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Sample records for real-time reverse-transcription loop-mediated

  1. 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 use...

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    D Wang

    2014-01-01

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

  4. 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...... vesicular diseases and from that of genetically related picornaviruses. Diagnostic sensitivity was validated by the amplification of reference FMDV strains and archival material from field cases of FMD. In comparison with the performance of the established diagnostic TaqMan (R) assay, RT-LAMP appears...

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

    Directory of Open Access Journals (Sweden)

    Joojin Jeong

    2015-09-01

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

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

    OpenAIRE

    D Wang; X Wang; Y Geng; C An

    2014-01-01

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

  7. 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

  8. Rapid detection of Prunus necrotic ringspot virus using magnetic nanoparticle-assisted reverse transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Zong, Xiaojuan; Wang, Wenwen; Wei, Hairong; Wang, Jiawei; Chen, Xin; Xu, Li; Zhu, Dongzi; Tan, Yue; Liu, Qingzhong

    2014-11-01

    Prunus necrotic ringspot virus (PNRSV) has seriously reduced the yield of Prunus species worldwide. In this study, a highly efficient and specific two-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect PNRSV. Total RNA was extracted from sweet cherry leaf samples using a commercial kit based on a magnetic nanoparticle technique. Transcripts were used as the templates for the assay. The results of this assay can be detected using agarose gel electrophoresis or by assessing in-tube fluorescence after adding SYBR Green I. The assay is highly specific for PNRSV, and it is more sensitive than reverse-transcription polymerase chain reaction (RT-PCR). Restriction enzyme digestion verified further the reliability of this RT-LAMP assay. To our knowledge, this is the first report of the application of RT-LAMP to PNRSV detection in Prunus species. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Hasiów-Jaroszewska, Beata; Budzyńska, Daria; Borodynko, Natasza; Pospieszny, Henryk

    2015-12-01

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

  10. Application of reverse transcription-PCR and real-time PCR in nanotoxicity research.

    Science.gov (United States)

    Mo, Yiqun; Wan, Rong; Zhang, Qunwei

    2012-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique to determine the expression level of target genes and is widely used in biomedical science research including nanotoxicology studies for semiquantitative analysis. Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT-PCR. Although a number of kits and reagents for RT-PCR and real-time PCR are commercially available, the basic principles are the same. Here, we describe the procedures for total RNA isolation by using TRI Reagent, for reverse transcription (RT) by M-MLV reverse transcriptase, and for PCR by GoTaq(®) DNA Polymerase. And real-time PCR will be performed on an iQ5 multicolor real-time PCR detection system by using iQ™ SYBR Green Supermix.

  11. Development of mRNA-based body fluid identification using reverse transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Satoh, Tetsuya; Kouroki, Seiya; Ogawa, Keita; Tanaka, Yorika; Matsumura, Kazutoshi; Iwase, Susumu

    2018-04-25

    Identifying body fluids from forensic samples can provide valuable evidence for criminal investigations. Messenger RNA (mRNA)-based body fluid identification was recently developed, and highly sensitive parallel identification using reverse transcription polymerase chain reaction (RT-PCR) has been described. In this study, we developed reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a simple, rapid assay for identifying three common forensic body fluids, namely blood, semen, and saliva, and evaluated its specificity and sensitivity. Hemoglobin beta (HBB), transglutaminase 4 (TGM4), and statherin (STATH) were selected as marker genes for blood, semen, and saliva, respectively. RT-LAMP could be performed in a single step including both reverse transcription and DNA amplification under an isothermal condition within 60 min, and detection could be conveniently performed via visual fluorescence. Marker-specific amplification was performed in each assay, and no cross-reaction was observed among five representative forensically relevant body fluids. The detection limits of the assays were 0.3 nL, 30 nL, and 0.3 μL for blood, semen, and saliva, respectively, and their sensitivities were comparable with those of RT-PCR. Furthermore, RT-LAMP assays were applicable to forensic casework samples. It is considered that RT-LAMP is useful for body fluid identification.

  12. 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.

  13. Visual detection of the human metapneumovirus using reverse transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye

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    Wang Xiang

    2012-07-01

    Full Text Available Abstract Background Human metapneumovirus (hMPV is a major cause of acute respiratory infections ranging from wheezing to bronchiolitis and pneumonia in children worldwide. The objective of this study is to develop a visual reverse transcription loop-mediated isothermal amplification (RT-LAMP assay for the detection of hMPV and applied to the clinical samples. Results In this study, visual RT-LAMP assay for hMPV was performed in one step with the addition of hydroxynaphthol blue (HNB, and were used to detect respiratory samples. Six primers, including two outer primers (F3 and B3, two inner primers (FIP, BIP and two loop primers (LF and LB, were designed for hMPV N gene by the online software. Moreover, the RT-LAMP assay showed good specificity and no cross-reactivity was observed with human rhinovirus (HRV, human respiratory syncytial Virus (RSV, or influenza virus A/PR/8/34 (H1N1. The detection limit of the RT-LAMP assay was approximately ten viral RNA copies, lower than that of traditional reverse transcriptase polymerase chain reaction (RT-PCR 100 RNA copies. In the 176 nasopharyngeal samples, 23 (13.1% were conformed as hMPV positive by RT-LAMP, but 18 (10.2% positive by RT-PCR. Conclusion Compared with conventional RT-PCR, the visual hMPV RT-LAMP assay performed well in the aspect of detect time, sensitivity, specificity and visibility. It is anticipated that the RT-LAMP will be used for clinical tests in hospital or field testing during outbreaks and in emergency.

  14. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2014-06-01

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

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

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    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.

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

    Science.gov (United States)

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

    2015-06-01

    Although widespread vaccination against canine distemper virus (CDV) has been conducted for many decades, several canine distemper outbreaks in vaccinated animals have been reported frequently. In order to detect and differentiate the wild-type and vaccine strains of the CDV from the vaccinated animals, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed. A set of four primers-two internal and two external-were designed to target the H gene for the specific detection of wild-type CDV variants. The CDV-H RT-LAMP assay rapidly amplified the target gene, within 60 min, using a water bath held at a constant temperature of 65°C. The assay was 100-fold more sensitive than conventional RT-PCR, with a detection limit of 10(-1)TCID50ml(-1). The system showed a preference for wild-type CDV, and exhibited less sensitivity to canine parvovirus, canine adenovirus type 1 and type 2, canine coronavirus, and canine parainfluenza virus. The assay was validated using 102 clinical samples obtained from vaccinated dog farms, and the results were comparable to a multiplex nested RT-PCR assay. The specific CDV-H RT-LAMP assay provides a simple, rapid, and sensitive tool for the detection of canines infected with wild-type CDV from canines vaccinated with attenuated vaccine. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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    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.

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

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    Hongmei Bao

    2014-01-01

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

  1. Development of a reverse transcription loop-mediated isothermal amplification method for the rapid detection of avian influenza virus subtype H7.

    Science.gov (United States)

    Bao, Hongmei; Wang, Xiurong; Zhao, Yuhui; Sun, Xiaodong; Li, Yanbing; Xiong, Yongzhong; Chen, Hualan

    2012-01-01

    A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7 avian influenza virus (H7 AIV) isotype was developed. The minimum detection limit of the RT-LAMP assay was 0.1-0.01 PFU per reaction for H7 AIV RNA, making this assay 100-fold more sensitive than the conventional RT-PCR method. This RT-LAMP assay also has the capacity to detect both high- and low-pathogenic H7 AIV strains. Using a pool of RNAs extracted from influenza viruses corresponding to all 15 HA subtypes (in addition to other avian pathogenic viruses), the RT-LAMP system was confirmed to amplify only H7 AIV RNA. Furthermore, specific pathogen free (SPF) chickens were infected artificially with H7 AIV, throat and cloacal swabs were collected, and viral shedding was examined using viral isolation, RT-PCR and RT-LAMP. Shedding was detected following viral isolation and RT-LAMP one day after infection, whereas viral detection using RT-PCR was effective only on day 3 post-infection. These results indicate that the RT-LAMP method could facilitate epidemiological surveillance and the rapid diagnosis of the avian influenza subtype H7. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Detection of Cucurbit chlorotic yellows virus from Bemisia tabaci captured on sticky traps using reverse transcription loop-mediated isothermal amplification (RT-LAMP) and simple template preparation.

    Science.gov (United States)

    Okuda, Mitsuru; Okuda, Shiori; Iwai, Hisashi

    2015-09-01

    Cucurbit chlorotic yellows virus (CCYV) of the genus Crinivirus within the family Closteroviridae is an emerging infectious agent of cucurbits leading to severe disease and significant economic losses. Effective detection and identification methods for this virus are urgently required. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect CCYV from its vector Bemisia tabaci. LAMP primer sets to detect CCYV were evaluated for their sensitivity and specificity, and a primer set designed from the HSP70h gene with corresponding loop primers were selected. The RT-LAMP assay was applied to detect CCYV from viruliferous B. tabaci trapped on sticky traps. A simple extraction procedure using RNAsecure™ was developed for template preparation. CCYV was detected in all of the B. tabaci 0, 1, 7 and 14 days after they were trapped. Although the rise of turbidity was delayed in reactions using RNA from B. tabaci trapped for 7 and 14 days compared with those from 0 and 1 day, the DNA amplification was sufficient to detect CCYV in all of the samples. These findings therefore present a simple template preparation method and an effective RT-LAMP assay, which can be easily and rapidly performed to monitor CCYV-viruliferous B. tabaci in the field. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2011-08-01

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

  4. A sensitive, reproducible, and economic real-time reverse transcription PCR detecting avian metapneumovirus subtypes A and B.

    Science.gov (United States)

    Franzo, G; Drigo, M; Lupini, C; Catelli, E; Laconi, A; Listorti, V; Bonci, M; Naylor, C J; Martini, M; Cecchinato, M

    2014-06-01

    Use of real-time PCR is increasing in the diagnosis of infectious disease due to its sensitivity, specificity, and speed of detection. These characteristics make it particularly suited for the diagnosis of viral infections, like avian metapneumovirus (AMPV), for which effective control benefits from continuously updated knowledge of the epidemiological situation. Other real-time reverse transcription (RT)-PCRs have been published based on highly specific fluorescent dye-labeled probes, but they have high initial cost, complex validation, and a marked susceptibility to the genetic variability of their target sequence. With this in mind, we developed and validated a SYBR Green I-based quantitative RT-PCR for the detection of the two most prevalent AMPV subtypes (i.e., subtypes A and B). The assay demonstrated an analytical sensitivity comparable with that of a previously published real-time RT-PCR and the ability to detect RNA equivalent to approximately 0.5 infectious doses for both A and B subtypes. The high efficiency and linearity between viral titer and crossing point displayed for both subtypes make it suited for viral quantification. Optimization of reaction conditions and the implementation of melting curve analysis guaranteed the high specificity of the assay. The stable melting temperature difference between the two subtypes indicated the possibility of subtyping through melting temperature analysis. These characteristics make our assay a sensitive, specific, and rapid tool, enabling contemporaneous detection, quantification, and discrimination of AMPV subtype A and B.

  5. Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification.

    Science.gov (United States)

    Wang, Jianchang; Wang, Jinfeng; Li, Ruiwen; Liu, Libing; Yuan, Wanzhe

    2017-08-15

    Canine distemper, caused by Canine distemper virus (CDV), is a highly contagious and fatal systemic disease in free-living and captive carnivores worldwide. Recombinase polymerase amplification (RPA), as an isothermal gene amplification technique, has been explored for the molecular detection of diverse pathogens. A real-time reverse transcription RPA (RT-RPA) assay for the detection of canine distemper virus (CDV) using primers and exo probe targeting the CDV nucleocapsid protein gene was developed. A series of other viruses were tested by the RT-RPA.Thirty-two field samples were further tested by RT-RPA, and the resuts were compared with those obtained by the real-time RT-PCR. The RT-RPA assay was performed successfully at 40 °C, and the results were obtained within 3 min-12 min. The assay could detect CDV, but did not show cross-detection of canine parvovirus-2 (CPV-2), canine coronavirus (CCoV), canine parainfluenza virus (CPIV), pseudorabies virus (PRV) or Newcastle disease virus (NDV), demonstrating high specificity. The analytical sensitivity of RT-RPA was 31.8 copies in vitro transcribed CDV RNA, which is 10 times lower than the real-time RT-PCR. The assay performance was validated by testing 32 field samples and compared to real-time RT-PCR. The results indicated an excellent correlation between RT-RPA and a reference real-time RT-PCR method. Both assays provided the same results, and R 2 value of the positive results was 0.947. The results demonstrated that the RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of CDV both in the laboratory and point-of-care facility, especially in the resource-limited settings.

  6. 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.

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    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.

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

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    Sanchita Bhadra

    Full Text Available The Middle East respiratory syndrome coronavirus (MERS-CoV, an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU (5 to 50 PFU/ml of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.

  8. Reference genes for gene expression analysis by real-time reverse transcription polymerase chain reaction of renal cell carcinoma.

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    Bjerregaard, Henriette; Pedersen, Shona; Kristensen, Søren Risom; Marcussen, Niels

    2011-12-01

    Differentiation between malignant renal cell carcinoma and benign oncocytoma is of great importance to choose the optimal treatment. Accurate preoperative diagnosis of renal tumor is therefore crucial; however, existing imaging techniques and histologic examinations are incapable of providing an optimal differentiation profile. Analysis of gene expression of molecular markers is a new possibility but relies on appropriate standardization to compare different samples. The aim of this study was to identify stably expressed reference genes suitable for the normalization of results extracted from gene expression analysis of renal tumors. Expression levels of 8 potential reference genes (ATP5J, HMBS, HPRT1, PPIA, TBP, 18S, GAPDH, and POLR2A) were examined by real-time reverse transcription polymerase chain reaction in tumor and normal tissue from removed kidneys from 13 patients with renal cell carcinoma and 5 patients with oncocytoma. The expression levels of genes were compared by gene stability value M, average gene stability M, pairwise variation V, and coefficient of variation CV. More candidates were not suitable for the purpose, but a combination of HMBS, PPIA, ATP5J, and TBP was found to be the best combination with an average gene stability value M of 0.9 and a CV of 0.4 in the 18 tumors and normal tissues. A combination of 4 genes, HMBS, PPIA, ATP5J, and TBP, is a possible reference in renal tumor gene expression analysis by reverse transcription polymerase chain reaction. A combination of four genes, HMBS, PPIA, ATP5J and TBP, being stably expressed in tissues from RCC is possible reference genes for gene expression analysis.

  9. 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

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    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.

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

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    Gregory L. Damhorst

    2015-09-01

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

  11. A real-time loop-mediated isothermal amplification assay for rapid detection of Shigella species.

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    Liew, P S; Teh, C S J; Lau, Y L; Thong, K L

    2014-12-01

    Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 10(5) CFU/ml, while PCR displayed a limit of 5.9 x 10(7) CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 10(4) CFU/g, whereas PCR was 3.6 x 10(5) CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.

  12. Evaluation of a duplex reverse-transcription real-time PCR assay for the detection of encephalomyocarditis virus.

    Science.gov (United States)

    Qin, Shaomin; Underwood, Darren; Driver, Luke; Kistler, Carol; Diallo, Ibrahim; Kirkland, Peter D

    2018-06-01

    We evaluated a fluorogenic probe-based assay for the detection of encephalomyocarditis virus (EMCV) by comparing a set of published primers and probe to a new set of primers and probe. The published reagents failed to amplify a range of Australian isolates and an Italian reference strain of EMCV. In contrast, an assay based on 2 new sets of primers and probes that were run in a duplex reverse-transcription real-time PCR (RT-rtPCR) worked well, with high amplification efficiency. The analytical sensitivity was ~100-fold higher than virus isolation in cell culture. The intra-assay variation was 0.21-4.90%. No cross-reactivity was observed with a range of other porcine viruses. One hundred and twenty-two clinical specimens were tested simultaneously by RT-rtPCR and virus isolation in cell culture; 72 specimens gave positive results by RT-rtPCR, and 63 of these were also positive by virus isolation. Of 245 archived cell culture isolates of EMCV that were tested in the RT-rtPCR, 242 samples were positive. The new duplex RT-rtPCR assay is a reliable tool for the detection of EMCV in clinical specimens and for use in epidemiologic investigations.

  13. Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription.

    Science.gov (United States)

    Johnston, Stephen; Gallaher, Zachary; Czaja, Krzysztof

    2012-05-15

    Quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2(-∆∆Ct) normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxin selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference β-III tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.

  14. Outbreak of hepatitis E virus infection in Darfur, Sudan: effectiveness of real-time reverse transcription-PCR analysis of dried blood spots.

    Science.gov (United States)

    Mérens, Audrey; Guérin, Philippe Jean; Guthmann, Jean-Paul; Nicand, Elisabeth

    2009-06-01

    Biological samples collected in refugee camps during an outbreak of hepatitis E were used to compare the accuracy of hepatitis E virus RNA amplification by real-time reverse transcription-PCR (RT-PCR) for sera and dried blood spots (concordance of 90.6%). Biological profiles (RT-PCR and serology) of asymptomatic individuals were also analyzed.

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

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    Ge Y

    2017-04-01

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

  16. 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.

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    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.

  17. Real-time reverse transcription-polymerase chain reaction assays for identification of wild poliovirus 1 & 3.

    Science.gov (United States)

    Sharma, Deepa K; Nalavade, Uma P; Deshpande, Jagadish M

    2015-10-01

    The poliovirus serotype identification and intratypic differentiation by real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay is suitable for serotype mixtures but not for intratypic mixtures of wild and vaccine poliovirus strains. This study was undertaken to develop wild poliovirus 1 and 3 (WPV1 and WPV3) specific rRT-PCR assays for use. Specific primers and probes for rRT-PCR were designed based on VP1 sequences of WPV1 and WPV3 isolated in India since 2000. The specificity of the rRT-PCR assays was evaluated using WPV1 and WPV3 of different genetic lineages, non-polio enteroviruses (NPEVs) and mixtures of wild/wild and wild/Sabin vaccine strains. The sensitivity of the assays was determined by testing serial 10-fold dilutions of wild poliovirus 1 and 3 stock suspensions of known titre. No cross-reactivity with Sabin strains, intertypic wild poliovirus isolates or 27 types of NPEVs across all the four Enterovirus species was found for both the wild poliovirus 1 and 3 rRT-PCR assays. All WPV1 and WPV3 strains isolated since 2000 were successfully amplified. The rRT-PCR assays detected 10 4.40 CCID 50 /ml of WPV1 and 10 4.00 CCID 50 /ml of WPV3, respectively either as single isolate or mixture with Sabin vaccine strains or intertypic wild poliovirus. rRT-PCR assays for WPV1 and WPV3 have been validated to detect all the genetic variations of the WPV1 and WPV3 isolated in India for the last decade. When used in combination with the current rRT-PCR assay testing was complete for confirmation of the presence of wild poliovirus in intratypic mixtures.

  18. Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection.

