucleic acid.Methods B19 virus of VP2 gene was down-loaded from the GenBank,then primers of LAMP designed using PrimerExplorer V3 software from conserved regions, while the test of several important parameters to optimize teaching and evaluation of its sensitivity and specificity.Re-sults LAMP method is more sensitive than PCR detection method,and specific on the detection of B19.Conclusion Loop-mediated isothermal amplification rapid detection methods has high sensitivity and specificity,not only suitable for laboratory testing,but can be widely used in clinical testing.

  12. 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.

  13. The Seneca Amplification Construction

    Directory of Open Access Journals (Sweden)

    Wallace Chafe

    2012-01-01

    Full Text Available The polysynthetic morphology of the Northern Iroquoian languages presents a challenge to studies of clause combining. The discussion here focuses on a Seneca construction that may appear within a single clause but may also straddle clause boundaries. It amplifies the information provided by a referent, here called the trigger, that is introduced by the pronominal prefix within a verb or occasionally in some other way. The particle neh signals that further information about that referent will follow. This construction is found at four levels of syntactic complexity. At the first level the trigger and its amplification occur within the same prosodic phrase and the amplification is a noun. At the second level the amplification occurs in a separate prosodic phrase but remains a noun. At the third level the amplification exhibits verb morphology but has been lexicalized with a nominal function. At the fourth level the amplification functions as a full clause and neh serves as a marker of clause combining. Several varieties of amplification are discussed, as are cases in which the speaker judges that no amplification is needed. It is suggested that the typologically similar Caddo language illustrates a situation in which this construction could never arise, simply because Caddo verbs lack the pronominal element that triggers the construction in Seneca.

  14. Research of the event-specific loop-mediated isothermal amplification method for detection of transgenic maize Bt176%转基因玉米Bt176品系特异性环介导等温扩增检测方法的研究

    Institute of Scientific and Technical Information of China (English)

    凌莉; 刘静宇; 易敏英; 吴少云; 刘津; 蔡颖; 李志勇; 高东微

    2013-01-01

    根据转基因玉米品系Bt176外源插入片段与玉米基因组序列设计和筛选品系特异性引物,并对反应体系和反应条件进行优化,最终建立转基因玉米品系Bt176的品系特异性LAMP检测方法.对该方法进行了灵敏度、特异性和稳定性评价.评价结果表明:该方法定性检测低限为0.5%,特异性和稳定性均达到100%.%According to the exogenous insert sequences of the transgenic maize event Bt176 connected with the maize genome sequences,the event specific primers were designed and screened as well as the reaction system and the parameters were optimized. An event specific loop - mediatec isothermal amplification method for the detection of transgenic maize event Bt176 was established in this paper.Moreover,an evaluation of this method on its specificity,sensitivity and stability was performed.The evaluation results showed the LOD of 0.5% ,and both the specificity and stability of 100%.

  15. Development of new type passive autocatalytic recombiner. Part 1. Characterization of monolithic catalyst

    International Nuclear Information System (INIS)

    For hydrogen mitigation, a new type passive autocatalytic recombiner has been developed, as one of safety accident managements at a nuclear power plant. This new recombiner has an automotive monolithic substrate catalyst in order to reduce weight and to improve hydrogen treating capacity, environmental resistance, and product quality. The activation energy has been experimentally estimated at 5.75 kJ/mol with stoichiometric composition gas flow. The fed hydrogen and oxygen to the monolithic catalyst were immediately recombined in both the dry and the humid atmospheres. Moreover, it has been found that gamma-ray irradiation up to 1.0 MGy could promote catalytic activity, because the specific area of the catalyst and the surface area of the precious metals increased. In addition, no significant difference was observed in the compositional distribution in the monolithic catalyst by EPMA. As the result, an automotive monolithic catalyst is applicable to practical installation of PAR into NPPs. (author)

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

    Energy Technology Data Exchange (ETDEWEB)

    Kelm, S.; Reinecke, E-A.; Jahn, W., E-mail: s.kelm@fz-juelich.de [Forschungszentrum Juelich GmbH, Juelich (Germany); Allelein, H-J. [RWTH Aachen Univ.. Aachen (Germany)

    2011-07-01

    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)

  17. Integrated hydrogen control solutions for severe accidents using passive autocatalytic recombiners

    Energy Technology Data Exchange (ETDEWEB)

    Bauer, M.; Tietsch, W.; Sabate Farnos, R.

    2012-07-01

    In a severe accident or a beyond-design-basis-accident, the reaction of water with zirconium alloy cladding, radiolysis of water, corium-concrete reactions and other corrosion phenomena generate hydrogen (H2). The detonation of this H2 in containment or in auxiliary buildings can result in damage to structures or loss of containment integrity. Identifying the generation and special distribution of hydrogen and controlling its concentration with Passive Autocatalytic Recombiners (PARs) solves this concern. Westinghouse's approach for hydrogen management starts by defining the quantities and transport/distribution of H{sub 2} in-containment and out of containment with analysis tools such as MAAP, MELCOR, GASFLOW or FATE. Based on the results of these analyses, an optimized H2 Control Strategy is proposed in terms of number and location of PARs, and efficient integration with other H{sub 2} management devices like e.g. existing igniters, H{sub 2} monitors, etc.

  18. Autocatalytic closure in a cognitive system A tentative scenario for the origin of culture

    CERN Document Server

    Gabora, L

    1999-01-01

    This paper presents a speculative model of the cognitive mechanisms underlying the transition from episodic to mimetic (or memetic) culture with the arrival of Homo erectus, which Donald [1991] claims paved the way for the unique features of human culture. The model draws on Kauffman's [1993] theory of how an information-evolving system emerges through the formation of an autocatalytic network. Though originally formulated to explain the origin of life, this theory also provides a plausible account of how discrete episodic memories become woven into an internal model of the world, or worldview, that both structures, and is structured by, self-triggered streams of thought. Social interaction plays a role in (and may be critical to) this process. Implications for cognitive development are explored.

  19. Effects of intrinsic noise on a cubic autocatalytic reaction-diffusion system.

    Science.gov (United States)

    Cooper, Fred; Ghoshal, Gourab; Pérez-Mercader, Juan

    2014-06-01

    Starting from our recent chemical master equation derivation of the model of an autocatalytic reaction-diffusion chemical system with reactions U+2V→[over λ_{0}]3V and V→[over μ]P, U→[over ν]Q, we determine the effects of intrinsic noise on the momentum-space behavior of its kinetic parameters and chemical concentrations. We demonstrate that the intrinsic noise induces n→n molecular interaction processes with n≥4, where n is the number of participating molecules of type U or V. The momentum dependences of the reaction rates are driven by the fact that the autocatalytic reaction (inelastic scattering) is renormalized through the existence of an arbitrary number of intermediate elastic scatterings, which can also be interpreted as the creation and subsequent decay of a three body composite state σ=ϕ_{u}ϕ_{v}^{2}, where ϕ_{i} corresponds to the fields representing the densities of U and V. Finally, we discuss the difference between representing σ as a composite or an elementary particle (molecule) with its own kinetic parameters. In one dimension, we find that while they show markedly different behavior in the short spatiotemporal scale, high-momentum (UV) limit, they are formally equivalent in the large spatiotemporal scale, low momentum (IR) regime. On the other hand, in two dimensions and greater, due to the effects of fluctuations, there is no way to experimentally distinguish between a fundamental and composite σ. Thus, in this regime, σ behaves as an entity unto itself, suggesting that it can be effectively treated as an independent chemical species.

  20. The Detection of Infectious Salmon Anaemia Virus Using Real-Time Fluorescent Loop-Mediated Isothermal Amplification%传染性鲑鱼贫血症病毒实时荧光环介导等温扩增检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    史秀杰; 于力; 王津津; 何俊强; 郑晓聪; 贾鹏兰; 文升; 杨锦舜; 刘荭

    2014-01-01

    根据ISAV的基因保守序列,利用LAMP Designer软件设计了6条引物,采用新型的环介导等温扩增设备进行扩增和检测,优化了反应条件,分析了所建立方法的特异性和灵敏度,并与RT-PCR和实时荧光RT-PCR进行比较。研究表明,该方法最适反应温度为64℃,反应10 min就可以观察到明显的扩增。该方法灵敏度高,检测限为78.4 fg RNA,比常规RT-PCR灵敏度高100倍,与实时荧光定量RT-PCR灵敏度相当;特异性好,与传染性胰腺坏死病毒(IPNV)、鲤春病毒血症病毒(SVCV)、出血性败血症病毒(VHSV)、鱼类病毒性神经坏死病病毒(VNNV)、鱼腹水病毒(YAV)等14种主要鱼类病毒没有交叉反应。结果表明,本研究建立了 ISAV 的实时荧光环介导等温扩增检测方法,实验能对整个扩增过程进行实时监测,提高检测灵敏度的同时,防止由于开盖跑电泳或加染料而导致的污染。%The infectious salmon anaemia virus (ISAV) is classified as an Orthomyxoviridae. Its genome consists of 8 single-stranded negative-sense RNA segments. ISAV is the pathogen of fatal ISA listed by the World Organization for Animal Health (OIE). It mainly affects salmon farming in Europe and Northern America, but there has been a high chance of its introduction into China due to the increased salmon importation. Therefore it is very important to establish a rapid and accurate method for ISAV detection. Conventional ISAV detection methods involve cell isolation followed by RT-PCR or real-time RT-PCR. Recently Japanese scientists have established a novel technique with high sensitivity and rapidity, namely Loop-mediated isothermal amplification (LAMP) assay. In this study, LAMP assay was developed for detecting infectious salmon anaemia virus (ISAV). Six specific primers were designed according to ISAV genes using LAMP Designer software. A novel LAMP instrument was applied for the amplification and detection. The

  1. Early amplification options.

    Science.gov (United States)

    Gabbard, Sandra Abbott; Schryer, Jennifer

    2003-01-01

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

  2. 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.

  3. Quantum Feedback Amplification

    Science.gov (United States)

    Yamamoto, Naoki

    2016-04-01

    Quantum amplification is essential for various quantum technologies such as communication and weak-signal detection. However, its practical use is still limited due to inevitable device fragility that brings about distortion in the output signal or state. This paper presents a general theory that solves this critical issue. The key idea is simple and easy to implement: just a passive feedback of the amplifier's auxiliary mode, which is usually thrown away. In fact, this scheme makes the controlled amplifier significantly robust, and furthermore it realizes the minimum-noise amplification even under realistic imperfections. Hence, the presented theory enables the quantum amplification to be implemented at a practical level. Also, a nondegenerate parametric amplifier subjected to a special detuning is proposed to show that, additionally, it has a broadband nature.

  4. Evaluation of Loop-mediated isothermal amplification for the rapid detection of M.tuberculosis in sputa%环介导等温扩增技术快速检测痰标本中结核分枝杆菌的初步评价

    Institute of Scientific and Technical Information of China (English)

    于霞; 梁倩; 马异峰; 付育红; 黄海荣

    2013-01-01

    目的 评价环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)技术在结核分枝杆菌诊断中的应用价值.方法 收集246份疑似肺结核患者的痰标本,分别进行涂片镜检,罗氏培养以及实时定量PCR和LAMP法检测.结果 涂片镜检,罗氏培养实时定量PCR和LAMP法对结核分枝杆菌的检出率分别为30.89%,45.53%,54.47%,57.32%.以罗氏培养结果作为金标准,LAMP法诊断的敏感性为96.19%(101/105),特异性为76.87%(103/134),两种方法检测结果的一致性是Κ=0.71,两法具有很好的吻合性.以实时定量法为标准,LAMP法的敏感性为90.07% (127/141),特异性为 93.33%(98/105),两法检测结果的一致性是κ=0.83,P<0.001.结论 LAMP法检测结核分枝杆菌具有较高的敏感性和特异性,与罗氏培养和RT-PCR有着较高的吻合性,很适合作为临床检测项目开展.%Objective To evaluate the diagnostic value of loop-mediated isothermal amplification(LAMP)in the diag nosis of Mycobacterium tuberculosis. Methods Collected 246 sputa from suspected tuberculosis patients and conducted smear microscopy,LJ culture,real time quantitative PCR (RT-PCR)and LAMP assay. Results The detection rate of smear microscopy, LJ culture, RT PCR and LAMP assay for Mycobacterium tuberculosis were 30. 89%, 45. 53%, 54.47% and 57.32% ,respectively. Comparing with culture method,the sensitivity and the specificity of the LAMP as say was 96. 19% (101/105) and 76. 87% (103/134), respectively. The two assays were in good consistency (κ= 0.71). Comparing with RT PCR,the sensitivity and specificity of the LAMP was 90.07% (127/141) and 93.33G(98/ 105) ,respectively. The two assays were in good consistency(κ= 0.83) . Conclusion LAMP assay for rapid detection of Mycobacterium tuberculosis with high sensitivity and specificity, has high consistency with culture and RT-PCR,and is very suitable for routine clinical test.

  5. Autocatalytic activation of the furin zymogen requires removal of the emerging enzyme's N-terminus from the active site.

    Directory of Open Access Journals (Sweden)

    Katarzyna Gawlik

    Full Text Available Before furin can act on protein substrates, it must go through an ordered process of activation. Similar to many other proteinases, furin is synthesized as a zymogen (profurin which becomes active only after the autocatalytic removal of its auto-inhibitory prodomain. We hypothesized that to activate profurin its prodomain had to be removed and, in addition, the emerging enzyme's N-terminus had to be ejected from the catalytic cleft.We constructed and analyzed the profurin mutants in which the egress of the emerging enzyme's N-terminus from the catalytic cleft was restricted. Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107 downward arrowAsp(108, but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75 downward arrowSer(76 cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108. These correctly processed furin mutants, however, remained self-inhibited by the constrained N-terminal sequence which continuously occupied the S' sub-sites of the catalytic cleft and interfered with the functional activity. Further, using the in vitro cleavage of the purified prodomain and the analyses of colon carcinoma LoVo cells with the reconstituted expression of the wild-type and mutant furins, we demonstrated that a three-step autocatalytic processing including the cleavage of the prodomain at the previously unidentified Arg-Leu-Gln-Arg(89 downward arrowGlu(90 site, is required for the efficient activation of furin.Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms. In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.

  6. [High titer ethanol production from an atmospheric glycerol autocatalytic organosolv pretreated wheat straw].

    Science.gov (United States)

    Wang, Liang; Liu, Jianquan; Zhang, Zhe; Zhang, Feiyang; Ren, Junli; Sun, Fubao; Zhang, Zhenyu; Ding, Cancan; Lin, Qiaowen

    2015-10-01

    The expensive production of bioethanol is because it has not yet reached the 'THREE-HIGH' (High-titer, high-conversion and high-productivity) technical levels of starchy ethanol production. To cope with it, it is necessary to implement a high-gravity mash bioethanol production (HMBP), in which sugar hydrolysates are thick and fermentation-inhibitive compounds are negligible. In this work, HMBP from an atmospheric glycerol autocatalytic organosolv pretreated wheat straw was carried out with different fermentation strategies. Under an optimized condition (15% substrate concentration, 10 g/L (NH4)2SO4, 30 FPU/g dry matter, 10% (V/V) inoculum ratio), HMBP was at 31.2 g/L with a shaking simultaneous saccharification and fermentation (SSF) at 37 degrees C for 72 h, and achieved with a conversion of 73% and a productivity of 0.43 g/(L x h). Further by a semi-SFF with pre-hydrolysis time of 24 h, HMBP reached 33.7 g/L, the conversion and productivity of which was 79% and 0.47 g/(L x h), respectively. During the SSF and semi-SSF, more than 90% of the cellulose in both substrates were hydrolyzed into fermentable sugars. Finally, a fed-batch semi-SFF was developed with an initial substrate concentration of 15%, in which dried substrate (= the weight of the initial substrate) was divided into three portions and added into the conical flask once each 8 h during the first 24 h. HMBP achieved at 51.2 g/L for 72 h with a high productivity of 0.71 g/(L x h) while a low cellulose conversion of 62%. Interestingly, the fermentation inhibitive compound was mainly acetic acid, less than 3.0 g/L, and there were no other inhibitors detected, commonly furfural and hydroxymethyl furfural existing in the slurry. The data indicate that the lignocellulosic substrate subjected to the atmospheric glycerol autocatalytic organosolv pretreatment is very applicable for HMBP. The fed-batch semi-SFF is effective and desirable to realize an HMBP. PMID:26964336

  7. Induction cascade with electro-explosive commutation of current for amplification of electric pulse power

    CERN Document Server

    Grabovskij, E V; Kuznetsov, V V; Lototskij, A P; Khaustov, E V; Khalimullin, Y A; Kasyanov, N Y; Kormilitsyn, A I; Filatov, V A; Shkolnikov, E Y

    2002-01-01

    Paper describes a circuit of power amplification induction cascade based on a two-loop solenoid and electrically exploded conductors serving as current breakers. Due to retention of the general magnetic flow current breaking in the first loop of accumulator results in current amplification in the second loop and in accelerated actuation of the second electrically exploded conductor. Current switching to load occurs with 20-fold reduction of charging current front duration and increase of its amplitude. Time to charge coil is selected within 300-350 mu s limits

  8. Necessary conditions for the emergence of homochirality via autocatalytic self-replication

    Science.gov (United States)

    Stich, Michael; Ribó, Josep M.; Blackmond, Donna G.; Hochberg, David

    2016-08-01

    We analyze a recent proposal for spontaneous mirror symmetry breaking based on the coupling of first-order enantioselective autocatalysis and direct production of the enantiomers that invokes a critical role for intrinsic reaction noise. For isolated systems, the racemic state is the unique stable outcome for both stochastic and deterministic dynamics when the system is in compliance with the constraints dictated by the thermodynamics of chemical reaction processes. In open systems, the racemic outcome also results for both stochastic and deterministic dynamics when driving the autocatalysis unidirectionally by external reagents. Nonracemic states can result in the latter only if the reverse reactions are strictly zero: these are kinetically controlled outcomes for small populations and volumes, and can be simulated by stochastic dynamics. However, the stability of the thermodynamic limit proves that the racemic outcome is the unique stable state for strictly irreversible externally driven autocatalysis. These findings contradict the suggestion that the inhibition requirement of the Frank autocatalytic model for the emergence of homochirality may be relaxed in a noise-induced mechanism.