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    Laurent Dacheux

    2016-07-01

    Full Text Available The definitive diagnosis of lyssavirus infection (including rabies in animals and humans is based on laboratory confirmation. The reference techniques for post-mortem rabies diagnosis are still based on direct immunofluorescence and virus isolation, but molecular techniques, such as polymerase chain reaction (PCR based methods, are increasingly being used and now constitute the principal tools for diagnosing rabies in humans and for epidemiological analyses. However, it remains a key challenge to obtain relevant specificity and sensitivity with these techniques while ensuring that the genetic diversity of lyssaviruses does not compromise detection. We developed a dual combined real-time reverse transcription polymerase chain reaction (combo RT-qPCR method for pan-lyssavirus detection. This method is based on two complementary technologies: a probe-based (TaqMan RT-qPCR for detecting the RABV species (pan-RABV RT-qPCR and a second reaction using an intercalating dye (SYBR Green to detect other lyssavirus species (pan-lyssa RT-qPCR. The performance parameters of this combined assay were evaluated with a large panel of primary animal samples covering almost all the genetic variability encountered at the viral species level, and they extended to almost all lyssavirus species characterized to date. This method was also evaluated for the diagnosis of human rabies on 211 biological samples (positive n = 76 and negative n = 135 including saliva, skin and brain biopsies. It detected all 41 human cases of rabies tested and confirmed the sensitivity and the interest of skin biopsy (91.5% and saliva (54% samples for intra-vitam diagnosis of human rabies. Finally, this method was successfully implemented in two rabies reference laboratories in enzootic countries (Cambodia and Morocco. This combined RT-qPCR method constitutes a relevant, useful, validated tool for the diagnosis of rabies in both humans and animals, and represents a promising tool for

  19. Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection.

    Science.gov (United States)

    Dacheux, Laurent; Larrous, Florence; Lavenir, Rachel; Lepelletier, Anthony; Faouzi, Abdellah; Troupin, Cécile; Nourlil, Jalal; Buchy, Philippe; Bourhy, Herve

    2016-07-01

    The definitive diagnosis of lyssavirus infection (including rabies) in animals and humans is based on laboratory confirmation. The reference techniques for post-mortem rabies diagnosis are still based on direct immunofluorescence and virus isolation, but molecular techniques, such as polymerase chain reaction (PCR) based methods, are increasingly being used and now constitute the principal tools for diagnosing rabies in humans and for epidemiological analyses. However, it remains a key challenge to obtain relevant specificity and sensitivity with these techniques while ensuring that the genetic diversity of lyssaviruses does not compromise detection. We developed a dual combined real-time reverse transcription polymerase chain reaction (combo RT-qPCR) method for pan-lyssavirus detection. This method is based on two complementary technologies: a probe-based (TaqMan) RT-qPCR for detecting the RABV species (pan-RABV RT-qPCR) and a second reaction using an intercalating dye (SYBR Green) to detect other lyssavirus species (pan-lyssa RT-qPCR). The performance parameters of this combined assay were evaluated with a large panel of primary animal samples covering almost all the genetic variability encountered at the viral species level, and they extended to almost all lyssavirus species characterized to date. This method was also evaluated for the diagnosis of human rabies on 211 biological samples (positive n = 76 and negative n = 135) including saliva, skin and brain biopsies. It detected all 41 human cases of rabies tested and confirmed the sensitivity and the interest of skin biopsy (91.5%) and saliva (54%) samples for intra-vitam diagnosis of human rabies. Finally, this method was successfully implemented in two rabies reference laboratories in enzootic countries (Cambodia and Morocco). This combined RT-qPCR method constitutes a relevant, useful, validated tool for the diagnosis of rabies in both humans and animals, and represents a promising tool for lyssavirus

  20. Screening suitable reference genes for normalization in reverse transcription quantitative real-time PCR analysis in melon.

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    Qiusheng Kong

    Full Text Available Melon (Cucumis melo. L is not only an economically important cucurbitaceous crop but also an attractive model for studying many biological characteristics. Screening appropriate reference genes is essential to reverse transcription quantitative real-time PCR (RT-qPCR, which is key to many studies involving gene expression analysis. In this study, 14 candidate reference genes were selected, and the variations in their expression in roots and leaves of plants subjected to biotic stress, abiotic stress, and plant growth regulator treatment were assessed by RT-qPCR. The stability of the expression of the selected genes was determined and ranked using geNorm and NormFinder. geNorm identified the two most stable genes for each set of conditions: CmADP and CmUBIep across all samples, CmUBIep and CmRPL in roots, CmRAN and CmACT in leaves, CmADP and CmRPL under abiotic stress conditions, CmTUA and CmACT under biotic stress conditions, and CmRAN and CmACT under plant growth regulator treatments. NormFinder determined CmRPL to be the best reference gene in roots and under biotic stress conditions and CmADP under the other experimental conditions. CmUBC2 and CmPP2A were not found to be suitable under many experimental conditions. The catalase family genes CmCAT1, CmCAT2, and CmCAT3 were identified in melon genome and used as target genes to validate the reliability of identified reference genes. The catalase family genes showed the most upregulation 3 days after inoculation with Fusarium wilt in roots, after which they were downregulated. Their levels of expression were significantly overestimated when the unsuitable reference gene was used for normalization. These results not only provide guidelines for the selection of reference genes for gene expression analyses in melons but may also provide valuable information for studying the functions of catalase family genes in stress responses.

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

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    Kiddle Guy

    2012-04-01

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

  2. Simplified Real-Time Multiplex Detection of Loop-Mediated Isothermal Amplification Using Novel Mediator Displacement Probes with Universal Reporters.

    Science.gov (United States)

    Becherer, Lisa; Bakheit, Mohammed; Frischmann, Sieghard; Stinco, Silvina; Borst, Nadine; Zengerle, Roland; von Stetten, Felix

    2018-04-03

    A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3-4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, n = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; n = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels.

  3. Enteroviruses in blood of patients with type 1 diabetes detected by integrated cell culture and reverse transcription quantitative real-time PCR.

    Science.gov (United States)

    Alidjinou, Enagnon Kazali; Sane, Famara; Lefevre, Christine; Baras, Agathe; Moumna, Ilham; Engelmann, Ilka; Vantyghem, Marie-Christine; Hober, Didier

    2017-11-01

    Enteroviruses (EV) have been associated with type 1 diabetes (T1D), but EV RNA detection has been reported in only a small proportion of T1D patients. We studied whether integrated cell culture and reverse transcription real-time PCR could improve EV detection in blood samples from patients with T1D. Blood was collected from 13 patients with T1D. The presence of EV RNA in blood was investigated by using real-time RT-PCR. In addition, plasma and white blood cells (WBC) were inoculated to BGM and Vero cell line cultures. Culture supernatants and cells collected on day 7 and day 14 were tested for EV RNA by real-time RT-PCR. Enterovirus identification was performed through sequencing of the VP4/VP2 region. Enterovirus RNA was detected in blood by using real-time RT-PCR in only one out of 13 patients. The detection of EV RNA in cultures inoculated with clinical samples (plasma and/or WBC) gave positive results in five other patients. The viral loads were low, ranging from 45 to 4420 copies/ng of total RNA. One isolate was successfully identified as coxsackievirus B1. Integrated cell culture and reverse transcription real-time PCR can improve the detection rate of EV in blood samples of patients with T1D and can be useful to investigate further the relationship between EV and the disease.

  4. Optimization of the elution buffer and concentration method for detecting hepatitis E virus in swine liver using a nested reverse transcription-polymerase chain reaction and real-time reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Son, Na Ry; Seo, Dong Joo; Lee, Min Hwa; Seo, Sheungwoo; Wang, Xiaoyu; Lee, Bog-Hieu; Lee, Jeong-Su; Joo, In-Sun; Hwang, In-Gyun; Choi, Changsun

    2014-09-01

    The aim of this study was to develop an optimal technique for detecting hepatitis E virus (HEV) in swine livers. Here, three elution buffers and two concentration methods were compared with respect to enhancing recovery of HEV from swine liver samples. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and nested RT-PCR were performed to detect HEV RNA. When phosphate-buffered saline (PBS, pH 7.4) was used to concentrate HEV in swine liver samples using ultrafiltration, real-time RT-PCR detected HEV in 6 of the 26 samples. When threonine buffer was used to concentrate HEV using polyethylene glycol (PEG) precipitation and ultrafiltration, real-time RT-PCR detected HEV in 1 and 3 of the 26 samples, respectively. When glycine buffer was used to concentrate HEV using ultrafiltration and PEG precipitation, real-time RT-PCR detected HEV in 1 and 3 samples of the 26 samples, respectively. When nested RT-PCR was used to detect HEV, all samples tested negative regardless of the type of elution buffer or concentration method used. Therefore, the combination of real-time RT-PCR and ultrafiltration with PBS buffer was the most sensitive and reliable method for detecting HEV in swine livers. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Porcine reproductive and respiratory syndrome virus: Interlaboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction detection methods

    DEFF Research Database (Denmark)

    Wernike, Kerstin; Bonilauri, Paolo; Dauber, Malte

    2012-01-01

    To compare the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays used for the diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV), a Europe-wide interlaboratory ring trial was conducted. A variety of PRRSV strains including North American...... (NA) and European (EU) genotype isolates were analyzed by the participants. Great differences regarding qualitative diagnostics as well as analytical sensitivity were observed between the individual RT-qPCR systems, especially when investigating strains from the EU genotype. None of the assays...

  6. Evaluation of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a screening method for the detection of influenza viruses in the fecal materials of water birds.

    Science.gov (United States)

    Yoshida, Hiromi; Sakoda, Yoshihiro; Endo, Mayumi; Motoshima, Masayuki; Yoshino, Fumi; Yamamoto, Naoki; Okamatsu, Masatoshi; Soejima, Takahiro; Senba, Syouhei; Kanda, Hidetoshi; Kida, Hiroshi

    2011-06-01

    Migratory water birds are a natural reservoir for influenza A viruses. Viruses replicate in the intestines of ducks and are shed with the fecal materials. Virus isolation from collected fecal materials, therefore, is an integral part of the surveillance of avian influenza in water birds. In the present study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) was assessed for its usefulness in detecting the RNA of influenza A viruses in fecal materials. It was found that, RT-LAMP specifically and sensitively detects the matrix gene of influenza A viruses. Influenza A viruses were isolated from the fecal materials in which viral RNA were detected by RT-LAMP in 35 min. The present findings indicate that RT-LAMP is useful as a high throughput screening method for field samples prior to virus isolation, allowing the processing of hundreds of samples per day.

  7. Development of field-based real-time reverse transcription-polymerase chain reaction assays for detection of Chikungunya and O'nyong-nyong viruses in mosquitoes.

    Science.gov (United States)

    Smith, Darci R; Lee, John S; Jahrling, Jordan; Kulesh, David A; Turell, Michael J; Groebner, Jennifer L; O'Guinn, Monica L

    2009-10-01

    Chikungunya (CHIK) and O'nyong-nyong (ONN) are important emerging arthropod-borne diseases. Molecular diagnosis of these two viruses in mosquitoes has not been evaluated, and the effects of extraneous mosquito tissue on assay performance have not been tested. Additionally, no real-time reverse transcription-polymerase chain reaction (RT-PCR) assay exists for detecting ONN virus (ONNV) RNA. We describe the development of sensitive and specific real-time RT-PCR assays for detecting CHIK and ONN viral RNA in mosquitoes, which have application for field use. In addition, we compared three methods for primer/probe design for assay development by evaluating their sensitivity and specificity. This comparison resulted in development of virus-specific assays that could detect less than one plaque-forming unit equivalent of each of the viruses in mosquitoes. The use of these assays will aid in arthropod-borne disease surveillance and in the control of the associated diseases.

  8. Variation in Bluetongue virus real-time reverse transcription polymerase chain reaction assay results in blood samples of sheep, cattle, and alpaca.

    Science.gov (United States)

    Brito, Barbara P; Gardner, Ian A; Hietala, Sharon K; Crossley, Beate M

    2011-07-01

    Bluetongue is a vector-borne viral disease that affects domestic and wild ruminants. The epidemiology of this disease has recently changed, with occurrence in new geographic areas. Various real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) assays are used to detect Bluetongue virus (BTV); however, the impact of biologic differences between New World camelids and domestic ruminant samples on PCR efficiency, for which the BTV real-time qRT-PCR was initially validated are unknown. New world camelids are known to have important biologic differences in whole blood composition, including hemoglobin concentration, which can alter PCR performance. In the present study, sheep, cattle, and alpaca blood were spiked with BTV serotypes 10, 11, 13, and 17 and analyzed in 10-fold dilutions by real-time qRT-PCR to determine if species affected nucleic acid recovery and assay performance. A separate experiment was performed using spiked alpaca blood subsequently diluted in 10-fold series in sheep blood to assess the influence of alpaca blood on performance efficiency of the BTV real-time qRT-PCR assay. Results showed that BTV-specific nucleic acid detection from alpaca blood was consistently 1-2 logs lower than from sheep and cattle blood, and results were similar for each of the 4 BTV serotypes analyzed.

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

    Directory of Open Access Journals (Sweden)

    Thaer YASEEN

    2015-12-01

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

  10. Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results.

    Directory of Open Access Journals (Sweden)

    Andreas Garcia-Bardon

    Full Text Available Real-time reverse transcription polymerase chain reaction (PCR is the gold standard for expression analysis. Designed to improve reproducibility and sensitivity, commercial kits are commonly used for the critical step of cDNA synthesis. The present study was designed to determine the impact of these kits. mRNA from mouse brains were pooled to create serial dilutions ranging from 0.0625 μg to 2 μg, which were transcribed into cDNA using four different commercial reverse-transcription kits. Next, we transcribed mRNA from brain tissue after acute brain injury and naïve mice into cDNA for qPCR. Depending on tested genes, some kits failed to show linear results in dilution series and revealed strong variations in cDNA yield. Absolute expression data in naïve and trauma settings varied substantially between these kits. Normalization with a housekeeping gene failed to reduce kit-dependent variations, whereas normalization eliminated differences when naïve samples from the same region were used. The study shows strong evidence that choice of commercial cDNA synthesis kit has a major impact on PCR results and, consequently, on comparability between studies. Additionally, it provides a solution to overcome this limitation by normalization with data from naïve samples. This simple step helps to compare mRNA expression data between different studies and groups.

  11. Laboratory evaluation of a quantitative real-time reverse transcription PCR assay for the detection and identification of the four subgroups of avian metapneumovirus.

    Science.gov (United States)

    Guionie, O; Toquin, D; Sellal, E; Bouley, S; Zwingelstein, F; Allée, C; Bougeard, S; Lemière, S; Eterradossi, N

    2007-02-01

    Avian metapneumovirus (AMPV) is an important pathogen causing respiratory diseases and egg drops in several avian species. Four AMPV subgroups have been identified. The laboratory diagnosis of AMPV infections relies on serological methods, on labour-intensive virus isolation procedures, and on recently developed subgroup specific reverse transcription PCR (RT-PCR) protocols. In the present study, both the specificity and sensitivity of a commercial real-time reverse transcription PCR (RRT-PCR) for the detection and identification of the four AMPV subgroups were evaluated. Fifteen non-AMPV avian viruses belonging to 7 genera and 32 AMPV belonging to the 4 subgroups were tested. No non-AMPV virus was detected, whereas all AMPV viruses were identified in agreement with their previous molecular and antigenic subgroup assignment. The sensitivity and quantitating ability of the RRT-PCR assay were determined using serial dilutions of RNA derived either from AMPV virus stocks or from runoff transcripts. In all cases, linear dose/responses were observed. The detection limits of the different subgroups ranged from 500 to 5000 RNA copies and from 0.03 to 3.16TCID50/ml. The results were reproducible under laboratory conditions, thus showing that quantitative RRT-PCR is a new and powerful tool for the rapid and sensitive detection, identification and quantitation of AMPVs.

  12. Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR "Double Check" Strategy

    DEFF Research Database (Denmark)

    Hoffmann, B.; Freuling, C. M.; Wakeley, P. R.

    2010-01-01

    To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus...... sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany......), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome...

  13. Rapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification (RT-LAMP.

    Directory of Open Access Journals (Sweden)

    Amanda E Calvert

    Full Text Available Zika virus (ZIKV has emerged as a major global public health concern in the last two years due to its link as a causative agent of human birth defects. Its rapid expansion into the Western Hemisphere as well as the ability to be transmitted from mother to fetus, through sexual transmission and possibly through blood transfusions has increased the need for a rapid and expansive public health response to this unprecedented epidemic. A non-invasive and rapid ZIKV diagnostic screening assay that can be performed in a clinical setting throughout pregnancy is vital for prenatal care of women living in areas of the world where exposure to the virus is possible. To meet this need we have developed a sensitive and specific reverse transcriptase loop-mediated isothermal amplification (RT-LAMP assay to detect ZIKV RNA in urine and serum with a simple visual detection. RT-LAMP results were shown to have a limit of detection 10-fold higher than qRT-PCR. As little as 1.2 RNA copies/μl was detected by RT-LAMP from a panel of 178 diagnostic specimens. The assay was shown to be highly specific for ZIKV RNA when tested with diagnostic specimens positive for dengue virus (DENV and chikungunya virus (CHIKV. The assay described here illustrates the potential for a fast, reliable, sensitive and specific assay for the detection of ZIKV from urine or serum that can be performed in a clinical or field setting with minimal equipment and technological expertise.

  14. Real-time reverse transcription polymerase chain reaction method for detection of Canine distemper virus modified live vaccine shedding for differentiation from infection with wild-type strains.

    Science.gov (United States)

    Wilkes, Rebecca P; Sanchez, Elena; Riley, Matthew C; Kennedy, Melissa A

    2014-01-01

    Canine distemper virus (CDV) remains a common cause of infectious disease in dogs, particularly in high-density housing situations such as shelters. Vaccination of all dogs against CDV is recommended at the time of admission to animal shelters and many use a modified live virus (MLV) vaccine. From a diagnostic standpoint for dogs with suspected CDV infection, this is problematic because highly sensitive diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) tests are able to detect MLV virus in clinical samples. Real-time PCR can be used to quantitate amount of virus shedding and can differentiate vaccine strains from wild-type strains when shedding is high. However, differentiation by quantitation is not possible in vaccinated animals during acute infection, when shedding is low and could be mistaken for low level vaccine virus shedding. While there are gel-based RT-PCR assays for differentiation of vaccine strains from field strains based on sequence differences, the sensitivity of these assays is unable to match that of the real-time RT-PCR assay currently used in the authors' laboratory. Therefore, a real-time RT-PCR assay was developed that detects CDV MLV vaccine strains and distinguishes them from wild-type strains based on nucleotide sequence differences, rather than the amount of viral RNA in the sample. The test is highly sensitive, with detection of as few as 5 virus genomic copies (corresponding to 10(-1) TCID(50)). Sequencing of the DNA real-time products also allows phylogenetic differentiation of the wild-type strains. This test will aid diagnosis during outbreaks of CDV in recently vaccinated animals.

  15. Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil

    Directory of Open Access Journals (Sweden)

    Marilia Farignoli Romeiro

    2016-06-01

    Full Text Available Abstract: INTRODUCTION: The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR method for the diagnosis of the Flavivirus genus. METHODS: We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. RESULTS: The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410 of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×107to 3.4×103copies per ml. CONCLUSIONS: The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies.

  16. 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.

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

    Science.gov (United States)

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

    2017-06-01

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

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

    Science.gov (United States)

    Nixon, Gavin J; Svenstrup, Helle F; Donald, Carol E; Carder, Caroline; Stephenson, Judith M; Morris-Jones, Stephen; Huggett, Jim F; Foy, Carole A

    2014-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Gavin J. Nixon

    2014-12-01

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

  20. 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

  1. Comparison of the Diagnostic Value Between Real-Time Reverse Transcription-Polymerase Chain Reaction Assay and Histopathologic Examination in Sentinel Lymph Nodes for Patients With Gastric Carcinoma.

    Science.gov (United States)

    Kwak, Yoonjin; Nam, Soo Kyung; Shin, Eun; Ahn, Sang-Hoon; Lee, Hee Eun; Park, Do Joong; Kim, Woo Ho; Kim, Hyung-Ho; Lee, Hye Seung

    2016-05-01

    Sentinel lymph node (SLN)-based diagnosis in gastric cancers has shown varied sensitivities and false-negative rates in several studies. Application of the reverse transcription-polymerase chain reaction (RT-PCR) in SLN diagnosis has recently been proposed. A total of 155 SLNs from 65 patients with cT1-2, N0 gastric cancer were examined. The histopathologic results were compared with results obtained by real-time RT-PCR for detecting molecular RNA (mRNA) of cytokeratin (CK)19, carcinoembryonic antigen (CEA), and CK20. The sensitivity and specificity of the multiple marker RT-PCR assay standardized against the results of the postoperative histological examination were 0.778 (95% confidence interval [CI], 0.577-0.914) and 0.781 (95% CI, 0.700-0.850), respectively. In comparison, the sensitivity and specificity of intraoperative diagnosis were 0.819 (95% CI, 0.619-0.937) and 1.000 (95% CI, 0.972-1.000), respectively. The positive predictive value of the multiple-marker RT-PCR assay was 0.355 (95% CI, 0.192-0.546) for predicting non-SLN metastasis, which was lower than that of intraoperative diagnosis (0.813, 95% CI, 0.544-0.960). The real-time RT-PCR assay could detect SLN metastasis in gastric cancer. However, the predictive value of the real-time RT-PCR assay was lower than that of precise histopathologic examination and did not outweigh that of our intraoperative SLN diagnosis. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Comparison of reverse-transcription real-time PCR and immunohistochemistry for the detection of canine distemper virus infection in raccoons in Ontario, Canada.

    Science.gov (United States)

    Nemeth, Nicole M; Oesterle, Paul T; Campbell, G Douglas; Ojkic, Davor; Jardine, Claire M

    2018-03-01

    Canine distemper virus (CDV) is a widespread morbillivirus that causes subclinical to fatal infections in domestic and wild carnivores. Raccoons ( Procyon lotor) are CDV reservoirs and suffer from associated disease. Aspects of pathogenesis may lead to difficulty in the interpretation of commonly used testing modalities, such as reverse-transcription real-time (RT-rt)PCR and immunohistochemistry (IHC). The reliance upon such tests is greater for wildlife, which are often submitted as carcasses with no clinical history. We compared CDV RT-rtPCR results to immunohistochemistry (the gold standard) in tissues from 74 raccoons. These tests had high kappa agreement (lymph node: 0.9335; lung: 0.8671) and a negative correlation between IHC score and threshold cycle (Ct) value for lymph node and lung (Spearman rank correlation coefficient [ r s ] = -0.8555 and -0.8179, respectively; p < 0.00001). An RT-rtPCR Ct value of 30 in lung and lymph node with sensitivity and specificity of 92.3 and 92.6% and 86.8 and 96.4%, respectively, was suitable for determining CDV involvement. Conjunctival swabs provide an alternative for distemper diagnosis, as there was a strong correlation between Ct values of conjunctival swabs and tissues ( r s = -0.8498, p < 0.00001, n = 46). This information will aid in more efficient and accurate diagnoses in individuals, small-scale outbreaks, and epidemiologic investigations in wildlife.

  3. Investigations on the frequency of norovirus contamination of ready-to-eat food items in Istanbul, Turkey, by using real-time reverse transcription PCR.

    Science.gov (United States)

    Yilmaz, Aysun; Bostan, Kamil; Altan, Eda; Muratoglu, Karlo; Turan, Nuri; Tan, Derya; Helps, Christopher; Yilmaz, Huseyin

    2011-05-01

    Investigation of norovirus (NoV) contamination of food items is important because many outbreaks occur after consumption of contaminated shellfish, vegetables, fruits, and water. The frequency of NoV contamination in food items has not previously been investigated in Turkey. The aim of this study was to investigate the frequency of human NoV genogroups (G) I and II in ready-to-eat tomatoes, parsley, green onion, lettuce, mixed salads, and cracked wheat balls. RNA was extracted with the RNeasy Mini Kit, and a real-time reverse transcription (RT) PCR assay was performed using primers specific for NoV GI and GII. Among the 525 samples analyzed, NoV GII was detected in 1 green onion sample and 1 tomato sample by both SYBR Green and TaqMan real-time RT-PCR assays; no GI virus was detected. The Enterobactericaeae and Escherichia coli levels in the NoV-positive green onion were 6.56 and 1.28 log CFU/g, and those in the tomato were 5.55 and 1.30 log CFU/g, respectively. No significant difference in the bacterial levels was found between the NoV-positive and NoV-negative samples. This study is the first in which NoV GII was found in ready-to-eat food collected from Istanbul, Turkey; thus, these foods may be considered a risk to human health. Epidemiological studies and measures to prevent NoV infection should be considered.

  4. Detection and differentiation of field and vaccine strains of canine distemper virus using reverse transcription followed by nested real time PCR (RT-nqPCR) and RFLP analysis.

    Science.gov (United States)

    Fischer, Cristine Dossin Bastos; Ikuta, Nilo; Canal, Cláudio Wageck; Makiejczuk, Aline; Allgayer, Mariangela da Costa; Cardoso, Cristine Hoffmeister; Lehmann, Fernanda Kieling; Fonseca, André Salvador Kazantzi; Lunge, Vagner Ricardo

    2013-12-01

    Canine distemper virus (CDV) is the cause of a severe and highly contagious disease in dogs. Practical diagnosis of canine distemper based on clinical signs and laboratory tests are required to confirm CDV infection. The present study aimed to develop a molecular assay to detect and differentiate field and vaccine CDV strains. Reverse transcription followed by nested real time polymerase chain reaction (RT-nqPCR) was developed, which exhibited analytical specificity (all the samples from healthy dogs and other canine infectious agents were not incorrectly detected) and sensitivity (all replicates of a vaccine strain were positive up to the 3125-fold dilution - 10(0.7) TCID50). RT-nqPCR was validated for CDV detection on different clinical samples (blood, urine, rectal and conjunctival swabs) of 103 animals suspected to have distemper. A total of 53 animals were found to be positive based on RT-nqPCR in at least one clinical sample. Blood resulted in more positive samples (50 out of 53, 94.3%), followed by urine (44/53, 83.0%), rectal (38/53, 71%) and conjunctival (27/53, 50.9%) swabs. A commercial immunochromatography (IC) assay had detected CDV in only 30 conjunctival samples of these positive dogs. Nucleoprotein (NC) gene sequencing of 25 samples demonstrated that 23 of them were closer to other Brazilian field strains and the remaining two to vaccine strains. A single nucleotide sequences difference, which creates an Msp I restriction enzyme digestion, was used to differentiate between field and vaccine CDV strains by restriction fragment length polymorphism (RFLP) analysis. The complete assay was more sensitive than was IC for the detection of CDV. Blood was the more frequently positive specimen and the addition of a restriction enzyme step allowed the differentiation of vaccine and Brazilian field strains. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Development of real-time reverse transcription polymerase chain reaction assays to quantify insulin-like growth factor receptor and insulin receptor expression in equine tissue

    Directory of Open Access Journals (Sweden)

    Stephen B. Hughes

    2013-08-01

    Full Text Available The insulin-like growth factor system (insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor and six insulin-like growth factor-binding proteins and insulin are essential to muscle metabolism and most aspects of male and female reproduction. Insulin-like growth factor and insulin play important roles in the regulation of cell growth, differentiation and the maintenance of cell differentiation in mammals. In order to better understand the local factors that regulate equine physiology, such as muscle metabolism and reproduction (e.g., germ cell development and fertilisation, real-time reverse transcription polymerase chain reaction assays for quantification of equine insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were developed. The assays were sensitive: 192 copies/µLand 891 copies/µL for insulin-like growth factor 1 receptor, messenger ribonucleic acid and insulin receptor respectively (95%limit of detection, and efficient: 1.01 for the insulin-like growth factor 1 receptor assay and 0.95 for the insulin receptor assay. The assays had a broad linear range of detection (seven logs for insulin-like growth factor 1 receptor and six logs for insulin receptor. This allowed for analysis of very small amounts of messenger ribonucleic acid. Low concentrations of both insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were detected in endometrium, lung and spleen samples, whilst high concentrations were detected in heart, muscle and kidney samples, this was most likely due to the high level of glucose metabolism and glucose utilisation by these tissues. The assays developed for insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid expression have been shown to work on equine tissue and will contribute to the understanding of insulin and insulin-like growth factor 1

  6. Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR.

    Science.gov (United States)

    Sung, Heungsup; Yong, Dongeun; Ki, Chang Seok; Kim, Jae Seok; Seong, Moon Woo; Lee, Hyukmin; Kim, Mi Na

    2016-09-01

    Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both Phomogenizing sputum samples prior to RNA extraction.

  7. Preliminary results on ghrelin mRNA quantification in buffalo calves during fasting and refeeding by real-time reverse transcription PCR assay

    Directory of Open Access Journals (Sweden)

    G. Neglia

    2010-02-01

    Full Text Available The aim of this trial was to evaluate ghrelin response to milk administration in 20 days old buffalo calves. The trial was carried out on 5 female buffalo calves with a mean age of 21.2±2.8 days. Five blood samples were collected from each animal into EDTA tubes, starting at 07.00 until 15.00, at 2-h intervals. At 09.00, after the second blood sample, replaced milk was administered to the calves. Blood samples were immediately placed at 4°C until processing, which was performed on the same day. We used real-time reverse transcription PCR system to detect the expression of ghrelin mRNA levels in blood of buffalo calves. Two calves showed a low ghrelin concentration at the start of the trial (Group A = low ghrelin concentration and three calves a high ghrelin concentration (Group B = high ghrelin concentration. Ghrelin expression was significantly higher either two hours (P<0.01 and just before feeding (P<0.05 in Group B vs. Group A. However, in both cases, a significant (P<0.05 difference was observed within each group between -2 and 6 hours after feeding. Therefore, ghrelin concentration tended to increase in animals that showed low levels and, similarly, it lowered in animals that showed high concentration. If these results will be confirmed, may represent the evidence that also in buffalo calves the ghrelin system may affect feed intake. Further studies are needed in order to better evaluate the ghrelin system in buffalo calves.

  8. Classical swine fever virus detection: results of a real-time reverse transcription polymerase chain reaction ring trial conducted in the framework of the European network of excellence for epizootic disease diagnosis and control

    DEFF Research Database (Denmark)

    Hoffmann, Bernd; Blome, Sandra; Bonilauri, Paolo

    2011-01-01

    and specificity values. Nevertheless, some in-house systems had unspecific reactions or suboptimal sensitivity with only a single CSFV genotype. Follow-up actions involved either improvement of suboptimal assays or replacement of specific laboratory assays with the FLI protocol, with or without modifications......The current study reports on a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) ring trial for the detection of Classical swine fever virus (CSFV) genomic RNA undertaken by 10 European laboratories. All laboratories were asked to use their routine in-house real-time RT...

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

    Science.gov (United States)

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

    2014-07-22

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

  10. Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search.

    Science.gov (United States)

    Piller, Nicolas; Decosterd, Isabelle; Suter, Marc R

    2013-07-10

    The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated.We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system - specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model. We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process

  11. Precipitation of PEG/Carboxyl-Modified Gold Nanoparticles with Magnesium Pyrophosphate: A New Platform for Real-Time Monitoring of Loop-Mediated Isothermal Amplification.

    Science.gov (United States)

    Qin, Ailin; Fu, Lok Tin; Wong, Jacky K F; Chau, Li Yin; Yip, Shea Ping; Lee, Thomas M H

    2017-03-29

    Gold nanoparticles have proven to be promising for decentralized nucleic acid testing by virtue of their simple visual readout and absorbance-based quantification. A major challenge toward their practical application is to achieve ultrasensitive detection without compromising simplicity. The conventional strategy of thermocycling amplification is unfavorable (because of both instrumentation and preparation of thermostable oligonucleotide-modified gold nanoparticle probes). Herein, on the basis of a previously unreported co-precipitation phenomenon between thiolated poly(ethylene glycol)/11-mercaptoundecanoic acid co-modified gold nanoparticles and magnesium pyrophosphate crystals (an isothermal DNA amplification reaction byproduct), a new ultrasensitive and simple DNA assay platform is developed. The binding mechanism underlying the co-precipitation phenomenon is found to be caused by the complexation of carboxyl and pyrophosphate with free magnesium ions. Remarkably, poly(ethylene glycol) does not hinder the binding and effectively stabilizes gold nanoparticles against magnesium ion-induced aggregation (without pyrophosphate). In fact, a similar phenomenon is observed in other poly(ethylene glycol)- and carboxyl-containing nanomaterials. When the gold nanoparticle probe is incorporated into a loop-mediated isothermal amplification reaction, it remains as a red dispersion for a negative sample (in the absence of a target DNA sequence) but appears as a red precipitate for a positive sample (in the presence of a target). This results in a first-of-its-kind gold nanoparticle-based DNA assay platform with isothermal amplification and real-time monitoring capabilities.

  12. Development of loop-mediated isothermal amplification and SYBR green real-time PCR methods for the detection of Citrus yellow mosaic badnavirus in citrus species.

    Science.gov (United States)

    Anthony Johnson, A M; Dasgupta, I; Sai Gopal, D V R

    2014-07-01

    Citrus yellow mosaic badnavirus (CMBV) is an important pathogen in southern India spread by infected citrus propagules. One of the measures to arrest the spread of CMBV is to develop methods to screen and certify citrus propagules as CMBV-free. The methods loop-mediated isothermal amplification (LAMP) and SYBR green real-time PCR (SGRTPCR) have been developed for the efficient detection of CMBV in citrus propagules. This paper compares the sensitivities of LAMP and SGRTPCR with polymerase chain reaction (PCR) for the detection of CMBV. Whereas PCR and LAMP were able to detect CMBV from a minimum of 10 ng of total DNA of infected leaf samples, SGRTPCR could detect the same from 1 ng of total DNA. Using SGRTPCR, the viral titres were estimated to be the highest in rough lemon and lowest in Nagpur Mandarin of the five naturally infected citrus species tested. The results will help in designing suitable strategies for the sensitive detection of CMBV from citrus propagules. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Kim, Mi-Ju; Kim, Hae-Yeong

    2018-04-25

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

  14. Detection of Tumor Cell-Specific mRNA in the Peripheral Blood of Patients with Breast Cancer — Evaluation of Several Markers with Real-Time Reverse Transcription-PCR

    Directory of Open Access Journals (Sweden)

    Ulrich Andergassen

    2013-01-01

    Full Text Available It is widely known that cells from epithelial tumors, e.g., breast cancer, detach from their primary tissue and enter blood circulation. We show that the presence of circulating tumor cells (CTCs in samples of patients with primary and metastatic breast cancer can be detected with an array of selected tumor-marker-genes by reverse transcription real-time PCR. The focus of the presented work is on detecting differences in gene expression between healthy individuals and adjuvant and metastatic breast cancer patients, not an accurate quantification of these differences. Therefore, total RNA was isolated from blood samples of healthy donors and patients with primary or metastatic breast cancer after enrichment of mononuclear cells by density gradient centrifugation. After reverse transcription real-time PCR was carried out with a set of marker genes (BCSP, CK8, Her2, MGL, CK18, CK19. B2M and GAPDH were used as reference genes. Blood samples from patients with metastatic disease revealed increased cytokine gene levels in comparison to normal blood samples. Detection of a single gene was not sufficient to detect CTCs by reverse transcription real-time PCR. Markers used here were selected based on a recent study detecting cancer cells on different protein levels. The combination of such a marker array leads to higher and more specific discovery rates, predominantly in metastatic patients. Identification of CTCs by PCR methods may lead to better diagnosis and prognosis and could help to choose an adequate therapy.

  15. Detection of live Salmonella enterica in fresh-cut vegetables by a TaqMan-based one-step reverse transcription real-time PCR.

    Science.gov (United States)

    Miao, Y J; Xiong, G T; Bai, M Y; Ge, Y; Wu, Z F

    2018-05-01

    Fresh-cut produce is at greater risk of Salmonella contamination. Detection and early warning systems play an important role in reducing the dissemination of contaminated products. One-step Reverse Transcription Polymerase Chain Reaction (RT-qPCR) targeting Salmonella tmRNA with or without a 6-h enrichment was evaluated for the detection of Salmonella in fresh-cut vegetables after 6-h storage. LOD of one-step RT-qPCR was 1·0 CFU per ml (about 100 copies tmRNA per ml) by assessed 10-fold serially diluted RNA from 10 6 CFU per ml bacteria culture. Then, one-step RT-qPCR assay was applied to detect viable Salmonella cells in 14 fresh-cut vegetables after 6-h storage. Without enrichment, this assay could detect 10 CFU per g for fresh-cut lettuce, cilantro, spinach, cabbage, Chinese cabbage and bell pepper, and 10 2 CFU per g for other vegetables. With a 6-h enrichment, this assay could detect 10 CFU per g for all fresh-cut vegetables used in this study. Moreover, this assay was able to discriminate viable cells from dead cells. This rapid detection assay may provide potential processing control and early warning method in fresh-cut vegetable processing to strengthen food safety assurance. Significance and Impact of the Study: Fresh-cut produce is at greater risk of Salmonella contamination. Rapid detection methods play an important role in reducing the dissemination of contaminated products. One-step RT-qPCR assay used in this study could detect 10 CFU per g Salmonella for 14 fresh-cut vegetables with a 6-h short enrichment. Moreover, this assay was able to discriminate viable cells from dead cells. This rapid detection assay may provide potential processing control and early warning method in fresh-cut vegetable processing to strengthen food safety assurance. © 2018 The Society for Applied Microbiology.

  16. A Novel Duplex Real-Time Reverse-Transcription PCR Assay for the Detection of Influenza A and the Novel Influenza A(H1N1 Strain

    Directory of Open Access Journals (Sweden)

    Theo P. Sloots

    2009-12-01

    Full Text Available Timely implementation of antiviral treatment and other public health based responses are dependent on accurate and rapid diagnosis of the novel pandemic influenza A(H1N1 strain. In this study we developed a duplex real-time PCR (RT-PCR (dFLU-TM assay for the simultaneous detection of a broad range of influenza A subtypes and specific detection of the novel H1N1 2009 pandemic strain. The assay was compared to the combined results of two previously described monoplex RT-PCR assays using 183 clinical samples and 10 seasonal influenza A isolates. Overall, the results showed that the dFLU-TM RT-PCR method is suitable for detection of influenza A, including the novel H1N1 pandemic strain, in clinical samples.

  17. Real-time quantitative reverse transcription-PCR analysis of expression stability of Actinobacillus pleuropneumoniae housekeeping genes during in vitro growth under iron-depleted conditions

    DEFF Research Database (Denmark)

    Nielsen, K. K.; Boye, Mette

    2005-01-01

    up-regulation under iron-restricted conditions compared to bacteria grown in medium with sufficient iron. The observed expression patterns of the genes of interest were consistent with previous observations. This study therefore lends further support to the use of real-time quantitative RT...... controls, as such controls have not been defined yet for this bacterium. Bacterial gene expression was studied during in vitro exponential and early stationary growth in medium with and without sufficient iron, respectively. First, the stability of expression of five genes, the glyA, tpiA, pykA, rec......F, and rhoAP genes involved in basic housekeeping, was evaluated on the basis of the mean pairwise variation. All the housekeeping genes included were stably expressed under the conditions investigated and consequently were included in the normalization procedure. Next, the geometric mean of the internal...