  9. Modeling of Auto-Catalytic Halogen Release and Ozone Depletion in Polar Regions

    International Nuclear Information System (INIS)

    This article concerns the modeling of the tropospheric ozone depletion event in polar spring where the aim is an improved understanding of the underlying physical and chemical processes. For this purpose, a model based on OpenFOAM 1.7.1 is developed, where two-dimensional compressible Navier-Stokes equations are solved numerically by finite volume method to predict the effects of turbulent mixing, advection of the fluid, and detailed chemical reactions. The present chemical reaction mechanism consists of 53 chemical reactions among 33 species to model the auto-catalytic process considering halogen species X, X2, XY, XO, HOX, where X and Y denote halogen atoms - here Br is studied. Large eddy simulation is applied to account for the turbulence and the Smagorinsky model is employed as sub-grid model. Good agreement with literature and experimental data is obtained for the profiles of the chemical species. In particular, the correct time scale of the phenomenon is captured. It is confirmed that the mixing ratio of ozone in the troposphere drops to a value near zero within several days. Moreover, it is shown that the well-mixed air is confined inside the boundary layer. A parameter study shows that the ozone depletion event happens even at reduced initial values of molecular bromine. Also, the study of coupled transport and chemistry shows that the turbulent mixing enhances the ozone depletion in the lowest layer above the earth's surface.

  10. 2D numerical simulation of passive autocatalytic recombiner for hydrogen mitigation

    Science.gov (United States)

    Gera, B.; Sharma, P. K.; Singh, R. K.

    2012-04-01

    Resolving hydrogen related safety issues, pertaining to nuclear reactor safety has been an important area of research world over for the past decade. The studies on hydrogen transport behavior and development of hydrogen mitigation systems are still being pursued actively in various research labs, including Bhabha Atomic Research Centre (BARC), in India. The passive autocatalytic recombiner (PAR) is one of such hydrogen mitigating device consisting of catalyst surfaces arranged in an open-ended enclosure. In the plate type recombiner design sheets made of stainless steel and coated with platinum catalyst material are arranged in parallel inside a flow channel. The catalyst elements are exposed to a constant flow of a mixture of air, hydrogen and steam, a catalytic reaction occurs spontaneously at the catalyst surfaces and the heat of reaction produces natural convection flow through the enclosure. Numerical simulation and experiments are required for an in-depth knowledge of such plate type PAR. Specific finite volume based in-house 2D computational fluid dynamics (CFD) code has been developed to model and analyse the working of these recombiners and has been used to simulate one literature quoted experiment. The validation results were in good agreement against literature quoted German REKO experiments. Parametric study has been performed for particular recombiner geometry for various inlet conditions. Salient features of the simplified CFD model developed at BARC and results of the present model calculations are presented in this paper.

  11. 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.

  12. 2D numerical simulation of passive autocatalytic recombiner for hydrogen mitigation

    Energy Technology Data Exchange (ETDEWEB)

    Gera, B.; Sharma, P.K.; Singh, R.K. [Bhabha Atomic Research Centre, Reactor Safety Division, Trombay, Mumbai (India)

    2012-04-15

    Resolving hydrogen related safety issues, pertaining to nuclear reactor safety has been an important area of research world over for the past decade. The studies on hydrogen transport behavior and development of hydrogen mitigation systems are still being pursued actively in various research labs, including Bhabha Atomic Research Centre (BARC), in India. The passive autocatalytic recombiner (PAR) is one of such hydrogen mitigating device consisting of catalyst surfaces arranged in an open-ended enclosure. In the plate type recombiner design sheets made of stainless steel and coated with platinum catalyst material are arranged in parallel inside a flow channel. The catalyst elements are exposed to a constant flow of a mixture of air, hydrogen and steam, a catalytic reaction occurs spontaneously at the catalyst surfaces and the heat of reaction produces natural convection flow through the enclosure. Numerical simulation and experiments are required for an in-depth knowledge of such plate type PAR. Specific finite volume based in-house 2D computational fluid dynamics (CFD) code has been developed to model and analyse the working of these recombiners and has been used to simulate one literature quoted experiment. The validation results were in good agreement against literature quoted German REKO experiments. Parametric study has been performed for particular recombiner geometry for various inlet conditions. Salient features of the simplified CFD model developed at BARC and results of the present model calculations are presented in this paper. (orig.)

  13. 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.

  14. 环介导等温扩增联合横向流动试纸条可视化检测海豚链球菌方法的建立%Visual Detection of Streptococcus iniae Based on Loop- mediated Isothermal Amplification Combined with a Lateral Flow Dipstick

    Institute of Scientific and Technical Information of China (English)

    王瑞娜; 周前进; 陈炯

    2014-01-01

    海豚链球菌(Streptococcus iniae)是一种革兰氏阳性球菌,呈β溶血,可感染多种淡水和海洋鱼类。本研究利用环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)进行核酸扩增,通过横向流动试纸条方法(lateral flow dipstick, LFD)实现检测,建立了一种可应用于海豚链球菌快速检测的LAMP-LFD技术。该技术以海豚链球菌促旋酶B亚单位(gyrase subunit B, gyrB)基因为检测靶标,设计3对引物进行由生物素标记的LAMP扩增反应,产物经异硫氰酸荧光素(fluorescein isothiocyanate, FITC)标记的探针杂交后,在LFD上完成检测。经优化的核酸扩增最适条件为65℃反应30 min,在此条件下,阳性扩增起始时间与模板浓度之间呈典型的线性相关性。从核酸扩增反应开始到LFD显色,整个检测时程只需40 min左右,比常规PCR技术缩短约2 h。LAMP-LFD能特异性地检出海豚链球菌,针对病原纯培养物的检测灵敏度为8.70×101 cfu/mL,是LAMP检测的10倍、常规PCR检测的100倍。以灵敏度浓度(8.70×101 cfu/mL)的海豚链球菌基因组DNA为模板进行的LAMP-LFD结果显示该方法具有良好的重复性。针对人工污染花鲈(Lateolabrax japonicus)肝组织的检测灵敏度为4.35×103 cfu/mL,同样为常规PCR检测方法的100倍。利用本方法可成功从患病花鲈的组织样品中检测出海豚链球菌,检测结果与常规的细菌分离鉴定方法结果一致。因此,利用LAMP-LFD能特异、准确、高效地检测出海豚链球菌,而且操作简单、费用低、耗时短,有望成为海豚链球菌的常规检测方法。%Streptococcus iniae is a species belonging to Gram-positive coccus and produces beta hemolysis, which can infect a broad range of freshwater and marine fish species and lead to seriously economic losses in the aquaculture industry worldwide. Thus, a diagnostic method for rapid and accurate detection of S. iniae was

  15. Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    OpenAIRE

    Poole, Catherine B.; Tanner, Nathan A.; Zhang, Yinhua; Thomas C Evans; Carlow, Clotilde K. S.

    2012-01-01

    Author Summary Brugian filariasis is a debilitating neglected tropical disease caused by infection with the filarial parasites Brugia malayi or Brugia timori. Adult worms live in the lymphatic system and produce large numbers of microfilariae that predominantly circulate in the blood at night. Bloodsucking mosquitoes spread the disease by ingesting microfilariae that develop into infective stage larvae in the insect. In rural areas, diagnosis still relies largely on microscopic examination of...

  16. Polyethersulfone improves isothermal nucleic acid amplification compared to current paper-based diagnostics.

    Science.gov (United States)

    Linnes, J C; Rodriguez, N M; Liu, L; Klapperich, C M

    2016-04-01

    Devices based on rapid, paper-based, isothermal nucleic acid amplification techniques have recently emerged with the potential to fill a growing need for highly sensitive point-of-care diagnostics throughout the world. As this field develops, such devices will require optimized materials that promote amplification and sample preparation. Herein, we systematically investigated isothermal nucleic acid amplification in materials currently used in rapid diagnostics (cellulose paper, glass fiber, and nitrocellulose) and two additional porous membranes with upstream sample preparation capabilities (polyethersulfone and polycarbonate). We compared amplification efficiency from four separate DNA and RNA targets (Bordetella pertussis, Chlamydia trachomatis, Neisseria gonorrhoeae, and Influenza A H1N1) within these materials using two different isothermal amplification schemes, helicase dependent amplification (tHDA) and loop-mediated isothermal amplification (LAMP), and traditional PCR. We found that the current paper-based diagnostic membranes inhibited nucleic acid amplification when compared to membrane-free controls; however, polyethersulfone allowed for efficient amplification in both LAMP and tHDA reactions. Further, observing the performance of traditional PCR amplification within these membranes was not predicative of their effects on in situ LAMP and tHDA. Polyethersulfone is a new material for paper-based nucleic acid amplification, yet provides an optimal support for rapid molecular diagnostics for point-of-care applications. PMID:26906904

  17. 实时荧光环介导等温扩增检测 HIV-1 DNA反应体系的建立%Establishment of real-time fluorescence loop-mediated isothermal amplification assay for rapid detec-tion of HIV-1 DNA

    Institute of Scientific and Technical Information of China (English)

    厉小玉; 张永乐; 潘克女; 吴亦栋; 陈刚; 周俊

    2013-01-01

      目的建立一种快速、敏感度高的实时荧光环介导等温定量检测HIV-1 DNA的方法。方法根据HIV病毒基因序列选择6个保守区域设计4条环介导等温扩增( LAMP)引物,建立环介导等温扩增体系,同时在体系中加入SYBR GreenⅠ荧光染料,并评价该方法的特异性和敏感度。结果成功合成实时荧光环介导等温定量检测HIV-1 DNA反应体系,检测200例HIV感染者外周淋巴细胞HIV-1 DNA,其中有195例阳性,并检测50例健康对照结果均阴性。其中有HIV-1 DNA定量结果显示最低含量为51拷贝/ml,最高含量为8.21×106拷贝/ml,平均值含量为5.78×105拷贝/ml,其中病毒含量比较集中在105数量级范围,共162例占83.08%。采用10倍稀释法发现该方法最低检出限为50拷贝/ml。对乙型肝炎病毒、疱疹病毒、丙型肝炎病毒进行交叉实验结果均为阴性。结论HIV感染者外周血中几乎都存在HIV-DNA,实时荧光环介导等温扩增是一种快速、敏感度高、特异性强的方法,适合各级医院的临床检测。%Objective To establish a rapid and high sensitive assay of real-time fluorescence loop-mediated isothermal amplification assay for clinical detection of HIV-1 DNA.Methods Four loop primers were constructed for loop-mediated isothermal amplification ( LAMP) assay based on six conserved regions selected from HIV gene sequence .SYBR Green Ⅰdye was added into the established LAMP assay and its specificity and sensitivity were evaluated .Results The real-time fluorescence LAMP assay for the detection of HIV-1 DNA was successfully established .It was used for the detection of HIV-1 DNA among 200 patients with HIV infection, of which 195 cases were positive.Moreover, 50 healthy subjects were found HIV-1 DNA negative tested by the real-time fluorescence LAMP assay .Quantitative testing for HIV-1 DNA showed that the lowest and the highest detectable amount were 51 copies

  18. Flux amplification in SSPX

    Science.gov (United States)

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

    2007-11-01

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

  19. Rapid Detection of Vibrio vulnificus by Loop-mediated Isothermal Amplification Combined with Lateral Flow Dipstick Assay%环介导等温扩增联合横向流动试纸条快速检测创伤弧菌检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    王耀焕; 王瑞娜; 周前进; 陈炯

    2014-01-01

    环介导等温扩增技术(LAMP)与横向流动试纸条(LFD)检测联合应用,建立了一种新的快速、便捷的创伤弧菌检测方法。针对创伤弧菌的外膜蛋白TolC基因设计6条特异性引物和1条异硫氰酸荧光素(FITC)标记的探针。生物素标记的LAMP扩增产物能够特异性地与FITC标记的探针杂交,杂交产物经LFD检测。优化后的扩增温度和时间为63℃反应35 min,加上细菌基因组DNA提取步骤,完成检测仅需要80 min。LAMP-LFD方法可特异性地检出创伤弧菌,对哈维氏弧菌等9种水产品常见病原菌的检测均呈阴性;对纯细菌培养物的检测灵敏度为3.7×102 CFU/mL或7.4 CFU/反应,是利用外引物建立的常规PCR检测的100倍。结果表明,该方法能够准确、快速、灵敏地检出创伤弧菌,可应用于创伤弧菌污染的水产品的检测。%A novel and rapid loop-mediated isothermal amplification(LAMP)combined with chromatographic lateral flow dipstick(LFD) assay was developed to detect Vibrio vulnificus. A set of six primers and a fluorescein isothiocyanate(FITC)-labeled probe that recognized V. vulnificus outer membrane protein TolC gene were designed. Biotinylated LAMP amplicons were hybridized exclusively with the FITC-labeled probe and detected by LFD assay. The assay was optimized and could detect V. vulnificus by incubation at 63℃for only 35 min, and the whole detection procedure from extraction of bacterial genomic DNA to the visualization of the amplicons by LFD last 80 min. V. vulnificus could be accurately detected by LAMP-LFD, and no amplification could be observed when another 9 bacterial genomic DNA were used. The sensitivity for V. vulnificus detection in pure culture was 3.7×102 CFU/mL or equivalent to 7.4 CFU per reaction, which is 100 times higher than that of PCR assay. The results indicate that LAMP-LFD is an accurate, rapid and sensitive tool for V. vulnificus detection and can be used for

  20. Development and testing of passive autocatalytic recombiners cooled by heat pipes

    International Nuclear Information System (INIS)

    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.

  1. 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.

  2. Modelling of the operational behaviour of passive autocatalytic recombiners; Modellierung des Betriebsverhaltens katalytischer Rekombinatioren

    Energy Technology Data Exchange (ETDEWEB)

    Schwarz, Ulrich

    2011-03-01

    Due to severe accidents in nuclear power plants, a significant amount of hydrogen can be produced. In pressurized water reactors, a possible and wide-spread measurement is the use of auto-catalytic recombiners. There are numerous numerical models describing the operational behaviour of recombiners for containment codes. The numerical model REKO-DIREKT was developed at the Forschungszentrum Juelich. This model describes the chemical reaction on the catalytic sheets by a physical model, as opposed to the usual codes based on empirical correlations. Additionally, there have been experimental studies concerning the catalytic recombination of hydrogen since the 1990s. The aim of this work is the further development of the program REKO-DIREKT to an independent recombiner model for severe accident and containment codes. Therefore, the catalyst model already existed has been submitted by a parameter optimization with an experimental database expanded during this work. In addition, a chimney model has been implemented which allows the calculation of the free convection flow through the recombiner housing due to the exothermal reaction. This model has been tested by experimental data gained by a recently built test facility. The complete recombiner model REKO-DIREKT has been validated by data from literature. Another aim of this work is the derivation of the reaction kinetics for recombiner designs regarding future reactor concepts. Therefore, experimental studies both on single catalytic coated meshes as well as on two meshes installed in a row have been performed in laboratory scale. By means of the measured data, a theoretical approach for the determination of the reaction rate has been derived.

  3. Implementation of passive autocatalytic recombiner system as a hydrogen mitigation system in Korean nuclear power plants

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chang Hyun; Sung, Je Joong; Ha, Sang Jun [Korea Hydro and Nuclear Power Co. Ltd., Central Research Institute, Daejeon (Korea, Republic of); Yeo, In Seon [KEPCO Engineering and Construction Co. Ltd., Gyeonggi-do (Korea, Republic of)

    2015-08-15

    Ensuring the containment integrity during a severe accident in nuclear power reactor by maintaining the hydrogen concentration below an acceptable level has been recognized to be of critical importance since Three Mile Island and Fukushima Daiichi nuclear power plant accidents. Although there exist various mitigation measures for hydrogen risk, a passive autocatalytic recombiner (PAR) has been emphasized as a viable option for the mitigation of hydrogen risk under the extended station blackout conditions due to its passive operation characteristics for the hydrogen removal. To enhance the capability of hydrogen control, the hydrogen mitigation system with various types of PARs has been implemented for all nuclear power plants in Korea. This paper presents an implementation procedure of PAR system and the analysis results to determine the location and capacity of PAR in OPR1000. Various accident scenarios have been adopted considering important event sequences from a combination of probabilistic methods, deterministic methods and sound engineering judgment. A MAAP 4.0.6+ with a multi-compartment model has been used as an analysis tool with conservative hydrogen generation and removal models. The detailed analyses have been performed for selected severe accident scenarios including sensitivity analysis with/without operations of various safety systems. The possibility of global flame acceleration (FA) and deflagration-to-detonation transient (DDT) has been assessed with sigma (flame acceleration potential) and 7-lambda (DDT potential) criterion. It is concluded that the newly designed hydrogen mitigation system with twenty-four (24) PARs can effectively remove hydrogen in the containment atmosphere and prevent global FA and DDT.