  18. Sensitivity and specificity of real-time reverse transcription polymerase chain reaction, histopathology, and immunohistochemical labeling for the detection of Rift Valley fever virus in naturally infected cattle and sheep.

    Science.gov (United States)

    Odendaal, Lieza; Fosgate, Geoffrey T; Romito, Marco; Coetzer, Jacobus A W; Clift, Sarah J

    2014-01-01

    Real-time reverse transcription polymerase chain reaction (real-time RT-PCR), histopathology, and immunohistochemical labeling (IHC) were performed on liver specimens from 380 naturally infected cattle and sheep necropsied during the 2010 Rift Valley fever (RVF) epidemic in South Africa. Sensitivity (Se) and specificity (Sp) of real-time RT-PCR, histopathology, and IHC were estimated in a latent-class model using a Bayesian framework. The Se and Sp of real-time RT-PCR were estimated as 97.4% (95% confidence interval [CI] = 95.2-98.8%) and 71.7% (95% CI = 65-77.9%) respectively. The Se and Sp of histopathology were estimated as 94.6% (95% CI = 91-97.2%) and 92.3% (95% CI = 87.6-95.8%), respectively. The Se and Sp of IHC were estimated as 97.6% (95% CI = 93.9-99.8%) and 99.4% (95% CI = 96.9-100%), respectively. Decreased Sp of real-time RT-PCR was ascribed to cross-contamination of samples. Stratified analysis of the data suggested variations in test accuracy with fetuses and severely autolyzed specimens. The Sp of histopathology in fetuses (83%) was 9.3% lower than the sample population (92.3%). The Se of IHC decreased from 97.6% to 81.5% in the presence of severe autolysis. The diagnostic Se and Sp of histopathology was higher than expected, confirming the value of routine postmortem examinations and histopathology of liver specimens. Aborted fetuses, however, should be screened using a variety of tests in areas endemic for RVF, and results from severely autolyzed specimens should be interpreted with caution. The most feasible testing option for countries lacking suitably equipped laboratories seems to be routine histology in combination with IHC.

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

    Science.gov (United States)

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

    2017-08-01

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

  20. Evaluation of the PrimerDesign™ genesig real-time reverse transcription-polymerase chain reaction assay and the INFINITI® Respiratory Viral Panel Plus assay for the detection of human metapneumovirus in Kuwait.

    Science.gov (United States)

    Al-Turab, Mariam; Chehadeh, Wassim; Al-Mulla, Fahd; Al-Nakib, Widad

    2012-04-01

    Human metapneumovirus (hMPV) is a respiratory pathogen that was discovered in 2001 and is considered a major cause of both upper and lower respiratory tract infections. A sensitive, fast, and high-throughput diagnostic test is needed for the detection of hMPV that may assist in the clinical management as well as in the reduction of inappropriate therapy. Therefore, a comparison assessment was performed in this study between the PrimerDesign™ genesig real-time reverse transcription-polymerase chain reaction (RT-PCR) Assay and the INFINITI(®) Respiratory Viral Panel Plus Assay (RVP-Plus) for the detection of hMPV infection in patients with respiratory tract infections. A total of 200 respiratory samples were collected from 185 hospitalized patients, during the winter season in Kuwait. Of 185 patients, 10 (5.4%) were positive for hMPV RNA by the in-house RT-PCR assay, while 7 (4%) were positive for hMPV RNA by the real-time RT-PCR assay and 9 (5%) were positive for hMPV RNA by the INFINITI(®) RVP-Plus assay. The high incidence rate (60%) of hMPV infection was in January 2011. The sensitivity of the real-time RT-PCR and INFINITI(®) RVP-Plus assays was 70% and 90%, respectively, with specificity of 100% for both assays. hMPV types A and B could be identified in this study; however, discordant genotyping results were found between the direct sequencing method and the INFINITI(®) RVP-Plus assay in 33% of hMPV-positive patients. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction.

    Science.gov (United States)

    Grau, Frederic R; Schroeder, Megan E; Mulhern, Erin L; McIntosh, Michael T; Bounpheng, Mangkey A

    2015-03-01

    African swine fever (ASF), classical swine fever (CSF), and foot-and-mouth disease (FMD) are highly contagious animal diseases of significant economic importance. Pigs infected with ASF and CSF viruses (ASFV and CSFV) develop clinical signs that may be indistinguishable from other diseases. Likewise, various causes of vesicular disease can mimic clinical signs caused by the FMD virus (FMDV). Early detection is critical to limiting the impact and spread of these disease outbreaks, and the ability to perform herd-level surveillance for all 3 diseases rapidly and cost effectively using a single diagnostic sample and test is highly desirable. This study assessed the feasibility of simultaneous ASFV, CSFV, and FMDV detection by multiplex reverse transcription real-time polymerase chain reaction (mRT-qPCR) in swine oral fluids collected through the use of chewing ropes. Animal groups were experimentally infected independently with each virus, observed for clinical signs, and oral fluids collected and tested throughout the course of infection. All animal groups chewed on the ropes readily before and after onset of clinical signs and before onset of lameness or serious clinical signs. ASFV was detected as early as 3 days postinoculation (dpi), 2-3 days before onset of clinical disease; CSFV was detected at 5 dpi, coincident with onset of clinical disease; and FMDV was detected as early as 1 dpi, 1 day before the onset of clinical disease. Equivalent results were observed in 4 independent studies and demonstrate the feasibility of oral fluids and mRT-qPCR for surveillance of ASF, CSF, and FMD in swine populations. © 2015 The Author(s).

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

    Science.gov (United States)

    Zhang, Xin; Zhang, He; Pu, Jinji; Qi, Yanxiang; Yu, Qunfang; Xie, Yixian; Peng, Jun

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Xin Zhang

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

  4. Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP) for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals.

    Science.gov (United States)

    Zhou, Qian-Jin; Wang, Lei; Chen, Jiong; Wang, Rui-Na; Shi, Yu-Hong; Li, Chang-Hong; Zhang, De-Min; Yan, Xiao-Jun; Zhang, Yan-Jun

    2014-09-01

    Rapid, low-cost, and user-friendly strategies are urgently needed for early disease diagnosis and timely treatment, particularly for on-site screening of pathogens in aquaculture. In this study, we successfully developed a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP), which was capable of simultaneously detecting 10 pathogenic bacteria in aquatic animals, i.e., Nocardia seriolae, Pseudomonas putida, Streptococcus iniae, Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio rotiferianus, and Vibrio vulnificus. The assay provided a nearly-automated approach, with only a single pipetting step per chip for sample dispensing. This technique could achieve limits of detection (LOD) ranging from 0.40 to 6.42pg per 1.414μL reaction in less than 30 min. The robust reproducibility was demonstrated by a little variation among duplications for each bacterium with the coefficient of variation (CV) for time to positive (Tp) value less than 0.10. The clinical sensitivity and specificity of this on-chip LAMP assay in detecting field samples were 96.2% and 93.8% by comparison with conventional microbiological methods. Compared with other well-known techniques, on-chip LAMP assay provides low sample and reagent consumption, ease-of-use, accelerated analysis, multiple bacteria and on-site detection, and high reproducibility, indicating that such a technique would be applicable for on-site detection and routine monitoring of multiple pathogens in aquaculture. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all hemagglutinin and neuraminidase genes of avian influenza viruses and comparison to standard serological subtyping tests

    Science.gov (United States)

    Tsukamoto, K.; Javier, P.C.; Shishido, M.; Noguchi, D.; Pearce, J.; Kang, H.-M.; Jeong, O.M.; Lee, Y.-J.; Nakanishi, K.; Ashizawa, T.

    2012-01-01

    Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10 1.5, 10 2.3, and 10 3.1 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples. Copyright ?? 2012, American Society for Microbiology. All Rights Reserved.

  6. Properties of the reverse transcription reaction in mRNA quantification

    DEFF Research Database (Denmark)

    Ståhlberg, Anders; Håkansson, Joakim; Xian, Xiaojie

    2004-01-01

    BACKGROUND: In most measurements of gene expression, mRNA is first reverse-transcribed into cDNA. We studied the reverse transcription reaction and its consequences for quantitative measurements of gene expression. METHODS: We used SYBR green I-based quantitative real-time PCR (QPCR) to measure...... the properties of reverse transcription reaction for the beta-tubulin, glyceraldehyde-3-phosphate dehydrogenase, Glut2, CaV1D, and insulin II genes, using random hexamers, oligo(dT), and gene-specific reverse transcription primers. RESULTS: Experimental variation in reverse transcription-QPCR (RT......-QPCR) was mainly attributable to the reverse transcription step. Reverse transcription efficiency depended on priming strategy, and the dependence was different for the five genes studied. Reverse transcription yields also depended on total RNA concentration. CONCLUSIONS: RT-QPCR gene expression measurements...

  7. Improved detection limit in rapid detection of human enterovirus 71 and coxsackievirus A16 by a novel reverse transcription-isothermal multiple-self-matching-initiated amplification assay.

    Science.gov (United States)

    Ding, Xiong; Nie, Kai; Shi, Lei; Zhang, Yong; Guan, Li; Zhang, Dan; Qi, Shunxiang; Ma, Xuejun

    2014-06-01

    Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-foot-and-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription-isothermal multiple-self-matching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R(2) values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R(2) values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71

  8. Development of one-step Loop-Mediated Isothermal Amplification (LAMP) for the detection of norovirus in oysters

    Science.gov (United States)

    The aim of this study was to develop a simple and rapid technique for detecting human norovirus (NoV). The loop-mediated isothermal amplification (LAMP) technique was evaluated and found to be sensitive, highly specific, and useful for routine oyster testing. Reverse transcription-LAMP (RT-LAMP) pri...

  9. Detection of feline Coronavirus in effusions of cats with and without feline infectious peritonitis using loop-mediated isothermal amplification.

    Science.gov (United States)

    Günther, Sonja; Felten, Sandra; Wess, Gerhard; Hartmann, Katrin; Weber, Karin

    2018-06-01

    Feline infectious peritonitis (FIP) is a fatal disease in cats worldwide. The aim of this study was to test two commercially available reaction mixtures in a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect feline Coronavirus (FCoV) in body cavity effusions of cats with and without FIP, in order to minimize the time from sampling to obtaining results. RNA was extracted from body cavity effusion samples of 71 cats, including 34 samples from cats with a definitive diagnosis of FIP, and 37 samples of control cats with similar clinical signs but other confirmed diseases. Two reaction mixtures (Isothermal Mastermix, OptiGene Ltd.and PCRun™ Molecular Detection Mix, Biogal) were tested using the same primers, which were designed to bind to a conserved region of the FCoV membrane protein gene. Both assays were conducted under isothermal conditions (61 °C-62 °C). Using the Isothermal Mastermix of OptiGene Ltd., amplification times ranged from 4 and 39 min with a sensitivity of 35.3% and a specificity of 94.6% for the reported sample group. Using the PCRun™ Molecular Detection Mix of Biogal, amplification times ranged from 18 to 77 min with a sensitivity of 58.8% and a specificity of 97.3%. Although the RT-LAMP assay is less sensitive than real time reverse transcription PCR (RT-PCR), it can be performed without the need of expensive equipment and with less hands-on time. Further modifications of primers might lead to a suitable in-house test and accelerate the diagnosis of FIP. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. 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.

  11. Semi-Nested Real-Time Reverse Transcription Polymerase Chain Reaction Methods for the Successful Quantitation of Cytokeratin mRNA Expression Levels for the Subtyping of Non-Small-Cell Lung Carcinoma Using Paraffin-Embedded and Microdissected Lung Biopsy Specimens

    International Nuclear Information System (INIS)

    Nakanishi, Yoko; Shimizu, Tetsuo; Tsujino, Ichiro; Obana, Yukari; Seki, Toshimi; Fuchinoue, Fumi; Ohni, Sumie; Oinuma, Toshinori; Kusumi, Yoshiaki; Yamada, Tsutomu; Takahashi, Noriaki; Hashimoto, Shu; Nemoto, Norimichi

    2013-01-01

    In patients with inoperable advanced non-small cell lung carcinomas (NSCLCs), histological subtyping using small-mount biopsy specimens was often required to decide the indications for drug treatment. The aim of this study was to assess the utility of highly sensitive mRNA quantitation for the subtyping of advanced NSCLC using small formalin fixing and paraffin embedding (FFPE) biopsy samples. Cytokeratin (CK) 6, CK7, CK14, CK18, and thyroid transcription factor (TTF)-1 mRNA expression levels were measured using semi-nested real-time quantitative (snq) reverse-transcribed polymerase chain reaction (RT-PCR) in microdissected tumor cells collected from 52 lung biopsies. Our results using the present snqRT-PCR method showed an improvement in mRNA quantitation from small FFPE samples, and the mRNA expression level using snqRT-PCR was correlated with the immunohistochemical protein expression level. CK7, CK18, and TTF-1 mRNA were expressed at significantly higher levels (P<0.05) in adenocarcinoma (AD) than in squamous cell carcinoma (SQ), while CK6 and CK14 mRNA expression was significantly higher (P<0.05) in SQ than in AD. Each histology-specific CK, particularly CK18 in AD and CK6 in SQ, were shown to be correlated with a poor prognosis (P=0.02, 0.02, respectively). Our results demonstrated that a quantitative CK subtype mRNA analysis from lung biopsy samples can be useful for predicting the histology subtype and prognosis of advanced NSCLC

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

    Science.gov (United States)

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

    2013-10-01

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

  13. Reverse transcription loop-mediated isothermal amplification for species-specific detection of tomato chlorotic spot orthotospovirus

    Science.gov (United States)

    Tomato chlorotic spot virus (TCSV) is an emerging tospovirus that can cause severe disease on tomato plants. There are at least four tospoviruses infecting tomato, and mixed infection of various viruses in a field crop is quite common. With similarity in the symptomatology and cross serological reac...

  14. Real-time systems

    OpenAIRE

    Badr, Salah M.; Bruztman, Donald P.; Nelson, Michael L.; Byrnes, Ronald Benton

    1992-01-01

    This paper presents an introduction to the basic issues involved in real-time systems. Both real-time operating sys and real-time programming languages are explored. Concurrent programming and process synchronization and communication are also discussed. The real-time requirements of the Naval Postgraduate School Autonomous Under Vehicle (AUV) are then examined. Autonomous underwater vehicle (AUV), hard real-time system, real-time operating system, real-time programming language, real-time sy...

  15. Associated technologies ensures complete loop mediated ...

    African Journals Online (AJOL)

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

  16. Comparison of multiplex reverse transcription-PCR-enzyme ...

    African Journals Online (AJOL)

    Comparison of multiplex reverse transcription-PCR-enzyme hybridization assay with immunofluorescence techniques for the detection of four viral respiratory pathogens in pediatric community acquired pneumonia.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  18. Real Time Revisited

    Science.gov (United States)

    Allen, Phillip G.

    1985-12-01

    The call for abolishing photo reconnaissance in favor of real time is once more being heard. Ten years ago the same cries were being heard with the introduction of the Charge Coupled Device (CCD). The real time system problems that existed then and stopped real time proliferation have not been solved. The lack of an organized program by either DoD or industry has hampered any efforts to solve the problems, and as such, very little has happened in real time in the last ten years. Real time is not a replacement for photo, just as photo is not a replacement for infra-red or radar. Operational real time sensors can be designed only after their role has been defined and improvements made to the weak links in the system. Plodding ahead on a real time reconnaissance suite without benefit of evaluation of utility will allow this same paper to be used ten years from now.

  19. Intracytoplasmic maturation of the human immunodeficiency virus type 1 reverse transcription complexes determines their capacity to integrate into chromatin

    Directory of Open Access Journals (Sweden)

    Kashanchi Fatah

    2006-01-01

    Full Text Available Abstract Background The early events of the HIV-1 life cycle include entry of the viral core into target cell, assembly of the reverse transcription complex (RTCs performing reverse transcription, its transformation into integration-competent complexes called pre-integration complexes (PICs, trafficking of complexes into the nucleus, and finally integration of the viral DNA into chromatin. Molecular details and temporal organization of these processes remain among the least investigated and most controversial problems in the biology of HIV. Results To quantitatively evaluate maturation and nuclear translocation of the HIV-1 RTCs, nucleoprotein complexes isolated from the nucleus (nRTC and cytoplasm (cRTC of HeLa cells infected with MLV Env-pseudotyped HIV-1 were analyzed by real-time PCR. While most complexes completed reverse transcription in the cytoplasm, some got into the nucleus before completing DNA synthesis. The HIV-specific RNA complexes could get into the nucleus when reverse transcription was blocked by reverse transcriptase inhibitor, although nuclear import of RNA complexes was less efficient than of DNA-containing RTCs. Analysis of the RTC nuclear import in synchronized cells infected in the G2/M phase of the cell cycle showed enrichment in the nuclei of RTCs containing incomplete HIV-1 DNA compared to non-synchronized cells, where RTCs with complete reverse transcripts prevailed. Immunoprecipitation assays identified viral proteins IN, Vpr, MA, and cellular Ini1 and PML associated with both cRTCs and nRTCs, whereas CA was detected only in cRTCs and RT was diminished in nRTCs. Cytoplasmic maturation of the complexes was associated with increased immunoreactivity with anti-Vpr and anti-IN antibodies, and decreased reactivity with antibodies to RT. Both cRTCs and nRTCs carried out endogenous reverse transcription reaction in vitro. In contrast to cRTCs, in vitro completion of reverse transcription in nRTCs did not increase their

  20. Real Time Systems

    DEFF Research Database (Denmark)

    Christensen, Knud Smed

    2000-01-01

    Describes fundamentals of parallel programming and a kernel for that. Describes methods for modelling and checking parallel problems. Real time problems.......Describes fundamentals of parallel programming and a kernel for that. Describes methods for modelling and checking parallel problems. Real time problems....

  1. Real-time shadows

    CERN Document Server

    Eisemann, Elmar; Assarsson, Ulf; Wimmer, Michael

    2011-01-01

    Important elements of games, movies, and other computer-generated content, shadows are crucial for enhancing realism and providing important visual cues. In recent years, there have been notable improvements in visual quality and speed, making high-quality realistic real-time shadows a reachable goal. Real-Time Shadows is a comprehensive guide to the theory and practice of real-time shadow techniques. It covers a large variety of different effects, including hard, soft, volumetric, and semi-transparent shadows.The book explains the basics as well as many advanced aspects related to the domain

  2. Real time expert systems

    International Nuclear Information System (INIS)

    Asami, Tohru; Hashimoto, Kazuo; Yamamoto, Seiichi

    1992-01-01

    Recently, aiming at the application to the plant control for nuclear reactors and traffic and communication control, the research and the practical use of the expert system suitable to real time processing have become conspicuous. In this report, the condition for the required function to control the object that dynamically changes within a limited time is presented, and the technical difference between the real time expert system developed so as to satisfy it and the expert system of conventional type is explained with the actual examples and from theoretical aspect. The expert system of conventional type has the technical base in the problem-solving equipment originating in STRIPS. The real time expert system is applied to the fields accompanied by surveillance and control, to which conventional expert system is hard to be applied. The requirement for the real time expert system, the example of the real time expert system, and as the techniques of realizing real time processing, the realization of interruption processing, dispersion processing, and the mechanism of maintaining the consistency of knowledge are explained. (K.I.)

  3. Real-time radiography

    International Nuclear Information System (INIS)

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

    1981-01-01

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

  4. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  5. Lineage-Specific Real-Time RT-PCR for Yellow Fever Virus Outbreak Surveillance, Brazil.

    Science.gov (United States)

    Fischer, Carlo; Torres, Maria C; Patel, Pranav; Moreira-Soto, Andres; Gould, Ernest A; Charrel, Rémi N; de Lamballerie, Xavier; Nogueira, Rita Maria Ribeiro; Sequeira, Patricia C; Rodrigues, Cintia D S; Kümmerer, Beate M; Drosten, Christian; Landt, Olfert; Bispo de Filippis, Ana Maria; Drexler, Jan Felix

    2017-11-01

    The current yellow fever outbreak in Brazil prompted widespread yellow fever virus (YFV) vaccination campaigns, imposing a responsibility to distinguish between vaccine- and wild-type YFV-associated disease. We developed novel multiplex real-time reverse transcription PCRs that differentiate between vaccine and American wild-type YFV. We validated these highly specific and sensitive assays in an outbreak setting.

  6. Lineage-Specific Real-Time RT-PCR for Yellow Fever Virus Outbreak Surveillance, Brazil

    OpenAIRE

    Fischer, Carlo; Torres, Maria C.; Patel, Pranav; Moreira-Soto, Andres; Gould, Ernest A.; Charrel, Rémi N.; de Lamballerie, Xavier; Nogueira, Rita Maria Ribeiro; Sequeira, Patricia C.; Rodrigues, Cintia D.S.; Kümmerer, Beate M.; Drosten, Christian; Landt, Olfert; Bispo de Filippis, Ana Maria; Drexler, Jan Felix

    2017-01-01

    The current yellow fever outbreak in Brazil prompted widespread yellow fever virus (YFV) vaccination campaigns, imposing a responsibility to distinguish between vaccine- and wild-type YFV-associated disease. We developed novel multiplex real-time reverse transcription PCRs that differentiate between vaccine and American wild-type YFV. We validated these highly specific and sensitive assays in an outbreak setting.

  7. Detection of three porcine vesicular viruses using multiplex real-time primer-probe energy transfer

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Aguero, M.

    2006-01-01

    Rapid identification of the etiologic agent in infected animals is important for the control of an outbreak of vesicular disease in livestock. We have in the present study developed a multiplex real-time reverse transcription-PCR, based on primer-probe energy transfer (PriProET), for simultaneous...

  8. Route around real time

    International Nuclear Information System (INIS)

    Terrier, Francois

    1996-01-01

    The greater and greater autonomy and complexity asked to the control and command systems lead to work on introducing techniques such as Artificial Intelligence or concurrent object programming in industrial applications. However, while the critical feature of these systems impose to control the dynamics of the proposed solutions, their complexity often imposes a high adaptability to a partially modelled environment. The studies presented start from low level control and command systems to more complex applications at higher levels, such as 'supervision systems'. Techniques such as temporal reasoning and uncertainty management are proposed for the first studies, while the second are tackled with programming techniques based on the real time object paradigm. The outcomes of this itinerary crystallize on the ACCORD project which targets to manage - on the whole life cycle of a real time application - these two problematics, sometimes antagonistic: control of the dynamics and adaptivity. (author) [fr

  9. Requirements for DNA strand transfer during reverse transcription in mutant HIV-1 virions

    NARCIS (Netherlands)

    Berkhout, B.; van Wamel, J.; Klaver, B.

    1995-01-01

    Retroviruses convert their RNA genome into a DNA form by means of reverse transcription. According to the current model of reverse transcription, two strand transfer reactions are needed to synthesize a full-length DNA genome. Because reverse transcription is initiated close to the 5' end of the RNA

  10. Real time falling leaves

    OpenAIRE

    Vázquez Alcocer, Pere Pau; Balsa, Marcos

    2007-01-01

    There is a growing interest in simulating natural phenomena in computer graphics applications. Animating natural scenes in real time is one of the most challenging problems due to the inherent complexity of their structure, formed by millions of geometric entities, and the interactions that happen within. An example of natural scenario that is needed for games or simulation programs are forests. Forests are difficult to render because the huge amount of geometric entities and the large amount...

  11. Real Time Strategy Language

    OpenAIRE

    Hayes, Roy; Beling, Peter; Scherer, William

    2014-01-01

    Real Time Strategy (RTS) games provide complex domain to test the latest artificial intelligence (AI) research. In much of the literature, AI systems have been limited to playing one game. Although, this specialization has resulted in stronger AI gaming systems it does not address the key concerns of AI researcher. AI researchers seek the development of AI agents that can autonomously interpret learn, and apply new knowledge. To achieve human level performance, current AI systems rely on game...

  12. Real Time Processing

    CERN Multimedia

    CERN. Geneva; ANDERSON, Dustin James; DOGLIONI, Caterina

    2015-01-01

    The LHC provides experiments with an unprecedented amount of data. Experimental collaborations need to meet storage and computing requirements for the analysis of this data: this is often a limiting factor in the physics program that would be achievable if the whole dataset could be analysed. In this talk, I will describe the strategies adopted by the LHCb, CMS and ATLAS collaborations to overcome these limitations and make the most of LHC data: data parking, data scouting, and real-time analysis.

  13. Demonstration of a very inexpensive, turbidimetric, real-time, RT-LAMP detection platform using shrimp Laem-Singh virus (LSNV as a model.

    Directory of Open Access Journals (Sweden)

    Narong Arunrut

    Full Text Available Rapid and accurate detection of pathogens under field laboratory conditions is necessary for effective control of veterinary pathogens. Here we describe a prototype, portable, pathogen detection device developed for single tube, real-time, reverse transcription, loop-mediated isothermal amplification (RT-LAMP using Laem-Singh virus (LSNV as a model. LSNV is an RNA virus and a component cause of growth retardation in black tiger shrimp. We chose its RNA-dependent RNA polymerase (RdRp gene as the target for our tests. The basis for detection was measurement of turbidity arising from formation of a white, insoluble magnesium pyrophosphate precipitate byproduct upon amplification of the RdRp target sequence from 100 ng template RNA extracted from shrimp. The measurement device consisted of a heating block to maintain constant temperature in the RT-LAMP reaction for 8 Eppindorf sample tubes, a light-emitting diode (LED light source providing red light emission at 650 nm wavelength to pass through sample tubes, a light dependent resistance (LDR photo-detector and a software program to report turbidity events and could potentially be marketed for under US$3000. The device was connected to a computer to display real-time results in a variety of formats. The optimized protocol for LSNV detection consisted of incubation of the sample tubes at 65 °C for 1 h during which turbidity was continuously measured, and quantitative results could be obtained by reaction time measurement. The sensitivity of detection was comparable to that of conventional nested RT-PCR and there was no cross reaction with other common shrimp viruses. The device was used for quantitative measurement of relative copy numbers of LSNV RdRp in 8 shrimp tissues and they were found to be highest in the gills followed in order by the lymphoid organ and hemolymph (p ≤ 0.05. This platform can be easily adapted for detection of other pathogens under field laboratory settings.

  14. Real-time specifications

    DEFF Research Database (Denmark)

    David, A.; Larsen, K.G.; Legay, A.

    2015-01-01

    A specification theory combines notions of specifications and implementations with a satisfaction relation, a refinement relation, and a set of operators supporting stepwise design. We develop a specification framework for real-time systems using Timed I/O Automata as the specification formalism......, with the semantics expressed in terms of Timed I/O Transition Systems. We provide constructs for refinement, consistency checking, logical and structural composition, and quotient of specifications-all indispensable ingredients of a compositional design methodology. The theory is implemented in the new tool Ecdar...

  15. Real Time Text Analysis

    Science.gov (United States)

    Senthilkumar, K.; Ruchika Mehra Vijayan, E.

    2017-11-01

    This paper aims to illustrate real time analysis of large scale data. For practical implementation we are performing sentiment analysis on live Twitter feeds for each individual tweet. To analyze sentiments we will train our data model on sentiWordNet, a polarity assigned wordNet sample by Princeton University. Our main objective will be to efficiency analyze large scale data on the fly using distributed computation. Apache Spark and Apache Hadoop eco system is used as distributed computation platform with Java as development language

  16. Real time Faraday spectrometer

    Science.gov (United States)

    Smith, Jr., Tommy E.; Struve, Kenneth W.; Colella, Nicholas J.

    1991-01-01

    This invention uses a dipole magnet to bend the path of a charged particle beam. As the deflected particles exit the magnet, they are spatially dispersed in the bend-plane of the magnet according to their respective momenta and pass to a plurality of chambers having Faraday probes positioned therein. Both the current and energy distribution of the particles is then determined by the non-intersecting Faraday probes located along the chambers. The Faraday probes are magnetically isolated from each other by thin metal walls of the chambers, effectively providing real time current-versus-energy particle measurements.

  17. Real time Faraday spectrometer

    International Nuclear Information System (INIS)

    Smith, T.E.; Struve, K.W.; Colella, N.J.

    1991-01-01

    This patent describes an invention which uses a dipole magnet to bend the path of a charged particle beam. As the deflected particles exit the magnet, they are spatially dispersed in the bend-plane of the magnet according to their respective momenta and pass to a plurality of chambers having Faraday probes positioned therein. Both the current and energy distribution of the particles is then determined by the non-intersecting Faraday probes located along the chambers. The Faraday probes are magnetically isolated from each other by thin metal walls of the chambers, effectively providing real time current-versus-energy particle measurements

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

    OpenAIRE

    Ge, Yiyue; Zhou, Qiang; Zhao, Kangchen; Chi, Ying; Liu, Bin; Min, Xiaoyan; Shi, Zhiyang; Zou, Bingjie; Cui, Lunbiao

    2017-01-01

    Yiyue Ge,1 Qiang Zhou,2 Kangchen Zhao,1 Ying Chi,1 Bin Liu,3 Xiaoyan Min,4 Zhiyang Shi,1 Bingjie Zou,2 Lunbiao Cui1 1Institute of Pathogenic Microbiology, Key Laboratories of Enteric Pathogenic Microbiology (Ministry of Health), Jiangsu Provincial Center for Disease Control and Prevention, 2Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, 3Department of Biomedical Engineering, Nanjing Medical University, 4Department of Geriatrics, The First Affiliated Hospi...

  19. APOBEC3G inhibits elongation of HIV-1 reverse transcripts.

    Directory of Open Access Journals (Sweden)

    Kate N Bishop

    2008-12-01

    Full Text Available APOBEC3G (A3G is a host cytidine deaminase that, in the absence of Vif, restricts HIV-1 replication and reduces the amount of viral DNA that accumulates in cells. Initial studies determined that A3G induces extensive mutation of nascent HIV-1 cDNA during reverse transcription. It has been proposed that this triggers the degradation of the viral DNA, but there is now mounting evidence that this mechanism may not be correct. Here, we use a natural endogenous reverse transcriptase assay to show that, in cell-free virus particles, A3G is able to inhibit HIV-1 cDNA accumulation not only in the absence of hypermutation but also without the apparent need for any target cell factors. We find that although reverse transcription initiates in the presence of A3G, elongation of the cDNA product is impeded. These data support the model that A3G reduces HIV-1 cDNA levels by inhibiting synthesis rather than by inducing degradation.

  20. Real time spectrum analysis

    International Nuclear Information System (INIS)

    Blunden, A.; O'Prey, D.G.; Tait, W.H.

    1983-01-01

    A method is described for the separation of a composite pulse-height spectrum into its unresolved component parts, which belong to a set of measured library spectra. The method allows real-time estimation giving running estimates during acquisition of the spectrum, minimises computation space, especially for a number of parallel calculations, estimates in advance the rms errors, and produces a significance measure for the hypothesis that the composite contains only the library spectra. Least squares curve-fitting, and other methods, can be compared, with the formalism developed, allowing analytical comparison of the effect of detector energy resolution and detection efficiency. A rational basis for the choice between the various methods of spectrum analysis follows from the theory, minimising rms estimation errors. The method described is applicable for very low numbers of counts and poor resolution. (orig.)

  1. Real time production optimization

    Energy Technology Data Exchange (ETDEWEB)

    Saputelli, Luigi; Otavio, Joao; Araujo, Turiassu; Escorcia, Alvaro [Halliburton, Houston, TX (United States). Landmark Division

    2004-07-01

    Production optimization encompasses various activities of measuring, analyzing, modeling, prioritizing and implementing actions to enhance productivity of a field. We present a state-of-the-art framework for optimizing production on a continuous basis as new sensor data is acquired in real time. Permanently acquired data is modeled and analyzed in order to create predictive models. A model based control strategy is used to regulate well and field instrumentation. The optimum field operating point, which changes with time, satisfies the maximum economic return. This work is a starting point for further development in automatic, intelligent reservoir technologies which get the most out of the abilities of permanent, instrumented wells and remotely activated downhole completions. The strategy, tested with history-matched data from a compartmentalised giant field, proved to reduce operating costs while increasing oil recovery by 27% in this field. (author)

  2. Real time urbanism

    Directory of Open Access Journals (Sweden)

    Ana Ruiz Varona

    2012-12-01

    Full Text Available Nowadays, given the technological revolution of the society of information, the administrative management of the cities faces a new problem not as related to the projection of the urban space as to the capacity of controlling and measuring the process of direct and centralized production of the cities by part of some non-homogeneous social multitudes, in a hyper-accelerated time towards instantaneity. Against libertarian apologies of the new “participative urbanisms”, the article puts forward a discourse that shows the lost associated to the new problem of temporal instantaneity. In this regard we claim new process of mediation that allow administrations and urbanist monitoring the production of the city. To that end, a previous and necessary step will be the redefinition of the role of a new real time urbanist.

  3. Comparison of polymerase chain reaction (PCR) and loop-mediated ...

    African Journals Online (AJOL)

    Comparison of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) for diagnosis of Fusarium solani in human immunodeficiency virus (HIV) positive patients. ... The test was carried out in 1 h reaction at 65°C in a heater block. The specificity of the test was 100% and its sensitivity was a ...

  4. Loop-mediated isothermal amplification (LAMP) based detection of ...

    African Journals Online (AJOL)

    SAM

    2014-05-07

    May 7, 2014 ... 2 months for growing in a culture. Therefore, to control .... The LAMP reaction is carried out in a 25 µL reaction mixture containing ..... J. Fish Dis. 32(6):491-497. Goto M, Honda E, Ogura A, Nomoto A, Hanaki K (2009). Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy ...

  5. Development and application of a loop-mediated isothermal ...

    African Journals Online (AJOL)

    Haemophilus parasuis is the causative agent of Glässer's disease that has received much attention recently, due to the increasing economic losses this disease inflicts upon the pig industry worldwide. In this study, loop-mediated isothermal amplification method (LAMP) methodology was designed for diagnosing H.

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

    African Journals Online (AJOL)

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

  7. Development of Reverse Transcription Thermostable Helicase-Dependent DNA Amplification for the Detection of Tomato Spotted Wilt Virus.

    Science.gov (United States)

    Wu, Xinghai; Chen, Chanfa; Xiao, Xizhi; Deng, Ming Jun

    2016-11-01

    A protocol for the reverse transcription-helicase-dependent amplification (RT-HDA) of isothermal DNA was developed for the detection of tomato spotted wilt virus (TSWV). Specific primers, which were based on the highly conserved region of the N gene sequence in TSWV, were used for the amplification of virus's RNA. The LOD of RT-HDA, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP), and reverse transcriptase-polymerase chain reaction (RT-PCR) assays were conducted using 10-fold serial dilution of RNA eluates. TSWV sensitivity in RT-HDA and RT-LAMP was 4 pg RNA compared with 40 pg RNA in RT-PCR. The specificity of RT-HDA for TSWV was high, showing no cross-reactivity with other tomato and Tospovirus viruses including cucumber mosaic virus (CMV), tomato black ring virus (TBRV), tomato mosaic virus (ToMV), or impatiens necrotic spot virus (INSV). The RT-HDA method is effective for the detection of TSWV in plant samples and is a potential tool for early and rapid detection of TSWV.

  8. Ovation Prime Real-Time

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Ovation Prime Real-Time (OPRT) product is a real-time forecast and nowcast model of auroral power and is an operational implementation of the work by Newell et...

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

    Science.gov (United States)

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

    2015-02-01

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

  10. Development and evaluation of real-time loop-mediated isothermal amplification assay for rapid detection of cystic echinococcosis

    NARCIS (Netherlands)

    Ahmed, Mohamed E.; Eldigail, Mawahib H.; Elamin, Fatima M.; Ali, Ibtisam A.; Grobusch, Martin P.; Aradaib, Imadeldin E.

    2016-01-01

    Cystic echinococcosis (CE) or hydatidosis, caused by the larval stage of Echinococcus granulosus (EG)-complex, is a neglected parasitic disease of public health importance. The disease is endemic in many African and Mediterranean countries including the Sudan. The objective of the present study was

  11. Normalization of Reverse Transcription Quantitative PCR Data During Ageing in Distinct Cerebral Structures.

    Science.gov (United States)

    Bruckert, G; Vivien, D; Docagne, F; Roussel, B D

    2016-04-01

    Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a routine method in many laboratories. Normalization of data from experimental conditions is critical for data processing and is usually achieved by the use of a single reference gene. Nevertheless, as pointed by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, several reference genes should be used for reliable normalization. Ageing is a physiological process that results in a decline of many expressed genes. Reliable normalization of RT-qPCR data becomes crucial when studying ageing. Here, we propose a RT-qPCR study from four mouse brain regions (cortex, hippocampus, striatum and cerebellum) at different ages (from 8 weeks to 22 months) in which we studied the expression of nine commonly used reference genes. With the use of two different algorithms, we found that all brain structures need at least two genes for a good normalization step. We propose specific pairs of gene for efficient data normalization in the four brain regions studied. These results underline the importance of reliable reference genes for specific brain regions in ageing.

  12. Multiplex, Quantitative, Reverse Transcription PCR Detection of Influenza Viruses Using Droplet Microfluidic Technology

    Directory of Open Access Journals (Sweden)

    Ravi Prakash

    2014-12-01

    Full Text Available Quantitative, reverse transcription, polymerase chain reaction (qRT-PCR is facilitated by leveraging droplet microfluidic (DMF system, which due to its precision dispensing and sample handling capabilities at microliter and lower volumes has emerged as a popular method for miniaturization of the PCR platform. This work substantially improves and extends the functional capabilities of our previously demonstrated single qRT-PCR micro-chip, which utilized a combination of electrostatic and electrowetting droplet actuation. In the reported work we illustrate a spatially multiplexed micro-device that is capable of conducting up to eight parallel, real-time PCR reactions per usage, with adjustable control on the PCR thermal cycling parameters (both process time and temperature set-points. This micro-device has been utilized to detect and quantify the presence of two clinically relevant respiratory viruses, Influenza A and Influenza B, in human samples (nasopharyngeal swabs, throat swabs. The device performed accurate detection and quantification of the two respiratory viruses, over several orders of RNA copy counts, in unknown (blind panels of extracted patient samples with acceptably high PCR efficiency (>94%. The multi-stage qRT-PCR assays on eight panel patient samples were accomplished within 35–40 min, with a detection limit for the target Influenza virus RNAs estimated to be less than 10 RNA copies per reaction.

  13. Reverse transcriptase real-time PCR for detection and quantification of viable Campylobacter jejuni directly from poultry faecal samples

    DEFF Research Database (Denmark)

    Bui, Thanh Xuan; Wolff, Anders; Madsen, Mogens

    2012-01-01

    Campylobacter spp. is the most common cause of bacterial diarrhoea in humans worldwide. Therefore, rapid and reliable methods fordetection and quantification of this pathogen are required. In this study, we have developed a reverse transcription quantitative real-time PCR(RT-qPCR) for detection a...