  4. Study on detection of Bacillus anthracis and its quantitate based on Fluorescent Quantitative Loop-mediated Isothermal Amplification%使用环介导等温扩增技术检测炭疽芽孢杆菌及其定量检测的研究

    Institute of Scientific and Technical Information of China (English)

    易海华; 张阳; 房超; 孙涛; 徐政; 徐波; 刘继永

    2013-01-01

    Objective To develop a method of Fluorescent Quantitative and Qualitative Loop-mediated Isothermal Amplification (LAMP) to Bacillus anthracis rapidly ,specifically, sensitively and simply suited for used in the primary public health agency. Methods According to conserved nucleotide of Bacillus anthracis and principle of LAMP, a set of LAMP primers was designed and a LAMP and a real-time LAMP reaction system was set up. The specificity, sensitivity and repeatability of the method was evaluated. In addition, the linearity of the real-time LAMP between initial template copies Log value and reaction time was evaluated. Results The optimal assay showed that it was no cross-reaction with the other bacteria belong to Bacillus cereus group , and the threshold values of detection for Bacillus anthracis was 103 copies per reaction ,10 times more sensitive than PCR , and The Coefficient of Variance between tests was less than 5%. The kinetics curves showed a good linearity between initial template copies Log value and reaction time, which correlation coefficient was 0.9891. Conclusion These findings demonstrate that the fluorescent quantitative and qualitative LAMP has the potential clinical application for detection and differentiation of Bacillus anthracis in the primary hospital, inspection and quarantine institution for its sensitive, specific and simple feature.%目的 建立一种适合公共卫生机构现场使用的环介导等温扩增(LAMP)技术检测炭疽芽孢杆菌的方法.方法 根据LAMP方法的原理,建立LAMP以及荧光定量LAMP检测方法,对方法的特异性、灵敏度、重复性及模板浓度与反应时间之间的线性关系进行评估.结果 LAMP检测可在1h内完成,检出限为103拷贝/反应,灵敏度比经典PCR高10倍,与其他杆菌群细菌无交叉反应,平均试验间变异系数小于5%.反应时间与模板浓度有良好的线性关系(R2=0.9891).结论 该方法快速、灵敏、特异、操作简单、设备要求

  5. Development and application of loop-mediated isothermal amplification for detection of Streptococcus agalactiae in green terror Aequidens rivulatus%红尾皇冠鱼无乳链球菌病LAMP检测方法的建立与应用

    Institute of Scientific and Technical Information of China (English)

    姚学良; 徐晓丽; 李贺密; 包海岩; 任涵玮; 郑艳坤

    2014-01-01

    以cfb基因保守序列的6个区域设计4条特异性引物,利用链置换DNA聚合酶( BstDNA polymer:ase)在恒温(65℃)下保温40 min,建立了无乳链球菌 Streptococcus agalactiae 的 LAMP 技术。建立的LAMP方法能够特异性地检测无乳链球菌,检测灵敏度为3·7×101~3·7×102 cfu/mL,反应前加入显色剂钙黄绿素避免二次污染。现场应用中采用FTA卡采集红尾皇冠鱼Aequidens rivulatus病鱼肝脏组织病原菌,操作简便、快捷。研究表明,针对无乳链球菌cfb基因建立的LAMP检测方法具有较高的特异性及稳定性,尤其是具有快速、简便、成本低等优点,可用于无乳链球菌的野外现场快速检测。%The cfb gene of Streptococcus agalactiae was amplified by a set of four specific primers that recognize six distinct sequences of the target in 40 min by incubating all of the reagents in a single tube with BstDNA polymerase at a constant 65 ℃. A loop-mediated isothermal amplification ( LAMP) assay was developed and validated for the specific detection of Streptococcus agalactiae, with detection limits of 3. 7í101-3. 7í102 cfu/mL. The resulting am-plicons were visualized by adding calcein to the reaction tube before the reaction. FTA cards was used for collection and purification of nucleic acids from a liver of green terror Aequidens rivulatus with pathogenic bacteria in field ap-plications. The findings demonstrate that the LAMP-based assay is a sensitive and reliable means for the detection of Streptococcus agalactiae with rapidity, simplicity and low cost, especially suitable for on-site rapid diagnosis.

  6. Comparison of loop-mediated isothermal amplification and real-time PCR for the detection of Leptospira interrogans%环介导的等温扩增与实时荧光PCR检测问号钩端螺旋体的比较

    Institute of Scientific and Technical Information of China (English)

    方葆; 丁士标; 严杰; 林旭瑷

    2011-01-01

    目的 通过比较环介导的等温扩增技术(loop-mediated isothermal amplification,LAMP)与实时荧光PCR( real-time PCR)技术在检测问号钩端螺旋体的特异性及灵敏度方面的差异,寻找一种快速、灵敏且特异性强的问号钩端螺旋体检测方法。方法 根据问号钩端螺旋体lipL41基因序列设计LAMP及实时荧光PCR扩增所用的特异性引物,对我国15群15株问号钩端螺旋体参考菌株进行检测,比较二者在检测的特异性和灵敏度方面的差异。结果 LAMP反应大约可在30 min内完成,整个分析过程不超过1h。Real-time PCR的整个过程大约需要60 min。二者具有相同的检测灵敏度和特异性,最低检出限度均为100个拷贝,整个检测过程没有出现假阳性。结论 在检测速度、灵敏度和特异性方面,LAMP技术与real-time PCR技术相当,但LAMP检测技术是在恒温条件下进行,不需要特别昂贵的仪器设备,检测方法简单,在基层实验室及现场检测方面具有明显的优势,是可应用于问号钩端螺旋体检测的一种有效技术。%Objective To compare the sensitivity and specificity of Leptospira interrogans using loop-mediated isothermal amplification (LAMP) and real-time PCR technology, then looking for a rapid,sensitive and specific methods for the detection of Leptospira interrogans. Methods In accordance with lipL41 gene from Leptospira interrogans, primers for LAMP and real-time PCR were designed and used to detect Leptospira interrogans in cultured 15 reference strains of 15 serogroups in China, then compared the sensitivity and specificity of the two methods in the detection of Leptospira interrogans. Results The LAMP reaction could be completed within 30 min, and whole process of it less than 60 min. The whole real-time PCR reaction could be finished at about 60 min. Both of them had the same detection sensitivity and specificity,the lower detection limits in the reactions was

  7. Comparison of the roles of nucleotide synthesis, polymerization, and recombination in the origin of autocatalytic sets of RNAs.

    Science.gov (United States)

    Wu, Meng; Higgs, Paul G

    2011-11-01

    Ribozymes that act as polymerases and nucleotide synthases are known experimentally, even though no fully self-replicating system has yet been found. If the RNA World hypothesis is true, ribozymes must have arisen initially from within a random abiotic polymerization system. To investigate the origin of the RNA world, we studied a mathematical model of a chemical reaction system describing RNA polymerization. It is supposed that, in absence of ribozymes, polymerization occurs at a small spontaneous rate, and that in the presence of polymerase ribozymes, polymerization occurs at a faster rate that is proportional to the ribozyme concentration. Chains must be longer than a minimum threshold length in order to have the possibility of acting as ribozymes. The reaction system has two stable states that we term dead and living. The dead state is controlled by the small spontaneous rate and has negligible concentration of ribozymes. The living state has high concentration of ribozymes, and the reaction rates are determined by the ribozymes; thus, the system is autocatalytic. Concentration fluctuations in a finite volume can cause a transition to occur from the dead to the living state, that is, an origin of life occurs within this model. We also consider ribozymes that catalyze nucleotide synthesis. We show that living and dead states arise in the presence of synthase ribozymes in the same way as for polymerases. It has been proposed that recombination reactions are a way of generating long RNA chains in the early stages of life. We show that if the possibility of random reversible recombination reactions is added to our model, this does not lead to an increase in long polymer concentration. Thus, if recombination is fully reversible, there is no autocatalytic state controlled by recombination. Nevertheless, recombination can play an important role in ribozyme synthesis if there is an additional process that keeps the recombination reactions out of equilibrium. We modeled

  8. Loop-mediated isothermal amplification for rapid detection of dogs infected with Echinococcus species based on copro-DNAⅡ%环介导等温扩增技术(LAMP)检测棘球绦虫感染犬粪DNA的研究Ⅱ

    Institute of Scientific and Technical Information of China (English)

    陈璐; 吾拉木·马木提; 张德亭; 金一帮

    2014-01-01

    目的:为有效地控制预防流行区犬科动物的棘球绦虫感染情况,建立一种快速检测棘球绦虫感染犬的粪便DNA检测方法-环介导等温扩增技术(loop-mediated isothermal amplification .LAMP)。方法针对细粒棘球绦虫线粒体DNA ND2基因6个位点特异性的设计4条LAMP引物,利用LAMP法和普通PCR方法同时检测棘球绦虫、肥胖带绦虫以及犬肠道内的其它寄生虫DNA来验证LAMP方法的特异性;将转入ND2基因片段的质粒作为标准阳性对照,并做梯度稀释后同时用 LAMP和 PCR方法进行检测比较两者的敏感性。此外,将采集的46份犬粪便标本提取DNA ,分别运用LAMP和犬尸体剖检进行感染犬的初步检测评估。结果 E .g mtDNA ND2基因的LAMP引物能够鉴别多房棘球绦虫和细粒棘球绦虫这两个相近的种属,并不与其它待检寄生虫发生交叉反应,且最低检测限度为4×101拷贝(灵敏度比普通PCR高出103倍)。在初步对46份犬粪便DNA检测结果中 ND2 LAMP引物显示出较好的灵敏度和特异度,χ2检验结果该方法与犬尸体剖检方法的诊断效果无统计学差异。结论本研究初步建立了LAM P技术检测细粒棘球绦虫感染犬的新方法,在各项特异度、灵敏度和样本检测中展现出较好的效果。该方法不需要昂贵的仪器设备和繁琐的电泳分析过程,有望成为流行区和基层兽医站细粒棘球绦虫感染犬检测的新方法。%To control and prevent the Echinococcus in the place ,we established a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Echinococcus species specific DNA from dog faeces .Four primers which recognizing 6 dis-tinct regions on the NADH dehydrogenase subunit 2 (ND2) gene of Echinococcus granulosus were designed and used for LAMP assay .The specificity of LAMP assay was evaluated using DNA extracted from Echinococcus granulosus , Taenia saginata , and other dog

  9. Rollercoaster loop shapes

    Science.gov (United States)

    Pendrill, Ann-Marie

    2005-11-01

    Many modern rollercoasters feature loops. Although textbook loops are often circular, real rollercoaster loops are not. In this paper, we look into the mathematical description of various possible loop shapes, as well as their riding properties. We also discuss how a study of loop shapes can be used in physics education.

  10. Coherent white light amplification

    Science.gov (United States)

    Jovanovic, Igor; Barty, Christopher P.

    2004-05-25

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

  11. Hydrogen Recombination Rates of Plate-type Passive Auto-catalytic Recombiner

    International Nuclear Information System (INIS)

    The hydrogen mitigation system may include igniters, passive autocatalytic recombiner (PAR), and venting or dilution system. Recently PAR is commonly used as a main component of HMS in a NPP containment because of its passive nature. PARs are categorized by the shape and material of catalytic surface. Catalytic surface coated by platinum is mostly used for the hydrogen recombiners. The shapes of the catalytic surface can be grouped into plate type, honeycomb type and porous media type. Among them, the plate-type PAR is well tested by many experiments. PAR performance analysis can be approached by a multi-scale method which is composed of micro, meso and macro scales. The criterion of the scaling is the ratio of thickness of boundary layer developed on a catalytic surface to representative length of a computational domain. Mass diffusion in the boundary layer must be resolved in the micro scale analysis. In a lumped parameter (LP) analysis using a system code such as MAAP or MELCOR, the chamber of the PAR is much smaller than a computational node. The hydrogen depletion by a PAR is modeled as a source of mass and energy conservation equations. Te catalytic surface reaction of hydrogen must be modeled by a volume-averaged correlation. In this study, a micro scale analysis method is developed using libraries in OpenFOAM to evaluate a hydrogen depletion rate depending on parameters such as size and number of plates and plate arrangement. The analysis code is validated by simulating REKO-3 experiment. And hydrogen depletion analysis is conducted by changing the plate arrangement as a trial of the performance enhancement of a PAR. In this study, a numerical code for an analysis of a PAR performance in a micro scale has been developed by using OpenFOAM libraries. The physical and numerical models were validated by simulating the REKO-3 experiment. As a try to enhance the performance of the plate-type PAR, it was proposed to apply a staggered two-layer arrangement of the

  12. Hydrogen Recombination Rates of Plate-type Passive Auto-catalytic Recombiner

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jongtae; Hong, Seong-Wan [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Kim, Gun Hong [Kyungwon E-C Co., Seongnam (Korea, Republic of)

    2014-10-15

    The hydrogen mitigation system may include igniters, passive autocatalytic recombiner (PAR), and venting or dilution system. Recently PAR is commonly used as a main component of HMS in a NPP containment because of its passive nature. PARs are categorized by the shape and material of catalytic surface. Catalytic surface coated by platinum is mostly used for the hydrogen recombiners. The shapes of the catalytic surface can be grouped into plate type, honeycomb type and porous media type. Among them, the plate-type PAR is well tested by many experiments. PAR performance analysis can be approached by a multi-scale method which is composed of micro, meso and macro scales. The criterion of the scaling is the ratio of thickness of boundary layer developed on a catalytic surface to representative length of a computational domain. Mass diffusion in the boundary layer must be resolved in the micro scale analysis. In a lumped parameter (LP) analysis using a system code such as MAAP or MELCOR, the chamber of the PAR is much smaller than a computational node. The hydrogen depletion by a PAR is modeled as a source of mass and energy conservation equations. Te catalytic surface reaction of hydrogen must be modeled by a volume-averaged correlation. In this study, a micro scale analysis method is developed using libraries in OpenFOAM to evaluate a hydrogen depletion rate depending on parameters such as size and number of plates and plate arrangement. The analysis code is validated by simulating REKO-3 experiment. And hydrogen depletion analysis is conducted by changing the plate arrangement as a trial of the performance enhancement of a PAR. In this study, a numerical code for an analysis of a PAR performance in a micro scale has been developed by using OpenFOAM libraries. The physical and numerical models were validated by simulating the REKO-3 experiment. As a try to enhance the performance of the plate-type PAR, it was proposed to apply a staggered two-layer arrangement of the

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

    OpenAIRE

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

    2009-01-01

    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. Res...

  14. 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. PMID:26284787

  15. Derivation of an Analytical Solution to a Reaction-Diffusion Model for Autocatalytic Degradation and Erosion in Polymer Microspheres.

    Directory of Open Access Journals (Sweden)

    Ashlee N Ford Versypt

    Full Text Available 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.

  16. 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

  17. 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 extens...

  18. 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)

  19. Establishment of real-time PCR and loop-mediated isothermal amplification for detecting Cryptococcus neoformans CAP10 gene%荧光定量PCR和环介导等温扩增方法检测隐球菌CAP1O基因的比较研究

    Institute of Scientific and Technical Information of China (English)

    韩慧; 胡子有; 吴炳义

    2012-01-01

    目的 建立荧光定量PCR和环介导等温扩增技术(LAMP)检测隐球菌荚膜相关蛋白基因(CAP10)的方法,并比较和评价两种方法的优缺点.方法 针对新型隐球菌CAP10基因序列,使用在线软件设计引物,并构建质粒标准品,优化反应条件后用荧光定量PCR和LAMP两种方法检测定量的质粒,分析其敏感性和特异性,并检测临床标本.结果 荧光定量PCR检测CAP10质粒的敏感性为6.8×101拷贝,LAMP方法的敏感性为6.8×103拷贝,PCR检测阳性率比LAMP方法高.两种方法特异性好,对脑膜炎奈瑟菌、白色念珠菌、热带念珠菌、黄曲霉、黑曲霉和大肠埃希菌的检测结果均为阴性.结论 成功建立了检测CAP10基因的荧光定量PCR方法敏感性和特异性高,但是需要特殊仪器;LAMP方法敏感性较低,但其操作简单,无需特殊仪器和设备,加入核酸染料即能观察结果.两者均适用于新型隐球菌感染的检测.%Objective To establish real-time PCR and loop-mediated isothermal amplification (LAMP) systems for detecting Cryptococcus neoformans CAP10 gene.Methods Specific primers were designed targeting CAP10 gene of Cryptococcus neoformans,and the plasmid was constructed.After optimization of the reaction condition,the plasmid was quantitatively detected using real-time PCR and LAMP,and the detection sensitivity and specificity were evaluated.Clinical samples were also detected using the two methods.Results The detection sensitivity of real-time PCR and LAMP was 6.8×103 and 6.8×103 copies,respectively.Real-time PCR yielded a higher positivity rate than LAMP.Both of the two methods showed a high detection specificity and produced negative results in the detection of Neisseria meningitidis,Candida albicans,Candida tropicalis,Aspergillus flavus,Aspergillus niger and Escherichia coii.Conclusion Real-time PCR is highly sensitive and specific for detecting Cryptococcus neoformans CAP10 gene but requires sophisticated equipment

  20. A Novel Loop-Mediated Isothermal Amplification Assay Targeting DeoR Family Transcriptional Regulator Gene to Rapidly Detect Staphylococcus Epidermidis%利用环介导等温扩增技术针对DeoR家族转录调控因子基因进行表皮葡萄球菌快速鉴定

    Institute of Scientific and Technical Information of China (English)

    田怡婧; 刘有福; 宋玉竹

    2015-01-01

    表皮葡萄球菌是一种重要的机会致病性微生物,是院内交叉感染的一个重要原因。开发快速有效的检测技术对其防治具有重要意义。利用生物信息学方法进行分析,发现DeoR家族转录调控因子基因可用于表皮葡萄球菌鉴定。针对此基因设计引物,采用环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)进行检测,在65℃条件下反应进行60 min后,电泳检测可见明显的阶梯状条带。比较了3株表皮葡萄球菌临床分离株和8株其他细菌(金黄色葡萄球菌、溶血葡萄球菌、弗劳氏枸橼酸杆菌、肺炎克雷伯菌、铜绿假单胞菌、粪肠球菌、屎肠球菌和化脓性链球菌),结果显示该方法具有良好的特异性。构建DeoR家族转录调控因子基因质粒载体后考察了检测的灵敏度,结果显示其最低检测限为105拷贝/反应。由此针对DeoR家族转录调控因子基因建立的LAMP检测方法可快速、简便、特异以及敏感的鉴定表皮葡萄球菌。%Staphylococcus epidermidis is an opportunistic bacterium that causes a variety of infections including nos-ocomial infection.The development of effective techniques for rapid detection of this pathogen is essential for pre-vention and cure of its infection.In present study,a loop-mediated isothermal amplification(LAMP)assay targeting DeoR family transcriptional regulator gene to rapidly detect S.epidermidis is developed.This assay is performed in 60 min at an optimal temperature of 65℃and visualized as a ladder on a 1%agarosege.Specificity of the assay is evaluated by testing three S.epidermidis clinical isolates and eight non-S.epidermidis bacteria(Staphylococcus au-reus,Staphylococcus haemolyticus,Citrobacter freundii,Klebsiella pneumoniae,Pseudomonas aeruginosa,Enterococcus faecalis,Enterococcus Faecium,and Streptococcus pyogenes).No ladder pattern is seen with any of the other non-S.epidermidis bacteria