  14. Strand transfer and elongation of HIV-1 reverse transcription is facilitated by cell factors in vitro.

    Directory of Open Access Journals (Sweden)

    David Warrilow

    Full Text Available Recent work suggests a role for multiple host factors in facilitating HIV-1 reverse transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 reverse transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor reverse transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of reverse transcription. Instead, the cell factor(s enhanced first strand transfer and synthesis of late reverse transcription suggesting it stabilized the reverse transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous reverse transcription (ERT system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded reverse transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s suggesting a lack of involvement of the capsid in the in vitro assay. In addition, reverse transcription products were found to be resistant to exogenous DNase I activity when the active fraction was present in the ERT assay. These results indicate that the cell factor(s may improve reverse transcription by facilitating DNA strand transfer and DNA synthesis. It also had a protective function for the reverse transcription products, but it is unclear if this is related to improved DNA synthesis.

  15. Validated reverse transcription droplet digital PCR serves as a higher order method for absolute quantification of Potato virus Y strains.

    Science.gov (United States)

    Mehle, Nataša; Dobnik, David; Ravnikar, Maja; Pompe Novak, Maruša

    2018-05-03

    RNA viruses have a great potential for high genetic variability and rapid evolution that is generated by mutation and recombination under selection pressure. This is also the case of Potato virus Y (PVY), which comprises a high diversity of different recombinant and non-recombinant strains. Consequently, it is hard to develop reverse transcription real-time quantitative PCR (RT-qPCR) with the same amplification efficiencies for all PVY strains which would enable their equilibrate quantification; this is specially needed in mixed infections and other studies of pathogenesis. To achieve this, we initially transferred the PVY universal RT-qPCR assay to a reverse transcription droplet digital PCR (RT-ddPCR) format. RT-ddPCR is an absolute quantification method, where a calibration curve is not needed, and it is less prone to inhibitors. The RT-ddPCR developed and validated in this study achieved a dynamic range of quantification over five orders of magnitude, and in terms of its sensitivity, it was comparable to, or even better than, RT-qPCR. RT-ddPCR showed lower measurement variability. We have shown that RT-ddPCR can be used as a reference tool for the evaluation of different RT-qPCR assays. In addition, it can be used for quantification of RNA based on in-house reference materials that can then be used as calibrators in diagnostic laboratories.

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Fanfan Zhang

    2017-10-01

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

  18. Design and optimization of reverse-transcription quantitative PCR experiments.

    Science.gov (United States)

    Tichopad, Ales; Kitchen, Rob; Riedmaier, Irmgard; Becker, Christiane; Ståhlberg, Anders; Kubista, Mikael

    2009-10-01

    Quantitative PCR (qPCR) is a valuable technique for accurately and reliably profiling and quantifying gene expression. Typically, samples obtained from the organism of study have to be processed via several preparative steps before qPCR. We estimated the errors of sample withdrawal and extraction, reverse transcription (RT), and qPCR that are introduced into measurements of mRNA concentrations. We performed hierarchically arranged experiments with 3 animals, 3 samples, 3 RT reactions, and 3 qPCRs and quantified the expression of several genes in solid tissue, blood, cell culture, and single cells. A nested ANOVA design was used to model the experiments, and relative and absolute errors were calculated with this model for each processing level in the hierarchical design. We found that intersubject differences became easily confounded by sample heterogeneity for single cells and solid tissue. In cell cultures and blood, the noise from the RT and qPCR steps contributed substantially to the overall error because the sampling noise was less pronounced. We recommend the use of sample replicates preferentially to any other replicates when working with solid tissue, cell cultures, and single cells, and we recommend the use of RT replicates when working with blood. We show how an optimal sampling plan can be calculated for a limited budget. .

  19. HIV-1 reverse transcription initiation: a potential target for novel antivirals?

    NARCIS (Netherlands)

    Abbink, Truus E. M.; Berkhout, Ben

    2008-01-01

    Reverse transcription is an essential step in the retroviral life cycle, as it converts the genomic RNA into DNA. In this review, we describe recent developments concerning the initiation step of this complex, multi-step reaction. During initiation of reverse transcription, a cellular tRNA primer is

  20. Dependable Real-Time Systems

    Science.gov (United States)

    1991-09-30

    0196 or 413 545-0720 PI E-mail Address: krithi@nirvan.cs.umass.edu, stankovic(ocs.umass.edu Grant or Contract Title: Dependable Real - Time Systems Grant...Dependable Real - Time Systems " Grant or Contract Number: N00014-85-k-0398 L " Reporting Period: 1 Oct 87 - 30 Sep 91 , 2. Summary of Accomplishments ’ 2.1 Our...in developing a sound approach to scheduling tasks in complex real - time systems , (2) developed a real-time operating system kernel, a preliminary

  1. Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction.

    Science.gov (United States)

    Wilkes, Rebecca P; Tsai, Yun-Long; Lee, Pei-Yu; Lee, Fu-Chun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

    2014-09-09

    Canine distemper virus (CDV) has been associated with outbreaks of canine infectious respiratory disease in shelters and boarding kennel environments. POCKITTM Nucleic Acid Analyzer is a field-deployable device capable of generating automatically interpreted insulated isothermal polymerase chain reaction (iiPCR) results from extracted nucleic acid within one hour. In this study, reverse transcription iiPCR (RT-iiPCR) was developed to facilitate point-of-need diagnosis of CDV infection. Analytical sensitivity (limit of detection 95%) of the established CDV RT-iiPCR was about 11 copies of in vitro transcribed RNA per reaction. CDV RT-iiPCR generated positive signals from CDV, but not Bordetella bronchiseptica, canine parvovirus, canine herpesvirus, canine adenovirus 2, canine influenza virus (subtype H3N8), canine parainfluenza virus, and canine respiratory coronavirus. To evaluate accuracy of the established reaction in canine distemper clinical diagnosis, 110 specimens from dogs, raccoons, and foxes suspected with CDV infection were tested simultaneously by CDV RT-iiPCR and real-time RT-PCR. CDV RT-iiPCR demonstrated excellent sensitivity (100%) and specificity (100%), compared to real-time RT-PCR. The results indicated an excellent correlation between RT-iiPCR and a reference real time RT-PCR method. Working in a lyophilized format, the established method has great potential to be used for point-of-care diagnosis of canine distemper in animals, especially in resource-limited facilities.

  2. The reverse transcription inhibitor abacavir shows anticancer activity in prostate cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Francesca Carlini

    Full Text Available BACKGROUND: Transposable Elements (TEs comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1 and Human Endogenous Retroviruses (HERVs that code for their own endogenous reverse transcriptase (RT. Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC, a nucleoside reverse transcription inhibitor (NRTI, on PC3 and LNCaP prostate cancer cell lines. PRINCIPAL FINDINGS: ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. CONCLUSIONS: Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications.

  3. Real-Time Parameter Identification

    Data.gov (United States)

    National Aeronautics and Space Administration — Armstrong researchers have implemented in the control room a technique for estimating in real time the aerodynamic parameters that describe the stability and control...

  4. Prototyping real-time systems

    OpenAIRE

    Clynch, Gary

    1994-01-01

    The traditional software development paradigm, the waterfall life cycle model, is defective when used for developing real-time systems. This thesis puts forward an executable prototyping approach for the development of real-time systems. A prototyping system is proposed which uses ESML (Extended Systems Modelling Language) as a prototype specification language. The prototyping system advocates the translation of non-executable ESML specifications into executable LOOPN (Language of Object ...

  5. Real-time vision systems

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, R.; Hernandez, J.E.; Lu, Shin-yee [Lawrence Livermore National Lab., CA (United States)

    1994-11-15

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

  6. Reference genes for reverse transcription quantitative PCR in canine brain tissue

    NARCIS (Netherlands)

    Stassen, Quirine E M; Riemers, Frank M; Reijmerink, Hannah; Leegwater, Peter A J; Penning, Louis C

    2015-01-01

    BACKGROUND: In the last decade canine models have been used extensively to study genetic causes of neurological disorders such as epilepsy and Alzheimer's disease and unravel their pathophysiological pathways. Reverse transcription quantitative polymerase chain reaction is a sensitive and

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

    Science.gov (United States)

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

    2013-04-01

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

  8. Towards Real-Time Argumentation

    Directory of Open Access Journals (Sweden)

    Vicente JULIÁN

    2016-07-01

    Full Text Available In this paper, we deal with the problem of real-time coordination with the more general approach of reaching real-time agreements in MAS. Concretely, this work proposes a real-time argumentation framework in an attempt to provide agents with the ability of engaging in argumentative dialogues and come with a solution for their underlying agreement process within a bounded period of time. The framework has been implemented and evaluated in the domain of a customer support application. Concretely, we consider a society of agents that act on behalf of a group of technicians that must solve problems in a Technology Management Centre (TMC within a bounded time. This centre controls every process implicated in the provision of technological and customer support services to private or public organisations by means of a call centre. The contract signed between the TCM and the customer establishes penalties if the specified time is exceeded.

  9. 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.

  10. Real time automatic scene classification

    NARCIS (Netherlands)

    Verbrugge, R.; Israël, Menno; Taatgen, N.; van den Broek, Egon; van der Putten, Peter; Schomaker, L.; den Uyl, Marten J.

    2004-01-01

    This work has been done as part of the EU VICAR (IST) project and the EU SCOFI project (IAP). The aim of the first project was to develop a real time video indexing classification annotation and retrieval system. For our systems, we have adapted the approach of Picard and Minka [3], who categorized

  11. Real time freeway incident detection.

    Science.gov (United States)

    2014-04-01

    The US Department of Transportation (US-DOT) estimates that over half of all congestion : events are caused by highway incidents rather than by rush-hour traffic in big cities. Real-time : incident detection on freeways is an important part of any mo...

  12. Real Time Conference 2016 Overview

    Science.gov (United States)

    Luchetta, Adriano

    2017-06-01

    This is a special issue of the IEEE Transactions on Nuclear Science containing papers from the invited, oral, and poster presentation of the 20th Real Time Conference (RT2016). The conference was held June 6-10, 2016, at Centro Congressi Padova “A. Luciani,” Padova, Italy, and was organized by Consorzio RFX (CNR, ENEA, INFN, Università di Padova, Acciaierie Venete SpA) and the Istituto Nazionale di Fisica Nucleare. The Real Time Conference is multidisciplinary and focuses on the latest developments in real-time techniques in high-energy physics, nuclear physics, astrophysics and astroparticle physics, nuclear fusion, medical physics, space instrumentation, nuclear power instrumentation, general radiation instrumentation, and real-time security and safety. Taking place every second year, it is sponsored by the Computer Application in Nuclear and Plasma Sciences technical committee of the IEEE Nuclear and Plasma Sciences Society. RT2016 attracted more than 240 registrants, with a large proportion of young researchers and engineers. It had an attendance of 67 students from many countries.

  13. Designing Real Time Assistive Technologies

    DEFF Research Database (Denmark)

    Sonne, Tobias; Obel, Carsten; Grønbæk, Kaj

    2015-01-01

    activities and assists the child in maintaining attention. From a preliminary evaluation of CASTT with 20 children in several schools, we and found that: 1) it is possible to create a wearable sensor system for children with ADHD that monitors physical and physiological activities in real time; and that 2...

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

    Science.gov (United States)

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

    2016-10-01

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

  15. A novel CMOS image sensor system for quantitative loop-mediated isothermal amplification assays to detect food-borne pathogens.

    Science.gov (United States)

    Wang, Tiantian; Kim, Sanghyo; An, Jeong Ho

    2017-02-01

    Loop-mediated isothermal amplification (LAMP) is considered as one of the alternatives to the conventional PCR and it is an inexpensive portable diagnostic system with minimal power consumption. The present work describes the application of LAMP in real-time photon detection and quantitative analysis of nucleic acids integrated with a disposable complementary-metal-oxide semiconductor (CMOS) image sensor. This novel system works as an amplification-coupled detection platform, relying on a CMOS image sensor, with the aid of a computerized circuitry controller for the temperature and light sources. The CMOS image sensor captures the light which is passing through the sensor surface and converts into digital units using an analog-to-digital converter (ADC). This new system monitors the real-time photon variation, caused by the color changes during amplification. Escherichia coli O157 was used as a proof-of-concept target for quantitative analysis, and compared with the results for Staphylococcus aureus and Salmonella enterica to confirm the efficiency of the system. The system detected various DNA concentrations of E. coli O157 in a short time (45min), with a detection limit of 10fg/μL. The low-cost, simple, and compact design, with low power consumption, represents a significant advance in the development of a portable, sensitive, user-friendly, real-time, and quantitative analytic tools for point-of-care diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. ISTTOK real-time architecture

    Energy Technology Data Exchange (ETDEWEB)

    Carvalho, Ivo S., E-mail: ivoc@ipfn.ist.utl.pt; Duarte, Paulo; Fernandes, Horácio; Valcárcel, Daniel F.; Carvalho, Pedro J.; Silva, Carlos; Duarte, André S.; Neto, André; Sousa, Jorge; Batista, António J.N.; Hekkert, Tiago; Carvalho, Bernardo B.

    2014-03-15

    Highlights: • All real-time diagnostics and actuators were integrated in the same control platform. • A 100 μs control cycle was achieved under the MARTe framework. • Time-windows based control with several event-driven control strategies implemented. • AC discharges with exception handling on iron core flux saturation. • An HTML discharge configuration was developed for configuring the MARTe system. - Abstract: The ISTTOK tokamak was upgraded with a plasma control system based on the Advanced Telecommunications Computing Architecture (ATCA) standard. This control system was designed to improve the discharge stability and to extend the operational space to the alternate plasma current (AC) discharges as part of the ISTTOK scientific program. In order to accomplish these objectives all ISTTOK diagnostics and actuators relevant for real-time operation were integrated in the control system. The control system was programmed in C++ over the Multi-threaded Application Real-Time executor (MARTe) which provides, among other features, a real-time scheduler, an interrupt handler, an intercommunications interface between code blocks and a clearly bounded interface with the external devices. As a complement to the MARTe framework, the BaseLib2 library provides the foundations for the data, code introspection and also a Hypertext Transfer Protocol (HTTP) server service. Taking advantage of the modular nature of MARTe, the algorithms of each diagnostic data processing, discharge timing, context switch, control and actuators output reference generation, run on well-defined blocks of code named Generic Application Module (GAM). This approach allows reusability of the code, simplified simulation, replacement or editing without changing the remaining GAMs. The ISTTOK control system GAMs run sequentially each 100 μs cycle on an Intel{sup ®} Q8200 4-core processor running at 2.33 GHz located in the ATCA crate. Two boards (inside the ATCA crate) with 32 analog

  17. ISTTOK real-time architecture

    International Nuclear Information System (INIS)

    Carvalho, Ivo S.; Duarte, Paulo; Fernandes, Horácio; Valcárcel, Daniel F.; Carvalho, Pedro J.; Silva, Carlos; Duarte, André S.; Neto, André; Sousa, Jorge; Batista, António J.N.; Hekkert, Tiago; Carvalho, Bernardo B.

    2014-01-01

    Highlights: • All real-time diagnostics and actuators were integrated in the same control platform. • A 100 μs control cycle was achieved under the MARTe framework. • Time-windows based control with several event-driven control strategies implemented. • AC discharges with exception handling on iron core flux saturation. • An HTML discharge configuration was developed for configuring the MARTe system. - Abstract: The ISTTOK tokamak was upgraded with a plasma control system based on the Advanced Telecommunications Computing Architecture (ATCA) standard. This control system was designed to improve the discharge stability and to extend the operational space to the alternate plasma current (AC) discharges as part of the ISTTOK scientific program. In order to accomplish these objectives all ISTTOK diagnostics and actuators relevant for real-time operation were integrated in the control system. The control system was programmed in C++ over the Multi-threaded Application Real-Time executor (MARTe) which provides, among other features, a real-time scheduler, an interrupt handler, an intercommunications interface between code blocks and a clearly bounded interface with the external devices. As a complement to the MARTe framework, the BaseLib2 library provides the foundations for the data, code introspection and also a Hypertext Transfer Protocol (HTTP) server service. Taking advantage of the modular nature of MARTe, the algorithms of each diagnostic data processing, discharge timing, context switch, control and actuators output reference generation, run on well-defined blocks of code named Generic Application Module (GAM). This approach allows reusability of the code, simplified simulation, replacement or editing without changing the remaining GAMs. The ISTTOK control system GAMs run sequentially each 100 μs cycle on an Intel ® Q8200 4-core processor running at 2.33 GHz located in the ATCA crate. Two boards (inside the ATCA crate) with 32 analog

  18. Quantitative (real-time) PCR

    International Nuclear Information System (INIS)

    Denman, S.E.; McSweeney, C.S.

    2005-01-01

    Many nucleic acid-based probe and PCR assays have been developed for the detection tracking of specific microbes within the rumen ecosystem. Conventional PCR assays detect PCR products at the end stage of each PCR reaction, where exponential amplification is no longer being achieved. This approach can result in different end product (amplicon) quantities being generated. In contrast, using quantitative, or real-time PCR, quantification of the amplicon is performed not at the end of the reaction, but rather during exponential amplification, where theoretically each cycle will result in a doubling of product being created. For real-time PCR, the cycle at which fluorescence is deemed to be detectable above the background during the exponential phase is termed the cycle threshold (Ct). The Ct values obtained are then used for quantitation, which will be discussed later

  19. Real time psychrometric data collection

    International Nuclear Information System (INIS)

    McDaniel, K.H.

    1996-01-01

    Eight Mine Weather Stations (MWS) installed at the Waste Isolation Pilot Plant (WIPP) to monitor the underground ventilation system are helping to simulate real-time ventilation scenarios. Seasonal weather extremes can result in variations of Natural Ventilation Pressure (NVP) which can significantly effect the ventilation system. The eight MWS(s) (which previously collected and stored temperature, barometric pressure and relative humidity data for subsequent NVP calculations) were upgraded to provide continuous real-time data to the site wide Central monitoring System. This data can now be utilized by the ventilation engineer to create realtime ventilation simulations and trends which assist in the prediction and mitigation of NVP and psychrometric related events

  20. Real-time holographic endoscopy

    Science.gov (United States)

    Smigielski, Paul; Albe, Felix; Dischli, Bernard

    1992-08-01

    Some new experiments concerning holographic endoscopy are presented. The quantitative measurements of deformations of objects are obtained by the double-exposure and double- reference beam method, using either a cw-laser or a pulsed laser. Qualitative experiments using an argon laser with time-average holographic endoscopy are also presented. A video film on real-time endoscopic holographic interferometry was recorded with the help of a frequency-doubled YAG-laser working at 25 Hz for the first time.

  1. [Real time 3D echocardiography

    Science.gov (United States)

    Bauer, F.; Shiota, T.; Thomas, J. D.

    2001-01-01

    Three-dimensional representation of the heart is an old concern. Usually, 3D reconstruction of the cardiac mass is made by successive acquisition of 2D sections, the spatial localisation and orientation of which require complex guiding systems. More recently, the concept of volumetric acquisition has been introduced. A matricial emitter-receiver probe complex with parallel data processing provides instantaneous of a pyramidal 64 degrees x 64 degrees volume. The image is restituted in real time and is composed of 3 planes (planes B and C) which can be displaced in all spatial directions at any time during acquisition. The flexibility of this system of acquisition allows volume and mass measurement with greater accuracy and reproducibility, limiting inter-observer variability. Free navigation of the planes of investigation allows reconstruction for qualitative and quantitative analysis of valvular heart disease and other pathologies. Although real time 3D echocardiography is ready for clinical usage, some improvements are still necessary to improve its conviviality. Then real time 3D echocardiography could be the essential tool for understanding, diagnosis and management of patients.