  1. Establishment of method and modification of colorimetric judgment on HIV-1 virus detection by reverse transcription loop-mediated isothermal amplification%逆转录环介导等温扩增技术检测HIV-1方法的建立及颜色判定法的改良

    Institute of Scientific and Technical Information of China (English)

    丁雄; 聂凯; 曾亚岚; 王佶; 石磊; 马学军

    2013-01-01

    目的 基于逆转录环介导等温扩增(RT-LAMP)技术建立可用于现场检测HIV-1的方法.方法 构建实时荧光检测法时,先测试其特异性和灵敏度,并对其定量检测进行探索,然后用35份HIV-1阳性样本对该方法进行评定;在颜色判定法上,先对羟基萘酚蓝(HNB)进行改良,并验证其实际显色效果,然后选取效果明显的样本进行显色灵敏度测试和35份阳性样本显色评定.结果 实时荧光法对HIV-1有很好的检测特异性,灵敏度可达每反应管10 ~ 100拷贝数RNA.该方法对所有阳性临床样本均100% (35/35)检出,无一漏检,但其定量检测只局限于不低于每反应管103拷贝数.通过改良得到3种易分辨的HNB改良型显色剂,显色灵敏度等同于琼脂糖凝胶电泳级别,对35份HIV-1阳性样本的显色判定也无一漏检.结论 基于RT-LAMP技术的现场实时荧光法和改良型颜色判定法可以为真正实现现场快速、准确和灵敏检测HIV-1提供帮助.%Objective To establish the reverse transcription loop-mediated isothermal amplification (RT-LAMP) methods for on-site HIV-1 detection.Methods As for the real-time fluorescent RT-LAMP,we firstly tested the specificity and sensitivity,then explored its quantitative determination,and finally applied the method to the detection of 35 HIV-1 positive samples.For colorimetric judgment,after choosing different ameliorants to modify Hydroxynaphthol blue (HNB),we tested their real effects on coloration,and then picked out the modified dyes with obvious color change to test the sensitivity and the detection of the 35 HIV-1-positive samples.Results The real-time fluorescent RT-LAMP showed great specificity of HIV-1,and the sensitivity to detect HIV-1 RNA was between 10 and 100 copies per reaction.On testing 35 HIV-1-positive samples,the method could reach 100 percent detection rate.However,for the quantitative determination,the quantitative relation was not observed regarding the HIV-1

  2. Application of loop-mediated isothermal amplification assay in the rapid diagnosis of patients with tubercu-lous pleuritis%环介导等温扩增技术在结核性胸膜炎快速诊断中的应用

    Institute of Scientific and Technical Information of China (English)

    刘洋; 郭艳玲; 姜广路; 孙琦; 邢爱英; 张宗德

    2015-01-01

    Objective To explore the application of loop-mediated isothermal amplification ( LAMP) assay in the rapid diagnosis of patients with tuberculous pleuritis. Methods Mycobacterium tuberculosis in pleural effu-sion from 58 patients with tuberculous pleuritis and 32 lung cancer patients were tested by LAMP. The specificity of this assay was evaluated using H37Rv reference strains of mycobacterium tuberculosis, 8 species of non-tuberculous mycobacteria (NTM), eterobacter cloacae, and E. coli. Results H37Rv reference strain was successfully detected by this method, and there were no false-positive results of NTM, eterobacter cloacae and E. coli. The sensitivity of LAMP in detection of purified DNA from H37Rv was 320 aM. The detection sensitivity for the pleural effusion from 58 tuberculous pleuritis was 75. 8% (44/58), which was higher than smear microscopy (39. 6%). There was no significant difference between the sensitivity of LAMP and that of quantitative real-time PCR (χ2 =0. 25, P>0. 05). Conclusion Because of its speed, simplicity, sensitivity and specificity, the TB hspX LAMP assay is a potential gene diagnostic method for mycobacterium tuberculosis in patients with tuberculous pleuritis.%目的:探讨针对hspX 基因设计引物的环介导等温扩增技术( LAMP)对结核性胸膜炎患者结核分枝杆菌的诊断价值。方法采用LAMP技术对结核分枝杆菌标准株 H37Rv的DNA 样品进行检测,评价LAMP技术的检测限;同时对8种非结核分枝杆菌菌株及阴沟肠杆菌和大肠埃希菌标准菌株进行鉴定,评价该方法的特异性。对58例结核性胸膜炎患者和32例肺癌患者胸腔积液样本结核分枝杆菌进行检测,评价LAMP 技术的临床应用价值。结果 LAMP对倍比稀释的H37Rv的DNA 样品检测结果为阳性,检测限为320aM。八种非结核分枝杆菌和2种普通菌菌株检测结果为阴性。 LAMP对胸腔积液标本检测的灵敏度是75.8%(44/58),定量PCR 为79.3%(46/58),

  3. 基于颜色判定的环介导逆转录等温扩增技术检测柯萨奇病毒A6型%Colorimetric detection of coxsackievirus A6 by reverse transcription loop mediated isothermal amplification

    Institute of Scientific and Technical Information of China (English)

    关丽; 许松涛; 聂凯; 张丹; 李鑫娜; 许文波; 马学军

    2015-01-01

    目的 建立基于羟基萘酚蓝(hydroxy naphthol blue, HNB)颜色变化的简单、灵敏和快速的环介导逆转录等温扩增技术(RT-LAMP)检测方法,应用于柯萨奇病毒A6型(coxsackievirus A6,CV-A6)的检测.方法 针对CV-A6的VP1基因设计6条特异引物,在等温条件下(63℃)进行50 min扩增反应.扩增前在反应体系中加入HNB,通过观察颜色变化进行检测结果判定.使用多种肠道病毒进行特异性验证,使用梯度稀释的体外转录CV-A6全VP1基因RNA进行灵敏度分析,同时与实时荧光定量逆转录PCR(rRT-PCR)检测结果进行比较,并对92份手足口病患者临床标本进行检测.结果 本研究建立的RT-LAMP方法对除CV-A6外的23种肠道病毒的检测结果均为阴性,灵敏度为100拷贝/反应,与rRT-PCR方法相当.在对92份手足口病临床标本的检测中,检测结果与rRT-PCR方法相符,Kappa值为1,灵敏度和特异性均为100%.结论 本研究建立的针对CV-A6的LAMP检测方法,特异度高,灵敏度与rRT-PCR相当,有望应用于CV-A6感染的快速筛选,具有在基层医疗卫生机构和现场推广与应用的潜力.%Objective To develop a simple, rapid and sensitive colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of coxsackievirus A6 (CV-A6) based on the colour chang of hydroxy naphthol blue (HNB).Methods The method employed a set of six primers that recognized sequences of VP1 gene for amplification of nucleic acid under isothermal conditions at 63 ℃ for 50 min.The products were detected through visual inspection of color change by the pre-addition of HNB dye.The specificity was validated by detecting a collection of different human enteroviruses.The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of CV-A6 VP1 gene, and compared with real-time RT-PCR (rRT-PCR) in parallel.This assay was evaluated with 92 clinical specimens from

  4. Passive Autocatalytic Recombiner System to Mitigate Severe Accident Situations Design and Process Features and International Qualification and Implementation

    Energy Technology Data Exchange (ETDEWEB)

    Eckardt, B.; Losch, N.

    2011-07-01

    During severe accident situations large amounts of hydrogen can be released inside the reactor containment. Without countermeasures these releases could lead to a significant combustible gas accumulation within the containment, and an ignition of this gas mixture could exceed overpressure design values and jeopardize the integrity of the containment. The advantages and drawbacks of various hydrogen countermeasures to mitigate severe accidents, for example of igniters, Passive Autocatalytic Recombiners (PARs), post inertization, etc. have been investigated and ultimately the PAR system selected as the H2 mitigation technique to be implemented. A hydrogen reduction system based on Passive Autocatalytic Recombiners (PAR) can be a completely passive, efficient method to limit the rise of combustible gas concentration under severe accident conditions even in steam inerted containment atmosphere. A PAR system consists of a large number of passive individual autocatalytic recombiners - for example anywhere from 20 to 60 individual PARs - distributed inside the containment to accommodate a wide range of release scenarios. The effectiveness of such a PAR system has been demonstrated by analysis for different sets of severe accident sequences. To date more than 100 AREVA PAR systems have been contracted or most of them (> 80) are already installed in Western nuclear PWR and BWR's plants as well as in Russian-designed VVER nuclear power plants. Due to the different catalyst operational phenomena, including such possible poisoning of catalyst products, it is necessary to demonstrate the function of each specific PAR product under most representative testing conditions. For this reason at national and at international level large efforts have been undertaken for the evaluation and performing extensive PAR qualification programs. After finishing sets of single and multiple poison catalyst tests at small and large size test facilities located in different countries, it has

  5. 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)

  6. 环介导等温扩增联合横向流动试纸条可视化检测哈维氏弧菌的研究%Visual Detection ofVibrio harveyi Based on Loop-mediated Isothermal Amplification Combined with a Lateral Flow Dipstick

    Institute of Scientific and Technical Information of China (English)

    程蝶; 柴方超; 蔡怡; 周前进; 陈炯

    2016-01-01

    Based on nucleotide enrichment by a loop-mediated isothermal amplification(LAMP)and chromatographic visualization by a lateral flow dipstick(LFD)assay,this work aims to develop a novel LAMP-LFD method for the rapid detection of Vibrio harveyi. Three pairs of primers were designed using the hemolysin gene(vhhA)of V. harveyi as detection target,and used in LAMP reaction,among which the forward inner primer vhhA-FIP was biotinylated. Similarly,a fluorescein isothiocyanate(FITC)-labeled probe vhhA-HP was designed to specifically hybridize with LAMP products. And then the hybridized LAMP products were visually detected by LFD. The optimized LAMP was performed at 63℃ for 40 min;and visual detection via LFD took 50 min. The results indicated that LAMP-LFD was able to specifically identify V. harveyi from other 9 pathogenic bacteria commonly existing in the aquatic animals,such as V. vulnificus. The detection limit of LAMP-LFD was 1.0×102 CFU/mL for V. harveyi pure cultures(equivalent to 2 CFU per reaction),and 5×102 CFU/mL for V. harveyi contaminated tissues of large yellow croaker(equivalent to 20 CFU per reaction),both of which were 100 times lower than that of the conventional PCR method using both outer primers vhhA-F3/vhhA-B3. Therefore,this rapid and accurate LAMP-LFD method is a promising alternative in the surveillance and point-of-care test of V. harveyiin sea farming.%以哈维氏弧菌(Vibrio harveyi)为材料,利用环介导等温扩增技术(LAMP)进行核酸扩增,借助横向流动试纸条(LFD)完成产物检测,旨在建立一种可用于哈维氏弧菌快速检测的LAMP-LFD新技术。以哈维氏弧菌的溶血素基因(vhhA)为检测靶标设计了3对特异性引物(其中,上游内引物vhhA-FIP由生物素标记),进行由生物素标记的LAMP反应;同时设计1条异硫氰酸荧光素(FITC)标记的探针,与获得的LAMP产物进行特异性杂交,杂交产物经LFD完成检测。经

  7. 环介导等温扩增技术在检测痰标本结核分枝杆菌中的应用%Evaluation of loop-mediated isothermal amplification in direct detection mycobacterium tuberculosis DNA of sputum samples

    Institute of Scientific and Technical Information of China (English)

    王静; 张艳; 胡钰卿; 竺祖军; 岳永宁; 朱敏

    2013-01-01

      目的评价结核分枝杆菌环介导等温扩增技术(LAMP)快速检测试剂盒的临床应用效果。方法选取肺结核患者183例(肺结核组)和非肺结核患者120例(对照组)。采用涂片抗酸染色、罗氏培养法和LAMP对痰标本进行检测,采用结核分枝杆菌核酸扩增荧光检测试剂盒(TB- PCR)检测罗氏培养与LAMP结果不符的标本,分析LAMP与罗氏培养的检出率及两者的符合率。结果去除经鉴定为非结核分枝杆菌的10例标本,涂片抗酸染色、罗氏培养法和LAMP的阳性检出率分为64.1%(111/173)、64.7%(112/173)、78.6%(136/173)。LAMP与抗酸染色、罗氏培养阳性检出率的差异有统计学意义(P<0.01),抗酸染色与罗氏培养比较差异无统计学意义(P>0.05)。以罗氏培养为金标准,LAMP检测的灵敏度、特异度、阳性预测值和阴性预测值分别为88.5%(108/122)、82.3%(149/181)、77.1%(108/140)、91.4%(149/163),LAMP检测与罗氏培养的符合率为84.8%(257/303)。结论结核分枝杆菌环介导等温扩增技术的灵敏度和特异度高,具有简单易行、快速准确的特点,在肺结核患者的早期诊断中将发挥重要作用。%Objective To assess the clinical use of the loop- mediated isothermal amplification(LAMP) assay for detection mycobacterium tuberculosis in sputum samples. Methods LAMP with acid- fast bacilli (AFB)smear microscopy and Lowen-stein- Jensen (L- J)culture, were performed in 183 sputum specimens from pulmonary tuberculosis patients and 120 sputum specimens from non- pulmonary tuberculosis patients. The samples with different results between LAMP and L- J culture were tested by mycobacterium tuberculosis PCR fluorescence diagnostic kits. L- J culture positive strains were identified by p- Ni-trobenzoic acid medium. The sensitivity and specificity of LAMP were calculated according to the result of L- J culture. The de

  8. 环介导等温扩增快速检测临床常见曲霉菌方法的建立和应用%Establishment and application of loop-mediated isothermal amplification for detecting clinical isolates of Aspergillus spp

    Institute of Scientific and Technical Information of China (English)

    鲁勇; 汪一萍; 应建飞; 俞燕红; 贺明阳

    2014-01-01

    Objective To establish a method for detecting Aspergillus spp.by Loop-mediated isothermal amplification.Methods Aspergillus spp.specific primers were designed from the relative conservation region of the published sequence of 28S rRNA genes of A.fumigatus (GenBank accession number AY660917),A.terreus (GenBank accession number AF454183),A.flavus (GenBank accession number AF454158),and A.niger (GenBank accession number AF454169).Genomic DNA were extracted from Aspergillus standard strains,clinical control strains and clinical samples,and amplified by LAMP.The amplified results could be read with the naked eye by the coloring effect of fluorescent nucleotide dye without the DNA electrophoresis.Approximately 103 each Aspergillus conidia suspension was added to the serum from healthy volunteers,and detected these simulative clinical samples bv LAMP assay.Results The LAMP and PCR assays obtained positive results for all four Aspergillus species and 8 simulative clinical samples (double samples for each Aspergillus species) including 103 conidia,but negative in the remaining 15 non-Aspergillus species,human total blood genomic DNA,30 clinical serum samples infected with non-Aspergillus and 10 healthy volunteers.The LAMP assay had a minimum detection of 0.05-0.5pg,by means of detect different levels of 500,50,5,0.5,0.05 pg template in each reaction tube.Conclusion The results confirm that LAMP is a simple,rapid,sensitive and specific method,and can be used for detection of Aspergillus strains in clinical and environmental specimens.%目的 建立一种环介导等温扩增(LAMP)反应快速检测临床常见曲霉菌方法.方法 根据GenBank上提交的烟曲霉(登录号AY660917)、土曲霉(登录号AF454183)、黄曲霉(登录号AF454158)以及黑曲霉(登录号AF454169)菌的特异28S rRNA基因序列比对后的相对保守区设计特异LA MP引物,分别提取标准曲霉、临床对照菌株以及临床标本DNA,然后以LAMP技术扩增靶基因,通过反应

  9. 环介导等温扩增技术在检测细菌性肺炎常见致病菌中的应用%Value of loop-mediated isothermal amplification in detection of common pathogens of bacterial pneumonia

    Institute of Scientific and Technical Information of China (English)

    戴然然; 刘嘉琳; 万欢英

    2011-01-01

    Objective To investigate the value of loop-mediated isothermal amplification (LAMP) assay in the etiology diagnosis of bacterial pnuemonia through nucleic acid detection of common pathogens in pneumonia patients by LAMP method. Methods Sputum DNA was extracted from 75 pneumonia patients. DNA was amplified by LAMP. The fluorescence signals of products were detected by real-time PCR. The quantitative results were qualitatively analyzed at different cutoff values, and the data were compared with the results of sputum culture. Results The positive ratio of amplified products with LAMP assay at the cutoff value of 1 × 104 was 68.0% ,and the coincidence rate between LAMP assay and sputum culture was 56.0%. The positive ratio of amplified products with LAMP assay at the cutoff value of 1× 105 was 50.7%, and the coincidence rate between LAMP assay and sputum culture was 58.7%.The positive ratio of amplified products with LAMP assay at the cutoff value of 1 × 106 was 30.7%, and the coincidence rate between LAMP assay and sputum culture was 53.3%. There was no statistical significance on the positive rate between sputum culture and LAMP results at the cutoff value of 1 × 105.The detection rate of parts of bacteria by LAMP assay is higher than that by sputum culture at the cutoff value of 1 × 105 ,especially for the harsh bacteria. Conclusions DNA of common pathogens in sputum of patients with bacterial pneumonia can be easily and quickly amplified by LAMP method to identify pathogenic bacteria types. Compared with sputum culture, the bacterial detection rate is higher by LAMP assay at the cutoff value of 1 × 105. Especially for the harsh bacteria, LAMP method has significant advantages.%目的 应用环介导等温扩增(LAMP)方法对肺炎患者痰液常见致病菌进行核酸检测,研究LAMP方法在细菌性肺炎病原学诊断中的价值.方法 抽提75例肺炎患者痰液细菌DNA,根据肺炎常见8种致病细菌设计引物,应用LAMP技术扩增DNA,实

  10. K-loops: Loop Transformations for Reconfigurable Architectures

    NARCIS (Netherlands)

    Dragomir, O.S.

    2011-01-01

    The focus of this dissertation is on kernel loops (K-loops), which are loop nests that contain hardware mapped kernels in the loop body. In this thesis, we propose methods for improving the performance of such K-loops, by using standard loop transformations for exposing and exploiting the coarse gr

  11. Loop functions in thermal QCD

    OpenAIRE

    Vairo Antonio

    2014-01-01

    We discuss divergences of loop functions in thermal QCD and compute perturbatively the Polyakov loop, the Polyakov loop correlator and the cyclic Wilson loop. We show how these functions get mixed under renormalization.

  12. Loop functions in thermal QCD

    Directory of Open Access Journals (Sweden)

    Vairo Antonio

    2014-01-01

    Full Text Available We discuss divergences of loop functions in thermal QCD and compute perturbatively the Polyakov loop, the Polyakov loop correlator and the cyclic Wilson loop. We show how these functions get mixed under renormalization.

  13. Experiments of chimney flows for the validation of the behaviour of passive autocatalytic recombiners; Experimente zur Validierung eines Kaminmodells fuer die Simulation des Betriebsverhaltens eines Wasserstoffrekombinators

    Energy Technology Data Exchange (ETDEWEB)

    Simon, Berno [RWTH Aachen Univ. (Germany). Lehrstuhl fuer Reaktorsicherheit und -technik (LRST)

    2013-10-15

    For the validation of the computational code REKO-DIREKT simulating the operational behaviour of passive autocatalytic recombiners in severe accidents, experiments on chimney flows were performed in cooperation between RWTH Aachen and Forschungszentrum Juelich. This compact describes the experimental setup and shows the correlation between the H{sub 2} concentration, the catalyst temperature and the inlet velocity at various recombiner chimney heights. (orig.)

  14. Influence of DC plasma modification on the selected properties and the geometrical surface structure of polylactide prior to autocatalytic metallization

    Energy Technology Data Exchange (ETDEWEB)

    Moraczewski, Krzysztof, E-mail: kmm@ukw.edu.pl [Kazimierz Wielki University, Chodkiewicza 30, 85-064 Bydgoszcz (Poland); Rytlewski, Piotr [Kazimierz Wielki University, Chodkiewicza 30, 85-064 Bydgoszcz (Poland); Malinowski, Rafał [Institute for Engineering of Polymer Materials and Dyes, Marii Skłodowskiej-Curie 55, 87-100 Toruń (Poland); Tracz, Adam [Centre for Molecular and Macromolecular Studies of the Polish Academy of Sciences, Sienkiewicza 112, 90-363 Łódź (Poland); Żenkiewicz, Marian [Institute for Engineering of Polymer Materials and Dyes, Marii Skłodowskiej-Curie 55, 87-100 Toruń (Poland)

    2015-03-01

    The paper presents the results of studies to determine the applicability of plasma modification in the process of polylactide (PLA) surface preparation prior to the autocatalytic metallization. The polylactide plasma modification was carried out in an oxygen or nitrogen chemistry. The samples were tested with the following methods: scanning electron microscopy (SEM), atomic force microscopy (AFM), goniometry and electron spectrophotometry (XPS). Scanning electron microscopy and atomic force microscopy images were demonstrated. The results of surface free energy calculations, performed based on the results of the contact angle measurements have been presented. The results of the qualitative (degree of oxidation or nitridation) and quantitative analysis of the chemical composition of the polylactide surface layer have also been described. The results of the studies show that the DC plasma modification performed in the proposed condition is a suitable as a method of surface preparation for the polylactide metallization. - Highlights: • We modified polylactide surface layer with plasma generated in oxygen or nitrogen. • We tested selected properties and surface structure of modified samples. • DC plasma modification can be used to prepare the PLA surface for metallization. • For better results metallization should be preceded by sonication process.

  15. Experiment on large scale plume interaction with a stratified gas environment resembling the thermal activity of a autocatalytic recombiner

    Energy Technology Data Exchange (ETDEWEB)

    Mignot, G.; Kapulla, R.; Paladino, D.; Zboray, R. [Thermal Hydraulics Laboratory, Nuclear Energy and Safety Dept., Paul Scherrer Institut, 5232 Villigen PSI (Switzerland)

    2012-07-01

    Computational Fluid Dynamics codes (CFD) are increasingly being used to simulate containment conditions after various transient accident scenarios. Consequently, the reliability of such codes must be tested against experimental data. Such validation experiments related to gas mixing and hydrogen transport within containment compartments addressing the effect of heat source are presented in this paper. The experiments were conducted in the large-scale thermal-hydraulics PANDA facility located at the Paul-Scherrer-Inst. (PSI) in Switzerland, in the frame of the OECD/SETH-2 project. A 10 kW electric heater simulating the thermal activity of the autocatalytic recombiner was activated at full power in a containment vessel at the top of which a thick helium layer is initially present. The hot plume interacts with the bottom of the helium layer which is slowly eroded until complete break up at 1350 s. After final erosion of the layer a strong temperature and concentration gradient is maintained in the vessel below the heater inlet as well as in the adjacent vessel below the interconnecting pipe. A detailed characterization of the operating heater suggests the presence of cold gas ingress at the outlet that affects the flow in the chimney. This can be of concern if present in a real PAR unit. (authors)

  16. Loops and trees

    Science.gov (United States)

    Caron-Huot, S.

    2011-05-01

    We investigate relations between loop and tree amplitudes in quantum field theory that involve putting on-shell some loop propagators. This generalizes the so-called Feynman tree theorem which is satisfied at 1-loop. Exploiting retarded boundary conditions, we give a generalization to ℓ-loop expressing the loops as integrals over the on-shell phase space of exactly ℓ particles. We argue that the corresponding integrand for ℓ > 2 does not involve the forward limit of any physical tree amplitude, except in planar gauge theories. In that case we explicitly construct the relevant physical amplitude. Beyond the planar limit, abandoning direct integral representations, we propose that loops continue to be determined implicitly by the forward limit of physical connected trees, and we formulate a precise conjecture along this line. Finally, we set up technology to compute forward amplitudes in supersymmetric theories, in which specific simplifications occur.

  17. Diffusion of Wilson Loops

    OpenAIRE

    Brzoska, A. M.; Lenz, F.; Negele, J. W.; Thies, M.

    2004-01-01

    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 i...

  18. Optical chirped beam amplification and propagation

    Science.gov (United States)

    Barty, Christopher P.

    2004-10-12

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

  19. Double regenerative amplification of picosecond pulses

    Science.gov (United States)

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

    2012-04-01

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

  20. Neutron transport in irradiation loops (IRENE loop)

    International Nuclear Information System (INIS)

    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

  1. Loop Quantum Cosmology from Loop Quantum Gravity

    OpenAIRE

    Alesci, Emanuele; Cianfrani, Francesco

    2014-01-01

    We show how Loop Quantum Cosmology can be derived as an effective semiclassical description of Loop Quantum Gravity. Using the tools of QRLG, a gauge fixed version of LQG, we take the coherent states of the fundamental microscopic theory suitable to describe a Bianchi I Universe and we find a mapping between the expectation value of the Hamiltonian and the dynamics of LQC. Our results are in agreement with a lattice refinement framework for LQC, thus the so called ``old'' and ``improved-dynam...

  2. Comprehensive human genome amplification using multiple displacement amplification

    OpenAIRE

    Dean, Frank B.; Hosono, Seiyu; Fang, Linhua; Wu, Xiaohong; Faruqi, A. Fawad; Bray-Ward, Patricia; Zhenyu SUN; Zong, Qiuling; Du, Yuefen; Du, Jing; Driscoll, Mark; Song, Wanmin; Kingsmore, Stephen F.; Egholm, Michael; Lasken, Roger S.

    2002-01-01

    Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity. Because DNA yield from human samples is frequently limiting, much effort has been invested in developing methods for whole genome amplification (WGA) by random or degenerate oligonucleotide-primed PCR. However, existing WGA methods like degenerate oligonucleotide-primed PCR suffer from incomplete coverage and inadequate average DNA size. We describe a method, termed multi...

  3. Amplification of enantiomeric excess, mirror-image symmetry breaking and kinetic proofreading in Soai reaction models with different oligomeric orders.

    Science.gov (United States)

    Micheau, Jean-Claude; Coudret, Christophe; Cruz, José-Manuel; Buhse, Thomas

    2012-10-14

    A comprehensive kinetic analysis of three prototypical autocatalytic cycle models based on the absolute asymmetric Soai reaction is presented. The three models, which can give rise to amplification of enantiomeric excess and mirror-image symmetry breaking, vary by their monomeric, dimeric or trimeric order of the assumed catalytic species. Our numerical approach considered the entire chiral combinatorics of the diastereomeric interactions in the models as well as the multiplicity of coupled reversible reactions without applying fast equilibration or quasi-steady state approximations. For the simplest monomeric model, an extensive range of parameters was explored employing a random grid parameter scanning method that revealed the influence of the parameter values on the product distribution, the reaction-time, the attenuation or amplification of enantiomeric excess as well as on the presence or absence of mirror-image symmetry breaking. A symmetry breaking test was imposed on the three models showing that an increase in the catalytic oligomer size from one to three leads to a higher tolerance to poorer chiral recognition between the diastereoisomers and identifies the greater impact of the diastereoisomeric energy difference over an imperfect stereoselectivity in the catalytic step. This robustness is understood as a particular case of so-called kinetic proofreading in asymmetric autocatalysis.

  4. Water loop for training

    International Nuclear Information System (INIS)

    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.)

  5. What Controls DNA Looping?

    Directory of Open Access Journals (Sweden)

    Pamela J. Perez

    2014-08-01

    Full Text Available The looping of DNA provides a means of communication between sequentially distant genomic sites that operate in tandem to express, copy, and repair the information encoded in the DNA base sequence. The short loops implicated in the expression of bacterial genes suggest that molecular factors other than the naturally stiff double helix are involved in bringing the interacting sites into close spatial proximity. New computational techniques that take direct account of the three-dimensional structures and fluctuations of protein and DNA allow us to examine the likely means of enhancing such communication. Here, we describe the application of these approaches to the looping of a 92 base-pair DNA segment between the headpieces of the tetrameric Escherichia coli Lac repressor protein. The distortions of the double helix induced by a second protein—the nonspecific nucleoid protein HU—increase the computed likelihood of looping by several orders of magnitude over that of DNA alone. Large-scale deformations of the repressor, sequence-dependent features in the DNA loop, and deformability of the DNA operators also enhance looping, although to lesser degrees. The correspondence between the predicted looping propensities and the ease of looping derived from gene-expression and single-molecule measurements lends credence to the derived structural picture.

  6. Hybrid chirped pulse amplification system

    Science.gov (United States)

    Barty, Christopher P.; Jovanovic, Igor

    2005-03-29

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

  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)

    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

  8. Generic approach for designing and implementing a passive autocatalytic recombiner PAR-system in nuclear power plant containments

    International Nuclear Information System (INIS)

    PARSOAR project is one of the scientific projects on hydrogen risk in nuclear power plants co-sponsored by the European Commission in the Fifth Euratom Framework Program and the Swiss government. It is one of the components of the 'Safety Accident Management' (SAM) cluster that is focusing on review of severe accident risks and on development of countermeasures for defence-in-depth. The first objective is to carry out a state-of-the-art on passive autocatalytic recombiners (PAR) to constitute a database helpful for the PAR-designers, nuclear power plant designers and utilities, and safety authorities. The second objective is to elaborate a handbook guide that defines an approach for implementing catalytic recombiners in nuclear power plants, and that proposes recommended practices to support the detailed implementation by individual nuclear actors. The third objective is to identify the needs in terms of complementary experimental qualification or computer code developments about the hydrogen risk in nuclear power plants. It is also recommended that future experimental tests and numerical modelling should concentrate on the remaining issues of (a) lowering of existing uncertainties about hydrogen sources as core reflood or the reactions between boron carbide and steam or uranium and steam, (b) completing the experimental qualification of catalytic recombiners for existing and future nuclear power plant applications, and (c) performing well-qualified couplings between computational fluid dynamics computer codes and numerical models simulating PAR-behaviour (thanks to some global and well-instrumented experiments reproducing a complete reference accident sequence in presence of catalytic recombiners). Such areas to be considered for future work could be integrated in the scope of a European hydrogen network of excellence, in the framework of the Sixth Euratom Framework Program (2002-2006)

  9. Generic approach for designing and implementing a passive autocatalytic recombiner PAR-system in nuclear power plant containments

    Energy Technology Data Exchange (ETDEWEB)

    Bachellerie, E. E-mail: bachelle@tecatom.fr; Arnould, F.; Auglaire, M.; Boeck, B. de; Braillard, O.; Eckardt, B.; Ferroni, F.; Moffett, R

    2003-04-01

    PARSOAR project is one of the scientific projects on hydrogen risk in nuclear power plants co-sponsored by the European Commission in the Fifth Euratom Framework Program and the Swiss government. It is one of the components of the 'Safety Accident Management' (SAM) cluster that is focusing on review of severe accident risks and on development of countermeasures for defence-in-depth. The first objective is to carry out a state-of-the-art on passive autocatalytic recombiners (PAR) to constitute a database helpful for the PAR-designers, nuclear power plant designers and utilities, and safety authorities. The second objective is to elaborate a handbook guide that defines an approach for implementing catalytic recombiners in nuclear power plants, and that proposes recommended practices to support the detailed implementation by individual nuclear actors. The third objective is to identify the needs in terms of complementary experimental qualification or computer code developments about the hydrogen risk in nuclear power plants. It is also recommended that future experimental tests and numerical modelling should concentrate on the remaining issues of (a) lowering of existing uncertainties about hydrogen sources as core reflood or the reactions between boron carbide and steam or uranium and steam, (b) completing the experimental qualification of catalytic recombiners for existing and future nuclear power plant applications, and (c) performing well-qualified couplings between computational fluid dynamics computer codes and numerical models simulating PAR-behaviour (thanks to some global and well-instrumented experiments reproducing a complete reference accident sequence in presence of catalytic recombiners). Such areas to be considered for future work could be integrated in the scope of a European hydrogen network of excellence, in the framework of the Sixth Euratom Framework Program (2002-2006)

  10. Interpretation of test results performed with catalyst samples of passive autocatalytic recombiners (PAR) under realistic PWR severe accident conditions

    Energy Technology Data Exchange (ETDEWEB)

    Zeyen, Roland [European Commission Joint Research Centre, Saint-Paul-lez-Durance (France). Inst. for Energy (JRC/IE) de Cadarache; Payot, Frederic [Institut de Radioprotection et de Surete Nucleaire (IRSN), Saint-Paul-lez-Durance (France). Centre d' Etudes de Cadarache; Reinecke, Ernst-Arndt [Forschungszentrum Juelich GmbH (Germany). Inst. of Energy Research and Climate Research - Nuclear Waste Management and Reactor Safety (IEK-6)

    2013-06-15

    Passive Autocatalytic Recombiners (PAR) are used regularly in modern Pressurized Water Reactors as a particular feature in the event of severe accidents (core melting) accompanied by large releases of hydrogen into the reactor containment. One objective of the Phebus programme was to perform poisoning studies by inserting in the last test FPT3 a number of small recombiner coupons (30 x 30 mm) manufactured by among others AREVA-NP, NIS, AECL and IRSN/IRCELYON. The goal was to expose catalytic surfaces samples to the atmosphere of the Phebus model containment that collects fission products and aerosols released during the degradation of the Phebus FP pre-irradiated fuel bundle as representative as possible of a severe accidents, and in particular containing boron carbide resulting from the insertion of a B4C control rod into the small scale real irradiated fuel bundle. Before the Phebus FPT3 test, reference and qualification tests were performed in the H2PAR test facility at Cadarache in order to check if the coupon holding equipment was correctly designed with minimum thermal-hydraulic disturbance. The surface temperature of one coupon of each kind was measured by contact-miniature thermocouple during the experiment. This 'reference' temperature in the controlled H2PAR atmosphere was to be compared later to the temperatures reached in the Phebus FP conditions. The results coming from the coupon temperatures in FPT3 were quite un-expected. Therefore, supplementary small-scale tests were performed in the REKO-1 facility at Juelich to understand the relatively low temperatures and the particular behaviour of coupons coming from different manufacturers. (orig.)

  11. Operation behaviour of passive autocatalytic recombiners (PAR) for hydrogen mitigation in realistic containment conditions after a severe PWR accident

    Energy Technology Data Exchange (ETDEWEB)

    Zeyen, Roland [European Commission Joint Research Centre, Brussels (Belgium). Inst. for Energy (JRC/IE); Payot, Frederic [Institut de Radioprotection et de Surete Nucleaire (IRSN), Saint-Paul-Lez-Durance (France). Centre d' Etudes de Cadarache; Reinecke, Ernst-Arndt [Forschungszentrum Juelich (Germany). Inst. of Energy and Climate Research - Nuclear Waste Management and Reactor Safety (IEK-6)

    2012-11-01

    Passive Autocatalytic Recombiners (PAR) are used regularly in modern Pressurized Water Reactors as a particular feature in the event of severe accidents (core melting) accompanied by large releases of hydrogen into the reactor containment. One objective of the Phebus programme was to perform poisoning studies by inserting in the last test FPT3 a number of small recombiner coupons (30 x 30mm) manufactured by among others AREVANP, NIS, AECL and IRSN/IRCELYON. The goal was to expose catalytic surfaces samples to the atmosphere of the Phebus model containment that collects fission products and aerosols released during the degradation of the Phebus FP pre-irradiated fuel bundle as representative as possible of a severe accidents, and in particular containing boron carbide resulting from the insertion of a B4C control rod into the small scale real irradiated fuel bundle. Before the Phebus FPT3 test, reference and qualification tests were performed in the H2PAR test facility at Cadarache in order to check if the coupon holding equipment was correctly designed with minimum thermal-hydraulic disturbance. The surface temperature of one coupon of each kind was measured by contact-miniature thermocouple during the experiment. This ''reference'' temperature in the controlled H2PAR atmosphere was to be compared later to the temperatures reached in the Phebus FP conditions. The results coming from the coupon temperatures in FPT3 were quite un-expected: therefore it was thought that supplementary small-scale tests were needed (in 2010) to understand the relatively low temperatures and the particular behaviour of coupons coming from different manufacturers. The test results were supported and verified by dedicated code calculations. (orig.)