  2. 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.

  3. Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Dufva, I.H.; Dufva, Hans Martin

    2006-01-01

    oligonucleotides (pentadecamers) consistently, yielded at least 2 fold as much cDNA as did random hexamers using either-poly(A) RNA or an amplified version of messenger RNA (aRNA) as a template. The cDNA generated using pentadecamers did not differ in size distribution or the amount of incorporated label compared...... with cDNA generated with random hexamers. The increased efficiency of priming using random pentadecamers resulted in reverse transcription of > 80% of the template aRNA, while random hexamers induced reverse transcription of only 40% of the template aRNA. This suggests a better coverage...... that random pentadecamers can replace random hexamers in reverse transcription reactions on both poly(A) RNA and amplified RNA, resulting in higher cDNA yields and quality....

  4. Loop-mediated isothermal amplification (LAMP) based detection of ...

    African Journals Online (AJOL)

    Various diseases are caused by pathogenic bacteria and their diagnosis depends on accurate detection of pathogen from clinical samples. Several molecular methods have been developed including PCR, Real Time PCR or multiplex PCR which detects the pathogen accurately. However, every method has some ...

  5. The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.

    Directory of Open Access Journals (Sweden)

    Clément Monot

    2013-05-01

    Full Text Available L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP first uses its endonuclease (EN to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A tail, a process known as target-primed reverse transcription (TPRT. Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5'-TTTT/A-3' sites and frequently preceded by imperfect T-tracts. However, it is currently unclear whether--and to which degree--the liberated 3'-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3' end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3' overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

  6. The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.

    Science.gov (United States)

    Monot, Clément; Kuciak, Monika; Viollet, Sébastien; Mir, Ashfaq Ali; Gabus, Caroline; Darlix, Jean-Luc; Cristofari, Gaël

    2013-05-01

    L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A) tail, a process known as target-primed reverse transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5'-TTTT/A-3' sites) and frequently preceded by imperfect T-tracts. However, it is currently unclear whether--and to which degree--the liberated 3'-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3' end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3' overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

  7. The real time rolling shutter

    OpenAIRE

    Monaghan, David; O'Connor, Noel E.; Cleary, Anne; Connolly, Denis

    2015-01-01

    From an early age children are often told either, you are creative you should do art but stay away from science and maths. Or that you are mathematical you should do science but you're not that creative. Compounding this there also exist some traditional barriers of artistic rhetoric that say, "don't touch, don't think and don't be creative, we've already done that for you, you can just look...". The Real Time Rolling Shutter is part of a collaborative Art/Science partnership whose core tenet...

  8. Rapid Identification of Dengue Virus by Reverse Transcription-Polymerase Chain Reaction Using Field-Deployable Instrumentation

    National Research Council Canada - National Science Library

    McAvin, James C; Escamilla, Elizabeth M; Blow, James A; Turell, Micahel J; Quintana, Miguel; Bowles, David E; Swaby, James A; Barnes, William J; Huff, William B; Lahman, Kenton L

    2005-01-01

    ...) reverse transcription-polymerase chain reaction assays were developed for screening and seroype identification of infected mosquito vectors and human sera using a field-deployable, fluorometric thermocycler...

  9. Reliable gene expression analysis by reverse transcription-quantitative PCR: reporting and minimizing the uncertainty in data accuracy.

    Science.gov (United States)

    Remans, Tony; Keunen, Els; Bex, Geert Jan; Smeets, Karen; Vangronsveld, Jaco; Cuypers, Ann

    2014-10-01

    Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data. © 2014 American Society of Plant Biologists. All rights reserved.

  10. Candidate gene biodosimeters of mice and human exposure to ionizing radiation by quantitative reverse transcription polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Hamed Rezaeejam

    2015-01-01

    Full Text Available Understanding of cellular responses to ionizing radiation (IR is essential for the development of predictive markers useful for assessing human exposure. Biological markers of exposure to IR in human populations are of great interest for assessing normal tissue injury in radiation oncology and for biodosimetry in nuclear incidents and accidental radiation exposures. Traditional radiation exposure biomarkers based on cytogenetic assays (biodosimetry, are time-consuming and do not provide results fast enough and requires highly trained personnel for scoring. Hence, the development of rapid biodosimetry methods is one of the highest priorities. Exposure of cells to IR activates multiple signal transduction pathways, which result in complex alterations in gene-expression. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR has become the benchmark for the detection and quantification of RNA targets and is being utilized increasingly in monitoring the specific genes with more accurately and sensitively. This review evaluates the RT-qPCR as a biodosimetry method and we investigated the papers from 2000 up to now, which identified the genes-expression related the DNA repair, cell cycle checkpoint, and apoptosis induced by ionization radiation in peripheral blood and determined as biodosimeters. In conclusion, it could be say that RT-qPCR technique for determining the specific genes as biodosimeters could be a fully quantitative reliable and sensitive method. Furthermore, the results of the current review will help the researchers to recognize the most expressed genes induced by ionization radiation.

  11. Real time analysis under EDS

    International Nuclear Information System (INIS)

    Schneberk, D.

    1985-07-01

    This paper describes the analysis component of the Enrichment Diagnostic System (EDS) developed for the Atomic Vapor Laser Isotope Separation Program (AVLIS) at Lawrence Livermore National Laboratory (LLNL). Four different types of analysis are performed on data acquired through EDS: (1) absorption spectroscopy on laser-generated spectral lines, (2) mass spectrometer analysis, (3) general purpose waveform analysis, and (4) separation performance calculations. The information produced from this data includes: measures of particle density and velocity, partial pressures of residual gases, and overall measures of isotope enrichment. The analysis component supports a variety of real-time modeling tasks, a means for broadcasting data to other nodes, and a great degree of flexibility for tailoring computations to the exact needs of the process. A particular data base structure and program flow is common to all types of analysis. Key elements of the analysis component are: (1) a fast access data base which can configure all types of analysis, (2) a selected set of analysis routines, (3) a general purpose data manipulation and graphics package for the results of real time analysis. Each of these components are described with an emphasis upon how each contributes to overall system capability. 3 figs

  12. Structural features in the HIV-1 repeat region facilitate strand transfer during reverse transcription

    NARCIS (Netherlands)

    Berkhout, B.; Vastenhouw, N. L.; Klasens, B. I.; Huthoff, H.

    2001-01-01

    Two obligatory DNA strand transfers take place during reverse transcription of a retroviral RNA genome. The first strand transfer is facilitated by terminal repeat (R) elements in the viral genome. This strand-transfer reaction depends on base pairing between the cDNA of the 5'R and the 3'R. There

  13. Autonomous Real Time Requirements Tracing

    Science.gov (United States)

    Plattsmier, George; Stetson, Howard

    2014-01-01

    One of the more challenging aspects of software development is the ability to verify and validate the functional software requirements dictated by the Software Requirements Specification (SRS) and the Software Detail Design (SDD). Insuring the software has achieved the intended requirements is the responsibility of the Software Quality team and the Software Test team. The utilization of Timeliner-TLX(sup TM) Auto- Procedures for relocating ground operations positions to ISS automated on-board operations has begun the transition that would be required for manned deep space missions with minimal crew requirements. This transition also moves the auto-procedures from the procedure realm into the flight software arena and as such the operational requirements and testing will be more structured and rigorous. The autoprocedures would be required to meet NASA software standards as specified in the Software Safety Standard (NASASTD- 8719), the Software Engineering Requirements (NPR 7150), the Software Assurance Standard (NASA-STD-8739) and also the Human Rating Requirements (NPR-8705). The Autonomous Fluid Transfer System (AFTS) test-bed utilizes the Timeliner-TLX(sup TM) Language for development of autonomous command and control software. The Timeliner-TLX(sup TM) system has the unique feature of providing the current line of the statement in execution during real-time execution of the software. The feature of execution line number internal reporting unlocks the capability of monitoring the execution autonomously by use of a companion Timeliner-TLX(sup TM) sequence as the line number reporting is embedded inside the Timeliner-TLX(sup TM) execution engine. This negates I/O processing of this type data as the line number status of executing sequences is built-in as a function reference. This paper will outline the design and capabilities of the AFTS Autonomous Requirements Tracker, which traces and logs SRS requirements as they are being met during real-time execution of the

  14. Inter-laboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction methods used for detection of infectious pancreatic necrosis virus in Chile

    Directory of Open Access Journals (Sweden)

    David Tapia

    2017-07-01

    Conclusions: Overall, the ring trial showed high values of sensitivity and specificity, with some problems of repeatability and inter-laboratory variability. This last issue needs to be addressed in order to allow harmonized diagnostic of IPNV within the country. We recommend the use of the NRL methods as validated and reliable qRT-PCR protocols for the detection of IPNV.

  15. Evaluation of reference genes for reverse transcription quantitative real-time PCR (RT-qPCR) studies in Silene vulgaris considering the method of cDNA preparation

    Czech Academy of Sciences Publication Activity Database

    Koloušková, Pavla; Stone, James D.; Štorchová, Helena

    2017-01-01

    Roč. 12, č. 8 (2017), č. článku e0183470. E-ISSN 1932-6203 R&D Projects: GA MŠk(CZ) LH15075; GA ČR(CZ) GA16-09220S Institutional support: RVO:61389030 Keywords : CYTOPLASMIC MALE -STERILITY * SUITABLE REFERENCE GENES * INTERNAL CONTROL GENES Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Plant sciences, botany Impact factor: 2.806, year: 2016

  16. Mobile real time radiography system

    Energy Technology Data Exchange (ETDEWEB)

    Vigil, J.; Taggart, D.; Betts, S. [Los Alamos National Lab., NM (United States)] [and others

    1997-11-01

    A 450-keV Mobile Real Time Radiography (RTR) System was delivered to Los Alamos National Laboratory (LANL) in January 1996. It was purchased to inspect containers of radioactive waste produced at (LANL). Since its delivery it has been used to radiograph more than 600 drums of radioactive waste at various LANL sites. It has the capability of inspecting waste containers of various sizes from <1-gal. buckets up to standard waste boxes (SWB, dimensions 54.5 in. x 71 in. x 37 in.). It has three independent x-ray acquisition formats. The primary system used is a 12- in. image intensifier, the second is a 36-in. linear diode array (LDA) and the last is an open system. It is fully self contained with on board generator, HVAC, and a fire suppression system. It is on a 53-ft long x 8-ft. wide x 14-ft. high trailer that can be moved over any highway requiring only an easily obtainable overweight permit because it weights {approximately}38 tons. It was built to conform to industry standards for a cabinet system which does not require an exclusion zone. The fact that this unit is mobile has allowed us to operate where the waste is stored, rather than having to move the waste to a fixed facility.

  17. Mobile real time radiography system

    International Nuclear Information System (INIS)

    Vigil, J.; Taggart, D.; Betts, S.

    1997-01-01

    A 450-keV Mobile Real Time Radiography (RTR) System was delivered to Los Alamos National Laboratory (LANL) in January 1996. It was purchased to inspect containers of radioactive waste produced at (LANL). Since its delivery it has been used to radiograph more than 600 drums of radioactive waste at various LANL sites. It has the capability of inspecting waste containers of various sizes from <1-gal. buckets up to standard waste boxes (SWB, dimensions 54.5 in. x 71 in. x 37 in.). It has three independent x-ray acquisition formats. The primary system used is a 12- in. image intensifier, the second is a 36-in. linear diode array (LDA) and the last is an open system. It is fully self contained with on board generator, HVAC, and a fire suppression system. It is on a 53-ft long x 8-ft. wide x 14-ft. high trailer that can be moved over any highway requiring only an easily obtainable overweight permit because it weights ∼38 tons. It was built to conform to industry standards for a cabinet system which does not require an exclusion zone. The fact that this unit is mobile has allowed us to operate where the waste is stored, rather than having to move the waste to a fixed facility

  18. Real time 3D photometry

    Science.gov (United States)

    Fernandez-Balbuena, A. A.; Vazquez-Molini, D.; García-Botella, A.; Romo, J.; Serrano, Ana

    2017-09-01

    The photometry and radiometry measurement is a well-developed field. The necessity of measuring optical systems performance involves the use of several techniques like Gonio-photometry. The Gonio photometers are a precise measurement tool that is used in the lighting area like office, luminaire head car lighting, concentrator /collimator measurement and all the designed and fabricated optical systems that works with light. There is one disadvantage in this kind of measurements that obtain the intensity polar curves and the total flux of the optical system. In the industry, there are good Gonio photometers that are precise and reliable but they are very expensive and the measurement time is long. In industry the cost can be of minor importance but measuring time that is around 30 minutes is of major importance due to trained staff cost. We have designed a system to measure photometry in real time; it consists in a curved screen to get a huge measurement angle and a CCD. The system to be measured projects light onto the screen and the CCD records a video of the screen obtaining an image of the projected profile. A complex calibration permits to trace screen data (x,y,z) to intensity polar curve (I,αγ). This intensity is obtained in candels (cd) with an image + processing time below one second.

  19. A real time monitoring system

    International Nuclear Information System (INIS)

    Fontanini, Horacio; Galdoz, Erwin

    1989-01-01

    A real time monitoring system is described. It was initially developed to be used as a man-machine interface between a basic principles simulator of the Embalse Nuclear Power Plant and the operators. This simulator is under construction at the Bariloche Atomic Center's Process Control Division. Due to great design flexibility, this system can also be used in real plants. The system is designed to be run on a PC XT or AT personal computer with high resolution graphics capabilities. Three interrelated programs compose the system: 1) Graphics Editor, to build static image to be used as a reference frame where to show dynamically updated data. 2) Data acquisition and storage module. It is a memory resident module to acquire and store data in background. Data can be acquired and stored without interference with the operating system, via serial port or through analog-to-digital converter attached to the personal computer. 3) Display module. It shows the acquired data according to commands received from configuration files prepared by the operator. (Author) [es

  20. Scalable Real-Time Negotiation Toolkit

    National Research Council Canada - National Science Library

    Lesser, Victor

    2004-01-01

    ... to implement an adaptive distributed sensor network. These activities involved the development of a distributed soft, real-time heuristic resource allocation protocol, the development of a domain-independent soft, real time agent architecture...

  1. Hard Real-Time Networking on Firewire

    NARCIS (Netherlands)

    Zhang, Yuchen; Orlic, Bojan; Visser, Peter; Broenink, Jan

    2005-01-01

    This paper investigates the possibility of using standard, low-cost, widely used FireWire as a new generation fieldbus medium for real-time distributed control applications. A real-time software subsys- tem, RT-FireWire was designed that can, in combination with Linux-based real-time operating

  2. Modular specification of real-time systems

    DEFF Research Database (Denmark)

    Inal, Recep

    1994-01-01

    Duration Calculus, a real-time interval logic, has been embedded in the Z specification language to provide a notation for real-time systems that combines the modularisation and abstraction facilities of Z with a logic suitable for reasoning about real-time properties. In this article the notation...

  3. Students Collecting Real time Data

    Science.gov (United States)

    Miller, P.

    2006-05-01

    Students Collecting Real-Time Data The Hawaiian Islands Humpback Whale National Marine Sanctuary has created opportunities for middle and high school students to become Student Researchers and to be involved in real-time marine data collection. It is important that we expose students to different fields of science and encourage them to enter scientific fields of study. The Humpback Whale Sanctuary has an education visitor center in Kihei, Maui. Located right on the beach, the site has become a living classroom facility. There is a traditional Hawaiian fishpond fronting the property. The fishpond wall is being restored, using traditional methods. The site has the incredible opportunity of incorporating Hawaiian cultural practices with scientific studies. The Sanctuary offers opportunities for students to get involved in monitoring and data collection studies. Invasive Seaweed Study: Students are collecting data on invasive seaweed for the University of Hawaii. They pull a large net through the shallow waters. Seaweed is sorted, identified and weighed. The invasive seaweeds are removed. The data is recorded and sent to UH. Remote controlled monitoring boats: The sanctuary has 6 boogie board sized remote controlled boats used to monitor reefs. Boats have a camera with lights on the underside. The boats have water quality monitoring devices and GPS units. The video from the underwater camera is transmitted via a wireless transmission. Students are able to monitor the fish, limu and invertebrate populations on the reef and collect water quality data via television monitors or computers. The boat can also pull a small plankton tow net. Data is being compiled into data bases. Artificial Reef Modules: The Sanctuary has a scientific permit from the state to build and deploy artificial reef modules. High school students are designing and building modules. These are deployed out in the Fishpond fronting the Sanctuary site and students are monitoring them on a weekly basis

  4. 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

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

    Science.gov (United States)

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

    2018-01-02

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

  6. Identification of valid reference genes for gene expression studies of human stomach cancer by reverse transcription-qPCR

    Directory of Open Access Journals (Sweden)

    Lee Yeon-Su

    2010-05-01

    Full Text Available Abstract Background Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR is a powerful method for the analysis of gene expression. Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample. However, much effort has not been expended thus far in the search for reference genes suitable for the study of stomach cancer using RT-qPCR, although selection of optimal reference genes is critical for interpretation of results. Methods We assessed the suitability of six possible reference genes, beta-actin (ACTB, glyceraldehydes-3-phosphate dehydrogenase (GAPDH, hypoxanthine phosphoribosyl transferase 1 (HPRT1, beta-2-microglobulin (B2M, ribosomal subunit L29 (RPL29 and 18S ribosomal RNA (18S rRNA in 20 normal and tumor stomach tissue pairs of stomach cancer patients and 6 stomach cancer cell lines, by RT-qPCR. Employing expression stability analyses using NormFinder and geNorm algorithms we determined the order of performance of these reference genes and their variation values. Results This RT-qPCR study showed that there are statistically significant (p Conclusion This study validated RPL29 and RPL29-B2M as the best single reference genes and combination, for RT-qPCR analysis of 'all stomach tissues', and B2M and B2M-GAPDH as the best single reference gene and combination, for 'stomach cancer cell lines'. Use of these validated reference genes should provide more exact interpretation of differential gene expressions at transcription level in stomach cancer.

  7. Microchip capillary electrophoresis with laser-induced fluorescence combined with one-step duplex reverse-transcription polymerase chain reaction for the rapid detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens.