  12. 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.

  13. Spheromak Impedance and Current Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Fowler, T K; Hua, D D; Stallard, B W

    2002-01-31

    It is shown that high current amplification can be achieved only by injecting helicity on the timescale for reconnection, {tau}{sub REC}, which determines the effective impedance of the spheromak. An approximate equation for current amplification is: dI{sub TOR}{sup 2}/dt {approx} I{sup 2}/{tau}{sub REC} - I{sub TOR}{sup 2}/{tau}{sub closed} where I is the gun current, I{sub TOR} is the spheromak toroidal current and {tau}{sub CLOSED} is the ohmic decay time of the spheromak. Achieving high current amplification, I{sub TOR} >> I, requires {tau}{sub REC} <<{tau}{sub 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 {tau}{sub 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 {proportional_to} I, or I{sub TOR} {approx} I. Program implications are discussed.

  14. Natively unstructured loops differ from other loops.

    Directory of Open Access Journals (Sweden)

    Avner Schlessinger

    2007-07-01

    Full Text Available Natively unstructured or disordered protein regions may increase the functional complexity of an organism; they are particularly abundant in eukaryotes and often evade structure determination. Many computational methods predict unstructured regions by training on outliers in otherwise well-ordered structures. Here, we introduce an approach that uses a neural network in a very different and novel way. We hypothesize that very long contiguous segments with nonregular secondary structure (NORS regions differ significantly from regular, well-structured loops, and that a method detecting such features could predict natively unstructured regions. Training our new method, NORSnet, on predicted information rather than on experimental data yielded three major advantages: it removed the overlap between testing and training, it systematically covered entire proteomes, and it explicitly focused on one particular aspect of unstructured regions with a simple structural interpretation, namely that they are loops. Our hypothesis was correct: well-structured and unstructured loops differ so substantially that NORSnet succeeded in their distinction. Benchmarks on previously used and new experimental data of unstructured regions revealed that NORSnet performed very well. Although it was not the best single prediction method, NORSnet was sufficiently accurate to flag unstructured regions in proteins that were previously not annotated. In one application, NORSnet revealed previously undetected unstructured regions in putative targets for structural genomics and may thereby contribute to increasing structural coverage of large eukaryotic families. NORSnet found unstructured regions more often in domain boundaries than expected at random. In another application, we estimated that 50%-70% of all worm proteins observed to have more than seven protein-protein interaction partners have unstructured regions. The comparative analysis between NORSnet and DISOPRED2 suggested

  15. Natively unstructured loops differ from other loops.

    Science.gov (United States)

    Schlessinger, Avner; Liu, Jinfeng; Rost, Burkhard

    2007-07-01

    Natively unstructured or disordered protein regions may increase the functional complexity of an organism; they are particularly abundant in eukaryotes and often evade structure determination. Many computational methods predict unstructured regions by training on outliers in otherwise well-ordered structures. Here, we introduce an approach that uses a neural network in a very different and novel way. We hypothesize that very long contiguous segments with nonregular secondary structure (NORS regions) differ significantly from regular, well-structured loops, and that a method detecting such features could predict natively unstructured regions. Training our new method, NORSnet, on predicted information rather than on experimental data yielded three major advantages: it removed the overlap between testing and training, it systematically covered entire proteomes, and it explicitly focused on one particular aspect of unstructured regions with a simple structural interpretation, namely that they are loops. Our hypothesis was correct: well-structured and unstructured loops differ so substantially that NORSnet succeeded in their distinction. Benchmarks on previously used and new experimental data of unstructured regions revealed that NORSnet performed very well. Although it was not the best single prediction method, NORSnet was sufficiently accurate to flag unstructured regions in proteins that were previously not annotated. In one application, NORSnet revealed previously undetected unstructured regions in putative targets for structural genomics and may thereby contribute to increasing structural coverage of large eukaryotic families. NORSnet found unstructured regions more often in domain boundaries than expected at random. In another application, we estimated that 50%-70% of all worm proteins observed to have more than seven protein-protein interaction partners have unstructured regions. The comparative analysis between NORSnet and DISOPRED2 suggested that long

  16. Use of RAPD and PCR double amplification in the study of ancient DNA

    Directory of Open Access Journals (Sweden)

    F. Balzano

    2011-01-01

    Full Text Available This project analysed the DNA extracted from bones of ancient sheep which have been brought to light in Sardinian different archaeological sites. In order to better analyse this highly fragmented DNA, a double amplification technique was chosen. The first approach consisted of RAPD-PCR abd the second one in classic PCR. The RAPD-PCR amplified random fragments and allowed the production of numerous amplicons. The products of RAPD amplification have been amplified, more specifically, by the second PCR using primers for a sequence of 176 bp of mitochondrial D-loop region. These DNA fragments have been sequenced and the sequence analysis has confirmed that it belonged to Ovis aries. Consequently, this provedure can be considered a valid tool to perform amplification of degraded DNA, such as ancient DNA.

  17. Blind loop syndrome

    Science.gov (United States)

    ... operations for extreme obesity As a complication of inflammatory bowel disease Diseases such as diabetes or scleroderma may slow down movement in a segment of the intestine, leading to blind loop syndrome.

  18. Sensitivity Enhancement of Remotely Coupled NMR Detectors using Wirelessly Powered Parametric Amplification

    OpenAIRE

    Qian, Chunqi; Murphy-Boesch, Joseph; DODD, Stephen; Koretsky, Alan

    2012-01-01

    A completely wireless detection coil with an integrated parametric amplifier has been constructed to provide local amplification and transmission of MR signals. The sample coil is one element of a parametric amplifier using a zero-bias diode that mixes the weak MR signal with a strong pump signal that is obtained from an inductively coupled external loop. The NMR sample coil develops current gain via reduction in the effective coil resistance. Higher gain can be obtained by adjusting the leve...

  19. What Controls DNA Looping?

    OpenAIRE

    Perez, Pamela J.; Nicolas Clauvelin; Grosner, Michael A.; Colasanti, Andrew V.; Olson, Wilma K.

    2014-01-01

    The looping of DNA provides a means of communication between sequentially distant genomic sites that operate in tandem to express, copy, and repair the information encoded in the DNA base sequence. The short loops implicated in the expression of bacterial genes suggest that molecular factors other than the naturally stiff double helix are involved in bringing the interacting sites into close spatial proximity. New computational techniques that take direct account of the three-dimensional stru...

  20. Loop Quantum Gravity

    OpenAIRE

    Rovelli Carlo

    1997-01-01

    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: (...

  1. Quantum Reduced Loop Gravity

    OpenAIRE

    Alesci, Emanuele; Cianfrani, Francesco

    2015-01-01

    Quantum Reduced Loop Gravity provides a promising framework for a consistent characterization of the early Universe dynamics. Inspired by BKL conjecture, a flat Universe is described as a collection of Bianchi I homogeneous patches. The resulting quantum dynamics is described by the scalar constraint operator, whose matrix elements can be analytically computed. The effective semiclassical dynamics is discussed, and the differences with Loop Quantum Cosmology are emphasized.

  2. Reactor loops at Chalk River

    International Nuclear Information System (INIS)

    This report describes broadly the nine in-reactor loops, and their components, located in and around the NRX and NRU reactors at Chalk River. First an introduction and general description is given of the loops and their function, supplemented with a table outlining some loop specifications and nine simplified flow sheets, one for each individual loop. The report then proceeds to classify each loop into two categories, the 'main loop circuit' and the 'auxiliary circuit', and descriptions are given of each circuit's components in turn. These components, in part, are comprised of the main loop pumps, the test section, loop heaters, loop coolers, delayed-neutron monitors, surge tank, Dowtherm coolers, loop piping. Here again photographs, drawings and tables are included to provide a clearer understanding of the descriptive literature and to include, in tables, some specifications of the more important components in each loop. (author)

  3. Loops under Strategies ... Continued

    Directory of Open Access Journals (Sweden)

    René Thiemann

    2010-12-01

    Full Text Available While there are many approaches for automatically proving termination of term rewrite systems, up to now there exist only few techniques to disprove their termination automatically. Almost all of these techniques try to find loops, where the existence of a loop implies non-termination of the rewrite system. However, most programming languages use specific evaluation strategies, whereas loop detection techniques usually do not take strategies into account. So even if a rewrite system has a loop, it may still be terminating under certain strategies. Therefore, our goal is to develop decision procedures which can determine whether a given loop is also a loop under the respective evaluation strategy. In earlier work, such procedures were presented for the strategies of innermost, outermost, and context-sensitive evaluation. In the current paper, we build upon this work and develop such decision procedures for important strategies like leftmost-innermost, leftmost-outermost, (max-parallel-innermost, (max-parallel-outermost, and forbidden patterns (which generalize innermost, outermost, and context-sensitive strategies. In this way, we obtain the first approach to disprove termination under these strategies automatically.

  4. Choking loops on surfaces.

    Science.gov (United States)

    Feng, Xin; Tong, Yiying

    2013-08-01

    We present a method for computing "choking" loops--a set of surface loops that describe the narrowing of the volumes inside/outside of the surface and extend the notion of surface homology and homotopy loops. The intuition behind their definition is that a choking loop represents the region where an offset of the original surface would get pinched. Our generalized loops naturally include the usual 2g handles/tunnels computed based on the topology of the genus-g surface, but also include loops that identify chokepoints or bottlenecks, i.e., boundaries of small membranes separating the inside or outside volume of the surface into disconnected regions. Our definition is based on persistent homology theory, which gives a measure to topological structures, thus providing resilience to noise and a well-defined way to determine topological feature size. More precisely, the persistence computed here is based on the lower star filtration of the interior or exterior 3D domain with the distance field to the surface being the associated 3D Morse function. PMID:23744260

  5. Heralded amplification of photonic qubits.

    Science.gov (United States)

    Bruno, Natalia; Pini, Vittorio; Martin, Anthony; Verma, Varun B; Nam, Sae Woo; Mirin, Richard; Lita, Adriana; Marsili, Francesco; Korzh, Boris; Bussières, Félix; Sangouard, Nicolas; Zbinden, Hugo; Gisin, Nicolas; Thew, Rob

    2016-01-11

    We demonstrate postselection free heralded qubit amplification for Time-Bin qubits and single photon states in an all-fibre, telecom-wavelength, scheme that highlights the simplicity, stability and potential for fully integrated photonic solutions. Exploiting high-efficiency superconducting detectors, the gain, fidelity and the performance of the amplifier are studied as a function of loss. We also demonstrate the first heralded single photon amplifier with independent sources. This provides a significant advance towards demonstrating device-independent quantum key distribution as well as fundamental tests of quantum mechanics over extended distances. PMID:26832244

  6. 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.

  7. Dynamics and Control of DNA Sequence Amplification

    CERN Document Server

    Marimuthu, Karthikeyan

    2014-01-01

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

  8. Passive auto-catalytic recombiners operation in the presence of hydrogen and carbon monoxide: Experimental study and model development

    Energy Technology Data Exchange (ETDEWEB)

    Klauck, Michael, E-mail: klauck@lrst.rwth-aachen.de [RWTH Aachen University, Institute for Reactor Safety and Reactor Technology, 52072 Aachen (Germany); Reinecke, Ernst-Arndt, E-mail: e.reinecke@fz-juelich.de [Forschungszentrum Jülich GmbH, Institute of Energy and Climate Research – Nuclear Waste Management and Reactor Safety (IEK-6), 52425 Jülich (Germany); Kelm, Stephan, E-mail: s.kelm@fz-juelich.de [Forschungszentrum Jülich GmbH, Institute of Energy and Climate Research – Nuclear Waste Management and Reactor Safety (IEK-6), 52425 Jülich (Germany); Meynet, Nicolas, E-mail: nicolas.meynet@irsn.fr [Institut de Radioprotection et de Sûreté Nucléaire (IRSN), PSN-RES/SAG/BPhAG, BP 17 92262 Fontenay aux Roses (France); Bentaïb, Ahmed, E-mail: ahmed.bentaib@irsn.fr [Institut de Radioprotection et de Sûreté Nucléaire (IRSN), PSN-RES/SAG/BPhAG, BP 17 92262 Fontenay aux Roses (France); Allelein, Hans-Josef, E-mail: allelein@lrst.rwth-aachen.de [RWTH Aachen University, Institute for Reactor Safety and Reactor Technology, 52072 Aachen (Germany); Forschungszentrum Jülich GmbH, Institute of Energy and Climate Research – Nuclear Waste Management and Reactor Safety (IEK-6), 52425 Jülich (Germany)

    2014-01-15

    Highlights: • We studied the hydrogen conversion in the presence of carbon monoxide (CO). • CO recombines at a lower efficiency than hydrogen. • Under the given conditions, hydrogen conversion is not affected by CO. • We used three different numerical codes to simulate the experimental findings. • All codes are reproducing the experimental data well. -- Abstract: In a LWR severe accident, carbon monoxide (CO) may be generated inside the containment due to molten corium concrete interaction (MCCI). As a component of the accident atmosphere, CO will interact with passive auto-catalytic recombiners (PARs) which are installed inside LWR containments for hydrogen (H{sub 2}) removal. Depending on the boundary conditions, CO may either react with oxygen to carbon dioxide (CO{sub 2}) or act as catalyst poison, reducing the catalyst activity and hence the hydrogen conversion efficiency. A new experimental test programme performed in co-operation between JÜLICH and RWTH investigates these aspects aiming at providing data for model development for advanced severe accident analyses. In the first test series presented here, the parallel catalytic reaction of H{sub 2} and CO on the catalyst surface has been studied, i.e. the hydrogen recombination reaction was started before CO was injected. In total, 33 steady state measurements have been performed. The test series was jointly evaluated by JÜLICH, RWTH and IRSN. The test results show that under the given conditions the conversion of CO into CO{sub 2} has no negative impact on the parallel hydrogen conversion. The efficiency of the CO recombination in terms of molar rates is significantly smaller (by a factor of ∼2) than the corresponding H{sub 2} conversion efficiency. Due to the exothermal reaction, the parallel CO conversion may also have an impact on the possible ignition of the flammable gases at hot PAR surfaces. The authors have used three different numerical codes for the simulation of the parallel CO

  9. Isolation and Rapid Detection of Avian Borna Virus by a Reverse Transcription Loop-mediated Isothermal Amplification Assay for Outbreaks in Psittacine Birds%禽波纳病毒分离鉴定及其恒温扩增检测分析

    Institute of Scientific and Technical Information of China (English)

    田纯见; 唐羿; 周小明; 常彦磊; 吴晓薇; 朱道中; 王宏; 罗琼; 林志雄; 赵吟; 罗长保; 鱼海琼; 刘志玲; 陈茹

    2012-01-01

    利用腺胃扩张症(PDD)患病鹦鹉腺胃RT-PCR阳性病料,接种猪睾丸(ST)传代细胞,分离禽波纳病毒(ABV),建立实时RT-LAMP检测方法.将阳性病料接种ST细胞单层传代,出现细胞圆缩、脱落,ABV基质蛋白(M)基因扩增产物出现预计大小351 bp条带,测序后进化树分析显示为ABV5基因型.针对M基因设计ID37、ID30、ID19、ID6和ID1共5组引物,后3组引物RT-LAMP呈阳性反应.利用钙黄绿素建立实时RT-LAMP,分别在36(ID30)、38(ID37)和49(ID19)min出现扩增反应曲线,60 min内扩增达到峰值.对各种临床样品检测与RT-PCR结果一致,新城疫等类症病毒未见阳性反应,显示较高的特异性 ;对细胞培养物检测10-1~10-5为阳性,比较RT-PCR敏感性提高约100倍.RT-LAMP检测方法的建立为PDD防制提供新的检测方法,也是波纳病公共卫生研究有益的参考.%In this study an avian bornavirus (ABV) strain was isolated from sick parrots with proventricular dilatation disease(PDD). The virus grew in swine testicular (ST) cell monolayer with granulating, shrinking, rounding and falling off although classical Borna disease virus strains replicate very efficiently in cultured mammalian cells in which persistent, noncytolytic infections was readily established. Viruses were successfully isolated and demonstrated by reverse transcription-PCR analysis from the proventricular glands of parrot "glass 363" and "color" with confirmed PDD. The 351 bp product of the expected size bands of matrix protein (M) gene was cloned, the sequence and phylogenetic tree analysis showed that the isolated virus belonging to genotype ABV5. Five sets of M gene RT-LAMP primers ID1, ID6, ID19, ID30 and ID37 were designed using DNAStar and PrimerExplorer V5. 0 (network) and later three set reactions showed positive color reaction with specific electrophoretic bands. The amplification curves of of real-time RT-LAMP using fluorescent indicator calcein were shown in 36 (ID30), 38 (ID37

  10. Dynamics and control of DNA sequence amplification

    International Nuclear Information System (INIS)

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

  11. Dynamics and control of DNA sequence amplification

    Energy Technology Data Exchange (ETDEWEB)

    Marimuthu, Karthikeyan [Department of Chemical Engineering and Center for Advanced Process Decision-Making, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Chakrabarti, Raj, E-mail: raj@pmc-group.com, E-mail: rajc@andrew.cmu.edu [Department of Chemical Engineering and Center for Advanced Process Decision-Making, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Division of Fundamental Research, PMC Advanced Technology, Mount Laurel, New Jersey 08054 (United States)

    2014-10-28

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

  12. Risk Perception and Social Amplification

    International Nuclear Information System (INIS)

    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

  13. Classical Physics and Quantum Loops

    Energy Technology Data Exchange (ETDEWEB)

    Barry R. Holstein; John F. Donoghue

    2004-05-01

    The standard picture of the loop expansion associates a factor of h-bar with each loop, suggesting that the tree diagrams are to be associated with classical physics, while loop effects are quantum mechanical in nature. We discuss examples wherein classical effects arise from loop contributions and display the relationship between the classical terms and the long range effects of massless particles.

  14. Bootstrapping Null Polygon Wilson Loops

    OpenAIRE

    Gaiotto, Davide; Maldacena, Juan; Sever, Amit; Vieira, Pedro

    2010-01-01

    We derive the two loop expressions for polygonal Wilson loops by starting from the one loop expressions and applying an operator product expansion. We do this for polygonal Wilson loops in R^{1,1} and find a result in agreement with previous computations. We also discuss the spectrum of excitations around flux tube that connects two null Wilson lines.

  15. Direct construction of code loops

    OpenAIRE

    Nagy, Gabor P.

    2004-01-01

    Code loops were introduced by R. L. Griess. R.L. Griess and T. Hsu gave methods to construct the corresponding code loop from any given doubly even binary code; both these methods used some kind of induction. In this paper, we present a global construction of the loop, where we apply the correspondance between the concepts of Moufang loops and groups with triality.

  16. Genetic Programming with Simple Loops

    Institute of Scientific and Technical Information of China (English)

    QI Yuesheng; WANG Baozhong; KANG Lishan

    1999-01-01

    A kind of loop function LoopN inGenetic Programming (GP) is proposed.Different from other forms of loopfunction, such as While-Do and Repeat-Until, LoopNtakes only oneargument as its loop body and makes its loop body simply run N times,soinfinite loops will never happen. The problem of how to avoid too manylayers ofloops in Genetic Programming is also solved. The advantage ofLoopN in GP is shown bythe computational results in solving the mowerproblem.

  17. Tsunami Amplification due to Focusing

    Science.gov (United States)

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

    2012-12-01

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

  18. 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.

  19. Mechanisms of Metal-Induced Centrosome Amplification

    OpenAIRE

    Holmes, Amie L.; Wise, John Pierce

    2010-01-01

    Exposure to toxic and carcinogenic metals is widespread; however, their mechanisms of action remain largely unknown. One potential mechanism for metal-induced carcinogenicity and toxicity is centrosome amplification. Here, we review the mechanisms for metal-induced centrosome amplification, including arsenic, chromium, mercury and nano-titanium dioxide.

  20. 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.

  1. Wilson-loop instantons

    Science.gov (United States)

    Lee, Kimyeong; Holman, Richard; Kolb, Edward W.

    1987-01-01

    Wilson-loop symmetry breaking is considered on a space-time of the form M4 x K, where M4 is a four-dimensional space-time and K is an internal space with nontrivial and finite fundamental group. It is shown in a simple model that the different vacua obtained by breaking a non-Abelian gauge group by Wilson loops are separated in the space of gauge potentials by a finite energy barrier. An interpolating gauge configuration is then constructed between these vacua and shown to have minimum energy. Finally some implications of this construction are discussed.

  2. Loop quantum gravity

    Science.gov (United States)

    Chiou, Dah-Wei

    2015-12-01

    This paper presents an "in-a-nutshell" yet self-contained introductory review on loop quantum gravity (LQG) — a background-independent, nonperturbative approach to a consistent quantum theory of gravity. Instead of rigorous and systematic derivations, it aims to provide a general picture of LQG, placing emphasis on the fundamental ideas and their significance. The canonical formulation of LQG, as the central topic of the paper, is presented in a logically orderly fashion with moderate details, while the spin foam theory, black hole thermodynamics, and loop quantum cosmology are covered briefly. Current directions and open issues are also summarized.

  3. 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.

  4. On loop states in loop-quantum gravity

    Energy Technology Data Exchange (ETDEWEB)

    Dass, N D Hari [Hayama Center for Advanced Studies, Hayama, Kanagawa 240-0193 (Japan); Mathur, Manu [S N Bose National Centre for Basic Sciences, JD Block, Sector III, Salt Lake City, Calcutta 700 091 (India)

    2007-05-07

    We explicitly construct and characterize all possible independent loop states in (3 + 1)-dimensional loop-quantum gravity by regulating it on a 3D regular lattice in the Hamiltonian formalism. These loop states, characterized by the (dual) angular momentum quantum numbers, describe SU(2) rigid rotators on the links of the lattice. The loop states are constructed using the Schwinger bosons which are harmonic oscillators in the fundamental (spin half) representation of SU(2). Using the generalized Wigner-Eckart theorem, we compute the matrix elements of the volume operator in the loop basis. Some simple loop eigenstates of the volume operator are explicitly constructed.

  5. 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)

  6. Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion.

    Science.gov (United States)

    Yata, Tomoya; Takahashi, Yuki; Tan, Mengmeng; Hidaka, Kumi; Sugiyama, Hiroshi; Endo, Masayuki; Takakura, Yoshinobu; Nishikawa, Makiya

    2015-01-01

    The application of DNA as a functional material such as DNA hydrogel has attracted much attention. Despite an increasing interest, the high cost of DNA synthesis is a limiting factor for its utilization. To reduce the cost, we report here a highly efficient amplification technique for polypod-like structured DNA (polypodna) with adhesive ends that spontaneously forms DNA hydrogel. Two types of polypodna with three (tripodna) and four (tetrapodna) pods were selected, and a template oligodeoxynucleotide, containing a tandem sequence of a looped tripodna or tetrapodna, respectively, along with restriction enzyme (TspRI) sites, was designed. The template was circularized using T4 DNA ligase, and amplified by rolling circle amplification (RCA). The RCA product was highly viscous and resistant to restriction digestion. Observation under an electron microscope revealed microflower-like structures. These structures were composed of long DNA and magnesium pyrophosphate, and their treatment with EDTA followed by restriction digestion with TspRI resulted in numerous copies of polypodna with adhesive ends, which formed a DNA hydrogel. Thus, we believe this technique provides a new approach to produce DNA nanostructures, and helps in expanding their practical applications. PMID:26462616

  7. Numerical simulations of transverse oscillations in radiatively cooling coronal loops

    Science.gov (United States)

    Magyar, Norbert; Van Doorsselaere, Tom; Marcu, Alexandru

    2016-05-01

    We aim to study the influence of radiative cooling on the standing kink oscillations of coronal loops. To solve the 3D MHD ideal problem, we use the FLASH code. Our model consists of a straight, density enhanced and gravitationally stratified magnetic flux tube. We perturbed the system initially, leading to a transverse oscillation of the structure, and followed its evolution for a number of periods. A realistic radiative cooling is implemented. Results are compared to available analytical theory. We find that in the linear regime (i.e. low amplitude perturbation and slow cooling) the obtained period and damping time are in good agreement with theory. The cooling leads to an amplification of the oscillation amplitude. However, the difference between the cooling and non-cooling cases is small (around 6% after 6 oscillations). In high amplitude runs with realistic cooling, instabilities deform the loop, leading to increased damping. In this case, the difference between cooling and non-cooling is still negligible at around 12%. A set of simulations with higher density loops are also performed, to explore what happens when the cooling takes place in a very short time (t cool ≈ 100 s). In this case, the difference in amplitude after nearly 3 oscillation periods for the low amplitude case is 21% between cooling and non-cooling cases. We strengthen the results of previous analytical studies that state that the amplification due to cooling is ineffective, and its influence on the oscillation characteristics is small, at least for the cases shown here. Furthermore, the presence of a relatively strong damping in the high amplitude runs even in the fast cooling case indicates that it is unlikely that cooling could alone account for the observed, flare-related undamped oscillations of coronal loops. These results may be significant in the field of coronal seismology, allowing its application to coronal loop oscillations with observed fading-out or cooling behaviour.

  8. Loops: Twisting and Scaling

    Science.gov (United States)

    Walsh, R. W.

    2004-01-01

    Loop-like structures are the fundamental magnetic building blocks of the solar atmosphere. Recent space-based EUV and X-ray satellite observations (from Yohkoh SOHO and TRACE) have challenged the view that these features are simply static gravitationally stratified plasma pipes. Rather it is now surmised that each loop may consist of a bundle of fine plasma threads that are twisted around one another and can brighten independently. This invited review will outline the latest developments in ""untangling"" the topology of these features through three dimensional magnetohydrodynamic modelling and how their properties are being deduced through spectroscopic observations coupled to theoretical scaling laws. In particular recent interest has centred on how the observed thermal profile along loops can be employed as a tool to diagnose any localised energy input to the structure and hence constrain the presence of a particular coronal heating mechanism. The dynamic nature of loops will be highlighted and the implications of superior resolution plasma thread observations (whether spatial temporal or spectral) from future space missions (SolarB STEREO SDO and Solar Orbiter) will be discussed.

  9. Loop quantum gravity

    International Nuclear Information System (INIS)

    Loop quantum gravity is one of the approaches that are being studied to apply the rules of quantum mechanics to the gravitational field described by the theory of General Relativity . We present an introductory summary of the main ideas and recent results. (Author)

  10. 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.

  11. Linking Arctic amplification and local feedbacks

    Science.gov (United States)

    Balcerak, Ernie

    2011-11-01

    Climate simulations show that as the Earth warms, the Arctic warms more than the average global warming. However, models differ on how much more the Arctic warms, and although scientists have proposed a variety of mechanisms to explain the Arctic warming amplification, there is no consensus on the main reasons for it. To shed light on this issue, Hwang et al. investigated the relationship between Arctic amplification and poleward energy transport and local Arctic feedbacks, such as changes in cloud cover or ice loss, across a group of models. The researchers noted that differences in atmospheric energy transport did not explain the ranges of polar amplification; rather, models with more amplification showed less energy transport into high latitudes. The authors found that decreasing energy transport is due to a coupled relationship between Arctic amplification and energy transport: Arctic amplification reduces the equator-to-pole temperature gradient, which strongly decreases energy transport. They suggest that this coupled relationship should be taken into account in studies of Arctic amplification. (Geophysical Research Letters, doi:10.1029/2011GL048546, 2011)

  12. Quantum Amplitude Amplification and Estimation

    CERN Document Server

    Brassard, G; Mosca, M; Tapp, A; Brassard, Gilles; Hoyer, Peter; Mosca, Michele; Tapp, Alain

    2000-01-01

    Consider a Boolean function $\\chi: X \\to \\{0,1\\}$ that partitions set $X$ between its good and bad elements, where $x$ is good if $\\chi(x)=1$ and bad otherwise. Consider also a quantum algorithm $\\mathcal A$ such that $A \\ket{0} = \\sum_{x\\in X} \\alpha_x \\ket{x}$ is a quantum superposition of the elements of $X$, and let $a$ denote the probability that a good element is produced if $A \\ket{0}$ is measured. If we repeat the process of running $A$, measuring the output, and using $\\chi$ to check the validity of the result, we shall expect to repeat $1/a$ times on the average before a solution is found. *Amplitude amplification* is a process that allows to find a good $x$ after an expected number of applications of $A$ and its inverse which is proportional to $1/\\sqrt{a}$, assuming algorithm $A$ makes no measurements. This is a generalization of Grover's searching algorithm in which $A$ was restricted to producing an equal superposition of all members of $X$ and we had a promise that a single $x$ existed such tha...

  13. Loop Heat Pipes and Capillary Pumped Loops: An Applications Perspective

    Science.gov (United States)

    Butler, Dan; Ku, Jentung; Swanson, Theodore; Obenschain, Arthur F. (Technical Monitor)

    2001-01-01

    Capillary pumped loops (CPLS) and loop heat pipes (LHPS) are versatile two-phase heat transfer devices which have recently gained increasing acceptance in space applications. Both systems work based on the same principles and have very similar designs. Nevertheless, some differences exist in the construction of the evaporator and the hydro-accumulator, and these differences lead to very distinct operating characteristics for each loop. This paper presents comparisons of the two loops from an applications perspective, and addresses their impact on spacecraft design, integration, and test. Some technical challenges and issues for both loops are also addressed.

  14. On the extended loop calculus

    CERN Document Server

    Griego, J R

    1995-01-01

    Some features of extended loops are considered. In particular, the behaviour under diffeomorphism transformations of the wavefunctions with support on the extended loop space are studied. The basis of a method to obtain analytical expressions of diffeomorphism invariants via extended loops are settled. Applications to knot theory and quantum gravity are considered.

  15. Determinants of RNA hairpin loop-loop complex stability.

    Science.gov (United States)

    Gregorian, R S; Crothers, D M

    1995-05-19

    Complexes formed by RNA hairpin loops with complementary loop sequences derived from Escherichia coli RNA I and RNA II, which are involved in the control of DNA replication of plasmid ColE1, have been analyzed to determine the sequence and structural elements required to achieve full affinity. Of particular interest is the origin of the enhanced stability of the complex formed by hairpin loops whose loop sequences have been inverted 5' to 3' with respect to wild-type sequences. Full complementarity of the two interacting loops is required to achieve full or enhanced affinity, while the stems of the two hairpins can differ. The major determinant of enhanced affinity lies in the base-pairs formed at positions 1 and 7 of the loops, together with the two base-pairs of each stem which are closest to the loop. Sequence variation in the middle of the loops, or further down the stem away from the loops, exerts only a modest influence on complex stability. We incorporate these results into a model for the loop-loop interaction which accounts for the importance of positions one and seven and the first two nucleotides of the stem, while providing potentially unique structures for recognition by the RNA one modulator protein. PMID:7539081

  16. Phase locked loop

    OpenAIRE

    Beek, van, P.; Klumperink, Eric Antonius Maria; Nauta, Bram; Vaucher, Cicero Silveira

    2007-01-01

    A phase locked loop comprising a phase detector ( 100 ) for determining a phase difference between a reference signal (Ref) and mutually phase shifted signals (I, Q) to generate frequency control signals (U, D), the phase detector ( 100 ) comprising: means ( 10 ) for obtaining a first one of said frequency control signals (U, D) by binary multiplication of the reference signal (Ref) and one of the relative phase shifted signals (I, Q); and means ( 20 ) for obtaining a second one of said frequ...

  17. 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.

  18. 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.

  19. Loop structure of renormalizations

    International Nuclear Information System (INIS)

    Asymptotics in the internal momenta p and q is obtained for renormalized Feynman amplitudes recurrently to a number of loops. The asymptotics has the form of a polynomial in powers of these momenta. The renormalization method implies the exclusion of UV-''bad'' asymptotics which provides the p and q convergence of the integral (UV - ultraviolet divergences). It is pointed out the regularization of the integral performed here may be convenient for combined analysis of UV and infrared problem

  20. Lattice Loop Quantum Gravity

    OpenAIRE

    Aastrup, Johannes; Grimstrup, Jesper M.

    2009-01-01

    We present a separable version of Loop Quantum Gravity (LQG) based on an inductive system of cubic lattices. We construct semi-classical states for which the LQG operators -- the flux, the area and the volume operators -- have the right classical limits. Also, we present the Hamilton and diffeomorphism constraints as operator constraints and show that they have the right classical limit. Finally, we speculate whether the continuum limit, which these semi-classical states probe, can be defined...

  1. Isothermal DNA amplification in vitro: the helicase-dependent amplification system.

    Science.gov (United States)

    Jeong, Yong-Joo; Park, Kkothanahreum; Kim, Dong-Eun

    2009-10-01

    Since the development of polymerase chain reaction, amplification of nucleic acids has emerged as an elemental tool for molecular biology, genomics, and biotechnology. Amplification methods often use temperature cycling to exponentially amplify nucleic acids; however, isothermal amplification methods have also been developed, which do not require heating the double-stranded nucleic acid to dissociate the synthesized products from templates. Among the several methods used for isothermal DNA amplification, the helicase-dependent amplification (HDA) is discussed in this review with an emphasis on the reconstituted DNA replication system. Since DNA helicase can unwind the double-stranded DNA without the need for heating, the HDA system provides a very useful tool to amplify DNA in vitro under isothermal conditions with a simplified reaction scheme. This review describes components and detailed aspects of current HDA systems using Escherichia coli UvrD helicase and T7 bacteriophage gp4 helicase with consideration of the processivity and efficiency of DNA amplification. PMID:19629390

  2. 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...

  3. Can Anomalous Amplification be Attained without Postselection?

    Science.gov (United States)

    Martínez-Rincón, Julián; Liu, Wei-Tao; Viza, Gerardo I.; Howell, John C.

    2016-03-01

    We present a parameter estimation technique based on performing joint measurements of a weak interaction away from the weak-value-amplification approximation. Two detectors are used to collect full statistics of the correlations between two weakly entangled degrees of freedom. Without discarding of data, the protocol resembles the anomalous amplification of an imaginary-weak-value-like response. The amplification is induced in the difference signal of both detectors allowing robustness to different sources of technical noise, and offering in addition the advantages of balanced signals for precision metrology. All of the Fisher information about the parameter of interest is collected. A tunable phase controls the strength of the amplification response. We experimentally demonstrate the proposed technique by measuring polarization rotations in a linearly polarized laser pulse. We show that in the presence of technical noise the effective sensitivity and precision of a split detector is increased when compared to a conventional continuous-wave balanced detection technique.

  4. Can Anomalous Amplification be Attained Without Postselection?

    CERN Document Server

    Martínez-Rincón, Julián; Viza, Gerardo I; Howell, John C

    2015-01-01

    We present a parameter estimation technique based on performing joint measurements of a weak interaction away from the weak-value-amplification approximation. Two detectors are used to collect full statistics of the correlations between two weakly entangled degrees of freedom. Without the need of postselection, the protocol resembles the anomalous amplification of an imaginary-weak-value-like response. The amplification is induced in the difference signal of both detectors allowing robustness to different sources of technical noise, and offering in addition the advantages of balanced signals for precision metrology. All of the Fisher information about the parameter of interest is collected, and a phase controls the amplification response. We experimentally demonstrate the proposed technique by measuring polarization rotations in a linearly polarized laser pulse. The effective sensitivity and precision of a split detector is increased when compared to a conventional continuous-wave balanced detection technique...

  5. 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.

  6. 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.

  7. Numerical simulations of transverse oscillations in radiatively cooling coronal loops

    CERN Document Server

    Magyar, N; Marcu, A

    2015-01-01

    We aim to study the influence of radiative cooling on the standing kink oscillations of a coronal loop. Using the FLASH code, we solved the 3D ideal magnetohydrodynamic equations. Our model consists of a straight, density enhanced and gravitationally stratified magnetic flux tube. We perturbed the system initially, leading to a transverse oscillation of the structure, and followed its evolution for a number of periods. A realistic radiative cooling is implemented. Results are compared to available analytical theory. We find that in the linear regime (i.e. low amplitude perturbation and slow cooling) the obtained period and damping time are in good agreement with theory. The cooling leads to an amplification of the oscillation amplitude. However, the difference between the cooling and non-cooling cases is small (around 6% after 6 oscillations). In high amplitude runs with realistic cooling, instabilities deform the loop, leading to increased damping. In this case, the difference between cooling and non-cooling...

  8. Onshore seismic amplifications due to bathymetric features

    Science.gov (United States)

    Rodríguez-Castellanos, A.; Carbajal-Romero, M.; Flores-Guzmán, N.; Olivera-Villaseñor, E.; Kryvko, A.

    2016-08-01

    We perform numerical calculations for onshore seismic amplifications, taking into consideration the effect of bathymetric features on the propagation of seismic movements. To this end, the boundary element method is applied. Boundary elements are employed to irradiate waves and, consequently, force densities can be obtained for each boundary element. From this assumption, Huygens’ principle is applied, and since the diffracted waves are built at the boundary from which they are radiated, this idea is equivalent to Somigliana’s representation theorem. The application of boundary conditions leads to a linear system being obtained (Fredholm integral equations). Several numerical models are analyzed, with the first one being used to verify the proposed formulation, and the others being used to estimate onshore seismic amplifications due to the presence of bathymetric features. The results obtained show that compressional waves (P-waves) generate onshore seismic amplifications that can vary from 1.2 to 5.2 times the amplitude of the incident wave. On the other hand, the shear waves (S-waves) can cause seismic amplifications of up to 4.0 times the incident wave. Furthermore, an important result is that in most cases the highest seismic amplifications from an offshore earthquake are located on the shoreline and not offshore, despite the seafloor configuration. Moreover, the influence of the incident angle of seismic waves on the seismic amplifications is highlighted.

  9. Real-time DNA Amplification and Detection System Based on a CMOS Image Sensor.

    Science.gov (United States)

    Wang, Tiantian; Devadhasan, Jasmine Pramila; Lee, Do Young; Kim, Sanghyo

    2016-01-01

    In the present study, we developed a polypropylene well-integrated complementary metal oxide semiconductor (CMOS) platform to perform the loop mediated isothermal amplification (LAMP) technique for real-time DNA amplification and detection simultaneously. An amplification-coupled detection system directly measures the photon number changes based on the generation of magnesium pyrophosphate and color changes. The photon number decreases during the amplification process. The CMOS image sensor observes the photons and converts into digital units with the aid of an analog-to-digital converter (ADC). In addition, UV-spectral studies, optical color intensity detection, pH analysis, and electrophoresis detection were carried out to prove the efficiency of the CMOS sensor based the LAMP system. Moreover, Clostridium perfringens was utilized as proof-of-concept detection for the new system. We anticipate that this CMOS image sensor-based LAMP method will enable the creation of cost-effective, label-free, optical, real-time and portable molecular diagnostic devices. PMID:27302586

  10. Heat induces gene amplification in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Bin, E-mail: yanbin@mercyhealth.com [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 (United States); Ouyang, Ruoyun [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China); Huang, Chenghui [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 (China); Liu, Franklin [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Neill, Daniel [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Li, Chuanyuan [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, Mark [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)

    2012-10-26

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

  11. Heat induces gene amplification in cancer cells

    International Nuclear Information System (INIS)

    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.

  12. Rapid amplification of genetically modified organisms using a circular ferrofluid-driven PCR microchip.

    Science.gov (United States)

    Sun, Yi; Kwok, Yien-Chian; Foo-Peng Lee, Peter; Nguyen, Nam-Trung

    2009-07-01

    The use of genetically modified organisms (GMOs) as food and in food products is becoming more and more widespread. Polymerase chain reaction (PCR) technology is extensively used for the detection of GMOs in food products in order to verify compliance with labeling requirements. In this paper, we present a novel close-loop ferrofluid-driven PCR microchip for rapid amplification of GMOs. The microchip was fabricated in polymethyl methacrylate by CO2 laser ablation and was integrated with three temperature zones. PCR solution was contained in a circular closed microchannel and was driven by magnetic force generated by an external magnet through a small oil-based ferrofluid plug. Successful amplification of genetically modified soya and maize were achieved in less than 13 min. This PCR microchip combines advantages of cycling flexibility and quick temperature transitions associated with two existing microchip PCR techniques, and it provides a cost saving and less time-consuming way to conduct preliminary screening of GMOs.

  13. Hubungan Kekerabatan Sapi Aceh dengan Menggunakan Daerah Displacement-loop

    Directory of Open Access Journals (Sweden)

    Mohd. Agus Nashri Abdullah

    2008-10-01

    Full Text Available Relationship of aceh cattle using displacement-loop region ABSTRACT. The aims of this study were to describe relationship of D-loop of mtDNA Aceh cattle which is useful database for conducting conservation programme. The whole blood samples were collected (8 samples for D-loop analysis from four locations which were Aceh Besar, Pidie, North Aceh regencies and Banda Aceh city. Out group whole blood samples were collected from two samples from Bali cattles (Bali Island, Madura cattle (Madura Island, Pesisir cattle (West Sumatera respectively and one sample from PO cattle (West Java. Amplification of D-loop sequences of mtDNA with BIDLF and BIDLR primary have PCR product 980 bp. The Data were analyzed using Squint 1.02 and MEGA 4.0 programme. Result of analysis indicate that Aceh cattle have nearer relationship with zebu and there is items inset of genetik Bali cattle (Bos javanicus at the end sequences start ke-354 situs up to 483, so that the origin Aceh cattle was from Bos indicus which have hybridization with Bos javanicus.

  14. Loop expansion and the bosonic representation of loop quantum gravity

    CERN Document Server

    Bianchi, Eugenio; Hackl, Lucas; Yokomizo, Nelson

    2016-01-01

    We introduce a new loop expansion that provides a resolution of the identity in the Hilbert space of loop quantum gravity on a fixed graph. We work in the bosonic representation obtained by the canonical quantization of the spinorial formalism. The resolution of the identity gives a tool for implementing the projection of states in the full bosonic representation onto the space of solutions to the Gauss and area matching constraints of loop quantum gravity. This procedure is particularly efficient in the semiclassical regime, leading to explicit expressions for the loop expansions of coherent, heat kernel and squeezed states.

  15. 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

  16. Wilson Loop diagrams and Positroids

    OpenAIRE

    Agarwala, Susama; Amat, Eloi Marin

    2015-01-01

    In this paper, we study a new application of the positive Grassmanian to Wilson loop diagrams (or MHV diagrams) for scattering amplitudes in N=4 Super Yang-Mill theory ($N=4$ SYM). There has been much interest in studying this theory via the positive Grassmanians using BCFW recursion. This is the first attempt to study MHV diagrams for planar Wilson loop calculations (or planar amplitudes) in terms of positive Grassmannians. We codify Wilson loop diagrams completely in terms of matroids. This...

  17. Wilson Loop Renormalization Group Flows

    OpenAIRE

    Polchinski, Joseph; Sully, James

    2011-01-01

    The locally BPS Wilson loop and the pure gauge Wilson loop map under AdS/CFT duality to string world-sheet boundaries with standard and alternate quantizations of the world-sheet fields. This implies an RG flow between the two operators, which we verify at weak coupling. Many additional loop operators exist at strong coupling, with a rich pattern of RG flows.

  18. Combinatorial aspects of code loops

    OpenAIRE

    Vojtěchovský, Petr

    2007-01-01

    The existence and uniqueness (up to equivalence) of code loops was first established by R. Griess. Nevertheless, the explicit construction of code loops remained open until T. Hsu introduced the notion of symplectic cubic spaces and their Frattini extensions, and pointed out how the construction of code loops followed from the (purely combinatorial) result of O. Chein and E. Goodaire. Within this paper, we focus on their combinatorial construction and prove a more general result using the lan...

  19. Loop Heat Pipe Startup Behaviors

    Science.gov (United States)

    Ku, Jentung

    2016-01-01

    A loop heat pipe must start successfully before it can commence its service. The startup transient represents one of the most complex phenomena in the loop heat pipe operation. This paper discusses various aspects of loop heat pipe startup behaviors. Topics include the four startup scenarios, the initial fluid distribution between the evaporator and reservoir that determines the startup scenario, factors that affect the fluid distribution between the evaporator and reservoir, difficulties encountered during the low power startup, and methods to enhance the startup success. Also addressed are the pressure spike and pressure surge during the startup transient, and repeated cycles of loop startup and shutdown under certain conditions.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. On Arbitrary Phases in Quantum Amplitude Amplification

    CERN Document Server

    Hoyer, P

    2000-01-01

    We consider the use of arbitrary phases in quantum amplitude amplification which is a generalization of quantum searching. We prove that the phase condition in amplitude amplification is given by $\\tan(\\phi/2)=\\tan(\\phi/2)(1-2a)$, where $\\phi$ and $\\phi$ are the phases used and where $a$ is the success probability of the given algorithm. Thus the choice of phases depends nontrivially and nonlinearly on the success probability. Utilizing this condition, we give methods for constructing quantum algorithms that succeed with certainty and for implementing arbitrary rotations. We also conclude that phase errors of order up to $\\frac{1}{\\sqrt{a}}$ can be tolerated in amplitude amplification.

  5. Development of loop-mediated isothermal amplification (LAMP) for detection of Theileria equi.

    Science.gov (United States)

    Xie, Junren; Liu, Guangyuan; Tian, Zhancheng; Luo, Jin

    2013-09-01

    Several approaches have been developed for diagnosis of Theileria equi infection in horses and donkeys but all of them have limitations in practice. Due to numerous strengths including easy operation, cheapness and high sensitivity and specificity, LAMP has been already extensively used for surveillance of a number of diseases. We here set up a LAMP assay based on 18S rRNA gene for T. equi diagnosis. The approach was specific enough to differentiate T. equi from other evolutionary-related protozoa. Moreover, it was sensitive enough that LAMP was capable of detecting as much low as 10 copy target gene and 1 pg/μl blood genomic DNA. It was further demonstrated that LAMP was much more sensitive than canonical blood smear and comparable to PCR using test and field samples. The present results support an idea that LAMP developed in this study is reliable, reproducible and highly sensitive and specific, being a potential to be globally used for surveillance of T. equi infection in the field.

  6. Detection of mutation by allele-specific loop-mediated isothermal amplification (AS-LAMP).

    Science.gov (United States)

    Aonuma, Hiroka; Badolo, Athanase; Okado, Kiyoshi; Kanuka, Hirotaka

    2013-01-01

    For effective control of pathogen-transmitting mosquitoes, precise surveillance data of mosquito distribution are essential. Recently, an increase of insecticide resistance due to the kdr mutation in Anopheles gambiae, a mosquito that transmits the malaria parasite, has been reported. With the aim of developing a simple and effective method for surveying resistant mosquitoes, LAMP was applied to the allele-specific detection of the kdr gene in An. gambiae. Allele-specific LAMP (AS-LAMP) method successfully distinguished the kdr homozygote from the heterozygote and the wild type. The robustness of AS-LAMP suggests its usefulness for routine identification of insects, not only mosquitoes but also other vectors and agricultural pests. Here we describe the method of AS-LAMP to detect mutation in Anopheles mosquitoes. PMID:24026691

  7. Wilson Loop-Loop Correlators in AdS/QCD

    OpenAIRE

    Nian, J.; Pirner, H.J.

    2009-01-01

    We calculate the expectation value of one circular Wilson loop and the correlator of two concentric circular Wilson loops in AdS/QCD using the modified AdS_5-metric given in Ref.[1]. The confinement properties of this metric in AdS/QCD are analyzed and compared with QCD and Nambu-Goto theory in four dimensions.

  8. Dynamic PID loop control

    Energy Technology Data Exchange (ETDEWEB)

    Pei, L.; Klebaner, A.; Theilacker, J.; Soyars, W.; Martinez, A.; Bossert, R.; DeGraff, B.; Darve, C.; /Fermilab

    2011-06-01

    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.

  9. Inductance loop and partial

    CERN Document Server

    Paul, Clayton R

    2010-01-01

    "Inductance is an unprecedented text, thoroughly discussing "loop" inductance as well as the increasingly important "partial" inductance. These concepts and their proper calculation are crucial in designing modern high-speed digital systems. World-renowned leader in electromagnetics Clayton Paul provides the knowledge and tools necessary to understand and calculate inductance." "With the present and increasing emphasis on high-speed digital systems and high-frequency analog systems, it is imperative that system designers develop an intimate understanding of the concepts and methods in this book. Inductance is a much-needed textbook designed for senior and graduate-level engineering students, as well as a hands-on guide for working engineers and professionals engaged in the design of high-speed digital and high-frequency analog systems."--Jacket.

  10. 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.

  11. The Loop Algorithm

    Science.gov (United States)

    Evertz, Hans Gerd

    1998-03-01

    Exciting new investigations have recently become possible for strongly correlated systems of spins, bosons, and fermions, through Quantum Monte Carlo simulations with the Loop Algorithm (H.G. Evertz, G. Lana, and M. Marcu, Phys. Rev. Lett. 70, 875 (1993).) (For a recent review see: H.G. Evertz, xxx.lanl.gov/abs/cond-mat/9707221>cond- mat/9707221.) and its generalizations. A review of this new method, its generalizations and its applications is given, including some new results. The Loop Algorithm is based on a formulation of physical models in an extended ensemble of worldlines and graphs, and is related to Swendsen-Wang cluster algorithms. It performs nonlocal changes of worldline configurations, determined by local stochastic decisions. It overcomes many of the difficulties of traditional worldline simulations. Computer time requirements are reduced by orders of magnitude, through a corresponding reduction in autocorrelations. The grand-canonical ensemble (e.g. varying winding numbers) is naturally simulated. The continuous time limit can be taken directly. Improved Estimators exist which further reduce the errors of measured quantities. The algorithm applies unchanged in any dimension and for varying bond-strengths. It becomes less efficient in the presence of strong site disorder or strong magnetic fields. It applies directly to locally XYZ-like spin, fermion, and hard-core boson models. It has been extended to the Hubbard and the tJ model and generalized to higher spin representations. There have already been several large scale applications, especially for Heisenberg-like models, including a high statistics continuous time calculation of quantum critical exponents on a regularly depleted two-dimensional lattice of up to 20000 spatial sites at temperatures down to T=0.01 J.

  12. Continuous phase amplification with a Sagnac interferometer

    CERN Document Server

    Starling, David J; Williams, Nathan S; Jordan, Andrew N; Howell, John C

    2009-01-01

    We describe a weak value inspired phase amplification technique in a Sagnac interferometer. We monitor the relative phase between two paths of a slightly misaligned interferometer by measuring the average position of a split-Gaussian mode in the dark port. Although we monitor only the dark port, we show that the signal varies linearly with phase and that we can obtain similar sensitivity to balanced homodyne detection. We derive the source of the amplification both with classical wave optics and as an inverse weak value.

  13. Parametric Amplification For Detecting Weak Optical Signals

    Science.gov (United States)

    Hemmati, Hamid; Chen, Chien; Chakravarthi, Prakash

    1996-01-01

    Optical-communication receivers of proposed type implement high-sensitivity scheme of optical parametric amplification followed by direct detection for reception of extremely weak signals. Incorporates both optical parametric amplification and direct detection into optimized design enhancing effective signal-to-noise ratios during reception in photon-starved (photon-counting) regime. Eliminates need for complexity of heterodyne detection scheme and partly overcomes limitations imposed on older direct-detection schemes by noise generated in receivers and by limits on quantum efficiencies of photodetectors.

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

    Science.gov (United States)

    Cui, Wanling; Wang, Lei; Jiang, Wei

    2016-03-15

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

  15. RCD+: Fast loop modeling server.

    Science.gov (United States)

    López-Blanco, José Ramón; Canosa-Valls, Alejandro Jesús; Li, Yaohang; Chacón, Pablo

    2016-07-01

    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. PMID:27151199

  16. Loop groups and noncommutative geometry

    CERN Document Server

    Carpi, Sebastiano

    2015-01-01

    We describe the representation theory of loop groups in terms of K-theory and noncommutative geometry. This is done by constructing suitable spectral triples associated with the level l projective unitary positive-energy representations of any given loop group LG. The construction is based on certain supersymmetric conformal field theory models associated with LG.

  17. Wilson Loop Diagrams and Positroids

    Science.gov (United States)

    Agarwala, Susama; Marin-Amat, Eloi

    2016-07-01

    In this paper, we study a new application of the positive Grassmannian to Wilson loop diagrams (or MHV diagrams) for scattering amplitudes in N= 4 Super Yang-Mill theory (N = 4 SYM). There has been much interest in studying this theory via the positive Grassmannians using BCFW recursion. This is the first attempt to study MHV diagrams for planar Wilson loop calculations (or planar amplitudes) in terms of positive Grassmannians. We codify Wilson loop diagrams completely in terms of matroids. This allows us to apply the combinatorial tools in matroid theory used to identify positroids (non-negative Grassmannians) to Wilson loop diagrams. In doing so, we find that certain non-planar Wilson loop diagrams define positive Grassmannians. While non-planar diagrams do not have physical meaning, this finding suggests that they may have value as an algebraic tool, and deserve further investigation.

  18. Loop Heat Pipe Startup Behaviors

    Science.gov (United States)

    Ku, Jentung

    2014-01-01

    A loop heat pipe must start successfully before it can commence its service. The start-up transient represents one of the most complex phenomena in the loop heat pipe operation. This paper discusses various aspects of loop heat pipe start-up behaviors. Topics include the four start-up scenarios, the initial fluid distribution between the evaporator and reservoir that determines the start-up scenario, factors that affect the fluid distribution between the evaporator and reservoir, difficulties encountered during the low power start-up, and methods to enhance the start-up success. Also addressed are the thermodynamic constraint between the evaporator and reservoir in the loop heat pipe operation, the superheat requirement for nucleate boiling, pressure spike and pressure surge during the start-up transient, and repeated cycles of loop start-up andshutdown under certain conditions.

  19. Higher dimensional loop quantum cosmology

    Science.gov (United States)

    Zhang, Xiangdong

    2016-07-01

    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.

  20. Desert Amplification in a Warming Cli