    Science.gov (United States)

    Jia, Ruan; Chengjun, Sun; Heng, Chen; Chen, Zhou; Yuanqian, Li; Yongxin, Li

    2015-07-01

    Enterovirus 71 and Coxsackievirus A16 are the main pathogens causing hand-foot-mouth disease. In this paper, microchip capillary electrophoresis with laser-induced fluorescence combined with one-step duplex reverse transcript-polymerase chain reaction has been developed for the detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens. The specific reverse transcription-polymerase chain reaction amplicons labeled with SYBR Orange were separated by microchip capillary electrophoresis and detected by laser induced fluorescence detector within 7 min. The intraday and interday relative standard deviation of migration time for DNA Marker was in the range of 1.36-2.94 and 2.78-3.96%, respectively. The detection limits were as low as 2.06 × 10(3) copies/mL for Enterovirus 71 and 5 × 10(3) copies/mL for Coxsackievirus A16. No cross-reactivity was observed with rotavirus, astrovirus, norovirus, and adenovirus, which showed good specificity of the method. This assay was validated using 100 throat swab specimens that were detected by real-time reverse-transcript polymerase chain reaction in parallel and the two methods produced the same results. This study provided a rapid, sensitive and specific method for the detection of Enterovirus 71 and Coxsackievirus A16, which make a contribution to significant time and cost saving for the identification and treatment of patients. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. VERSE - Virtual Equivalent Real-time Simulation

    Science.gov (United States)

    Zheng, Yang; Martin, Bryan J.; Villaume, Nathaniel

    2005-01-01

    Distributed real-time simulations provide important timing validation and hardware in the- loop results for the spacecraft flight software development cycle. Occasionally, the need for higher fidelity modeling and more comprehensive debugging capabilities - combined with a limited amount of computational resources - calls for a non real-time simulation environment that mimics the real-time environment. By creating a non real-time environment that accommodates simulations and flight software designed for a multi-CPU real-time system, we can save development time, cut mission costs, and reduce the likelihood of errors. This paper presents such a solution: Virtual Equivalent Real-time Simulation Environment (VERSE). VERSE turns the real-time operating system RTAI (Real-time Application Interface) into an event driven simulator that runs in virtual real time. Designed to keep the original RTAI architecture as intact as possible, and therefore inheriting RTAI's many capabilities, VERSE was implemented with remarkably little change to the RTAI source code. This small footprint together with use of the same API allows users to easily run the same application in both real-time and virtual time environments. VERSE has been used to build a workstation testbed for NASA's Space Interferometry Mission (SIM PlanetQuest) instrument flight software. With its flexible simulation controls and inexpensive setup and replication costs, VERSE will become an invaluable tool in future mission development.

  9. 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.

  10. 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.

  11. Space Weather and Real-Time Monitoring

    Directory of Open Access Journals (Sweden)

    S Watari

    2009-04-01

    Full Text Available Recent advance of information and communications technology enables to collect a large amount of ground-based and space-based observation data in real-time. The real-time data realize nowcast of space weather. This paper reports a history of space weather by the International Space Environment Service (ISES in association with the International Geophysical Year (IGY and importance of real-time monitoring in space weather.

  12. Archtecture of distributed real-time systems

    OpenAIRE

    Wing Leung, Cheuk

    2013-01-01

    CRAFTERS (Constraint and Application Driven Framework for Tailoring Embedded Real-time System) project aims to address the problem of uncertainty and heterogeneity in a distributed system by providing seamless, portable connectivity and middleware. This thesis contributes to the project by investigating the techniques that can be used in a distributed real-time embedded system. The conclusion is that, there is a list of specifications to be meet in order to provide a transparent and real-time...

  13. Research Directions in Real-Time Systems.

    Science.gov (United States)

    1996-09-01

    This report summarizes a survey of published research in real time systems . Material is presented that provides an overview of the topic, focusing on...communications protocols and scheduling techniques. It is noted that real - time systems deserve special attention separate from other areas because of...formal tools for design and analysis of real - time systems . The early work on applications as well as notable theoretical advances are summarized

  14. A Real-Time Systems Symposium Preprint.

    Science.gov (United States)

    1983-09-01

    Real - Time Systems Symposium Preprint Interim Tech...estimate of the occurence of the error. Unclassii ledSECUqITY CLASSIF’ICA T" NO MI*IA If’ inDI /’rrd erter for~~ble. ’Corrputnqg A REAL - TIME SYSTEMS SYMPOSIUM...ABSTRACT This technical report contains a preprint of a paper accepted for presentation at the REAL - TIME SYSTEMS SYMPOSIUM, Arlington,

  15. Essays in real-time forecasting

    OpenAIRE

    Liebermann, Joelle

    2012-01-01

    This thesis contains three essays in the field of real-time econometrics, and more particularlyforecasting.The issue of using data as available in real-time to forecasters, policymakers or financialmarkets is an important one which has only recently been taken on board in the empiricalliterature. Data available and used in real-time are preliminary and differ from ex-postrevised data, and given that data revisions may be quite substantial, the use of latestavailable instead of real-time can s...

  16. Research in Distributed Real-Time Systems

    Science.gov (United States)

    Mukkamala, R.

    1997-01-01

    This document summarizes the progress we have made on our study of issues concerning the schedulability of real-time systems. Our study has produced several results in the scalability issues of distributed real-time systems. In particular, we have used our techniques to resolve schedulability issues in distributed systems with end-to-end requirements. During the next year (1997-98), we propose to extend the current work to address the modeling and workload characterization issues in distributed real-time systems. In particular, we propose to investigate the effect of different workload models and component models on the design and the subsequent performance of distributed real-time systems.

  17. Real-time Pricing in Power Markets

    DEFF Research Database (Denmark)

    Boom, Anette; Schwenen, Sebastian

    We examine welfare e ects of real-time pricing in electricity markets. Before stochastic energy demand is known, competitive retailers contract with nal consumers who exogenously do not have real-time meters. After demand is realized, two electricity generators compete in a uniform price auction...... to satisfy demand from retailers acting on behalf of subscribed customers and from consumers with real-time meters. Increasing the number of consumers on real-time pricing does not always increase welfare since risk-averse consumers dislike uncertain and high prices arising through market power...

  18. Real-time Pricing in Power Markets

    DEFF Research Database (Denmark)

    Boom, Anette; Schwenen, Sebastian

    We examine welfare eects of real-time pricing in electricity markets. Before stochastic energy demand is known, competitive retailers contract with nal consumers who exogenously do not have real-time meters. After demand is realized, two electricity generators compete in a uniform price auction...... to satisfy demand from retailers acting on behalf of subscribed customers and from consumers with real-time meters. Increasing the number of consumers on real-time pricing does not always increase welfare since risk-averse consumers dislike uncertain and high prices arising through market power...

  19. 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

    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...... 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...... and usually take a few hours to days to complete. In response to the demand for rapid on line or at site detection of pathogens, in this study, we describe for the first time an eight-chamber lab-on-a-chip (LOC) system with integrated magnetic beads-based sample preparation and loop-mediated isothermal...

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

    Science.gov (United States)

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

    2017-01-01

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

  1. Sequence-specific inhibition of duck hepatitis B virus reverse transcription by peptide nucleic acids (PNA)

    DEFF Research Database (Denmark)

    Robaczewska, Magdalena; Narayan, Ramamurthy; Seigneres, Beatrice

    2005-01-01

    BACKGROUND/AIMS: Peptide nucleic acids (PNAs) appear as promising new antisense agents, that have not yet been examined as hepatitis B virus (HBV) inhibitors. Our aim was to study the ability of PNAs targeting the duck HBV (DHBV) encapsidation signal epsilon to inhibit reverse transcription (RT...... in primary duck hepatocytes (PDH). RESULTS: Both PNAs reproducibly inhibited DHBV RT in a dose-dependent manner with IC(50) of 10nM, whereas up to 600-fold higher concentration of S-ODNs was required for similar inhibition. The PNA targeting the bulge and upper stem of epsilon appeared as more efficient RT...

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

    NARCIS (Netherlands)

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

    2016-01-01

    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,

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

    NARCIS (Netherlands)

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

    2018-01-01

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

  4. Real-time communication protocols: an overview

    NARCIS (Netherlands)

    Hanssen, F.T.Y.; Jansen, P.G.

    2003-01-01

    This paper describes several existing data link layer protocols that provide real-time capabilities on wired networks, focusing on token-ring and Carrier Sense Multiple Access based networks. Existing modifications to provide better real-time capabilities and performance are also described. Finally

  5. Towards exascale real-time RFI mitigation

    NARCIS (Netherlands)

    van Nieuwpoort, R.V.

    2016-01-01

    We describe the design and implementation of an extremely scalable real-time RFI mitigation method, based on the offline AOFlagger. All algorithms scale linearly in the number of samples. We describe how we implemented the flagger in the LOFAR real-time pipeline, on both CPUs and GPUs. Additionally,

  6. Real time refractive index measurement by ESPI

    International Nuclear Information System (INIS)

    Torroba, R.; Joenathan, C.

    1991-01-01

    In this paper a method to measure refractive index variations in real time is reported. A technique to introduce reference fringes in real time is discussed. Both the theoretical and experimental results are presented and an example with phase shifting is given. (author). 8 refs, 5 figs

  7. De toekomst van Real Time Intelligence

    NARCIS (Netherlands)

    Broek, J. van den; Berg, C.H. van den

    2013-01-01

    Al direct vanaf de start van de Nationale Politie is gewerkt aan het opzetten van tien real-time intelligence centra in Nederland. Van daaruit worden 24 uur per dag en zeven dagen in de week agenten op straat actief ondersteund met real-time informatie bij de melding waar ze op af gaan. In de visie

  8. Heterogeneous Embedded Real-Time Systems Environment

    Science.gov (United States)

    2003-12-01

    AFRL-IF-RS-TR-2003-290 Final Technical Report December 2003 HETEROGENEOUS EMBEDDED REAL - TIME SYSTEMS ENVIRONMENT Integrated...HETEROGENEOUS EMBEDDED REAL - TIME SYSTEMS ENVIRONMENT 6. AUTHOR(S) Cosmo Castellano and James Graham 5. FUNDING NUMBERS C - F30602-97-C-0259

  9. Validation of RNAi by real time PCR

    DEFF Research Database (Denmark)

    Josefsen, Knud; Lee, Ying Chiu

    2011-01-01

    Real time PCR is the analytic tool of choice for quantification of gene expression, while RNAi is concerned with downregulation of gene expression. Together, they constitute a powerful approach in any loss of function studies of selective genes. We illustrate here the use of real time PCR to verify...

  10. Real time programming environment for Windows

    Energy Technology Data Exchange (ETDEWEB)

    LaBelle, D.R. [LaBelle (Dennis R.), Clifton Park, NY (United States)

    1998-04-01

    This document provides a description of the Real Time Programming Environment (RTProE). RTProE tools allow a programmer to create soft real time projects under general, multi-purpose operating systems. The basic features necessary for real time applications are provided by RTProE, leaving the programmer free to concentrate efforts on his specific project. The current version supports Microsoft Windows{trademark} 95 and NT. The tasks of real time synchronization and communication with other programs are handled by RTProE. RTProE includes a generic method for connecting a graphical user interface (GUI) to allow real time control and interaction with the programmer`s product. Topics covered in this paper include real time performance issues, portability, details of shared memory management, code scheduling, application control, Operating System specific concerns and the use of Computer Aided Software Engineering (CASE) tools. The development of RTProE is an important step in the expansion of the real time programming community. The financial costs associated with using the system are minimal. All source code for RTProE has been made publicly available. Any person with access to a personal computer, Windows 95 or NT, and C or FORTRAN compilers can quickly enter the world of real time modeling and simulation.

  11. Storm real-time processing cookbook

    CERN Document Server

    Anderson, Quinton

    2013-01-01

    A Cookbook with plenty of practical recipes for different uses of Storm.If you are a Java developer with basic knowledge of real-time processing and would like to learn Storm to process unbounded streams of data in real time, then this book is for you.

  12. LabVIEW Real-Time

    CERN Multimedia

    CERN. Geneva; Flockhart, Ronald Bruce; Seppey, P

    2003-01-01

    With LabVIEW Real-Time, you can choose from a variety of RT Series hardware. Add a real-time data acquisition component into a larger measurement and automation system or create a single stand-alone real-time solution with data acquisition, signal conditioning, motion control, RS-232, GPIB instrumentation, and Ethernet connectivity. With the various hardware options, you can create a system to meet your precise needs today, while the modularity of the system means you can add to the solution as your system requirements grow. If you are interested in Reliable and Deterministic systems for Measurement and Automation, you will profit from this seminar. Agenda: Real-Time Overview LabVIEW RT Hardware Platforms - Linux on PXI Programming with LabVIEW RT Real-Time Operating Systems concepts Timing Applications Data Transfer

  13. MARTe: A Multiplatform Real-Time Framework

    Science.gov (United States)

    Neto, André C.; Sartori, Filippo; Piccolo, Fabio; Vitelli, Riccardo; De Tommasi, Gianmaria; Zabeo, Luca; Barbalace, Antonio; Fernandes, Horacio; Valcarcel, Daniel F.; Batista, Antonio J. N.

    2010-04-01

    Development of real-time applications is usually associated with nonportable code targeted at specific real-time operating systems. The boundary between hardware drivers, system services, and user code is commonly not well defined, making the development in the target host significantly difficult. The Multithreaded Application Real-Time executor (MARTe) is a framework built over a multiplatform library that allows the execution of the same code in different operating systems. The framework provides the high-level interfaces with hardware, external configuration programs, and user interfaces, assuring at the same time hard real-time performances. End-users of the framework are required to define and implement algorithms inside a well-defined block of software, named Generic Application Module (GAM), that is executed by the real-time scheduler. Each GAM is reconfigurable with a set of predefined configuration meta-parameters and interchanges information using a set of data pipes that are provided as inputs and required as output. Using these connections, different GAMs can be chained either in series or parallel. GAMs can be developed and debugged in a non-real-time system and, only once the robustness of the code and correctness of the algorithm are verified, deployed to the real-time system. The software also supplies a large set of utilities that greatly ease the interaction and debugging of a running system. Among the most useful are a highly efficient real-time logger, HTTP introspection of real-time objects, and HTTP remote configuration. MARTe is currently being used to successfully drive the plasma vertical stabilization controller on the largest magnetic confinement fusion device in the world, with a control loop cycle of 50 ?s and a jitter under 1 ?s. In this particular project, MARTe is used with the Real-Time Application Interface (RTAI)/Linux operating system exploiting the new ?86 multicore processors technology.

  14. Detection of ROS1 Gene Rearrangement in Lung Adenocarcinoma: Comparison of IHC, FISH and Real-Time RT-PCR

    OpenAIRE

    Shan, Ling; Lian, Fang; Guo, Lei; Qiu, Tian; Ling, Yun; Ying, Jianming; Lin, Dongmei

    2015-01-01

    Aims To compare fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and quantitative real-time reverse transcription-PCR (qRT-PCR) assays for detection of ROS1 fusion in a large number of ROS1-positive lung adenocatcinoma (ADC) patients. Methods Using IHC analysis, sixty lung ADCs including 16 cases with ROS1 protein expression and 44 cases without ROS1 expression were selected for this study. The ROS1 fusion status was examined by FISH and qRT-PCR assay. Results Among 60 ca...

  15. Research of real-time communication software

    Science.gov (United States)

    Li, Maotang; Guo, Jingbo; Liu, Yuzhong; Li, Jiahong

    2003-11-01

    Real-time communication has been playing an increasingly important role in our work, life and ocean monitor. With the rapid progress of computer and communication technique as well as the miniaturization of communication system, it is needed to develop the adaptable and reliable real-time communication software in the ocean monitor system. This paper involves the real-time communication software research based on the point-to-point satellite intercommunication system. The object-oriented design method is adopted, which can transmit and receive video data and audio data as well as engineering data by satellite channel. In the real-time communication software, some software modules are developed, which can realize the point-to-point satellite intercommunication in the ocean monitor system. There are three advantages for the real-time communication software. One is that the real-time communication software increases the reliability of the point-to-point satellite intercommunication system working. Second is that some optional parameters are intercalated, which greatly increases the flexibility of the system working. Third is that some hardware is substituted by the real-time communication software, which not only decrease the expense of the system and promotes the miniaturization of communication system, but also aggrandizes the agility of the system.

  16. Making real-time reactive systems reliable

    Science.gov (United States)

    Marzullo, Keith; Wood, Mark

    1990-01-01

    A reactive system is characterized by a control program that interacts with an environment (or controlled program). The control program monitors the environment and reacts to significant events by sending commands to the environment. This structure is quite general. Not only are most embedded real time systems reactive systems, but so are monitoring and debugging systems and distributed application management systems. Since reactive systems are usually long running and may control physical equipment, fault tolerance is vital. The research tries to understand the principal issues of fault tolerance in real time reactive systems and to build tools that allow a programmer to design reliable, real time reactive systems. In order to make real time reactive systems reliable, several issues must be addressed: (1) How can a control program be built to tolerate failures of sensors and actuators. To achieve this, a methodology was developed for transforming a control program that references physical value into one that tolerates sensors that can fail and can return inaccurate values; (2) How can the real time reactive system be built to tolerate failures of the control program. Towards this goal, whether the techniques presented can be extended to real time reactive systems is investigated; and (3) How can the environment be specified in a way that is useful for writing a control program. Towards this goal, whether a system with real time constraints can be expressed as an equivalent system without such constraints is also investigated.

  17. Real time sensor for therapeutic radiation delivery

    International Nuclear Information System (INIS)

    Bliss, M.; Craig, R.A.; Reeder, P.L.

    1998-01-01

    The invention is a real time sensor for therapeutic radiation. A probe is placed in or near the patient that senses in real time the dose at the location of the probe. The strength of the dose is determined by either an insertion or an exit probe. The location is determined by a series of vertical and horizontal sensing elements that gives the operator a real time read out dose location relative to placement of the patient. The increased accuracy prevents serious tissue damage to the patient by preventing overdose or delivery of a dose to a wrong location within the body. 14 figs

  18. Real time detecting system for turning force

    Energy Technology Data Exchange (ETDEWEB)

    Xiaobin, Yue [China Academy of Engineering Physics, Mianyang (China). Inst. of Machinery Manufacturing Technology

    2001-07-01

    How to get the real-time value of forces dropped on the tool in the course of processing by piezoelectric sensors is introduced. First, the analog signals of the cutting force were achieved by these sensors, amplified and transferred into digital signals by A/D transferring card. Then real-time software reads the information, put it into its own coordinate, drew the curve of forces, displayed it on the screen by the real time and saved it for the technicians to analyze the situation of the tool. So the cutting parameter can be optimized to improve surface quality of the pieces.

  19. Multiprocessor scheduling for real-time systems

    CERN Document Server

    Baruah, Sanjoy; Buttazzo, Giorgio

    2015-01-01

    This book provides a comprehensive overview of both theoretical and pragmatic aspects of resource-allocation and scheduling in multiprocessor and multicore hard-real-time systems.  The authors derive new, abstract models of real-time tasks that capture accurately the salient features of real application systems that are to be implemented on multiprocessor platforms, and identify rules for mapping application systems onto the most appropriate models.  New run-time multiprocessor scheduling algorithms are presented, which are demonstrably better than those currently used, both in terms of run-time efficiency and tractability of off-line analysis.  Readers will benefit from a new design and analysis framework for multiprocessor real-time systems, which will translate into a significantly enhanced ability to provide formally verified, safety-critical real-time systems at a significantly lower cost.

  20. Coordinating Transit Transfers in Real Time

    Science.gov (United States)

    2016-05-06

    Transfers are a major source of travel time variability for transit passengers. Coordinating transfers between transit routes in real time can reduce passenger waiting times and travel time variability, but these benefits need to be contrasted with t...

  1. Real-Time Penetrating Particle Analyzer (PAN)

    Science.gov (United States)

    Wu, X.; Ambrosi, G.; Bertucci, B.

    2018-02-01

    The PAN can measure penetrating particles with great precision to study energetic particles, solar activities, and the origin and propagation of cosmic rays. The real-time monitoring of penetrating particles is crucial for deep space human travel.

  2. Frequent dual initiation of reverse transcription in murine leukemia virus-based vectors containing two primer-binding sites

    International Nuclear Information System (INIS)

    Voronin, Yegor A.; Pathak, Vinay K.

    2003-01-01

    Retroviruses package two copies of viral RNA into each virion. Although each RNA contains a primer-binding site for initiation of DNA synthesis, it is unknown whether reverse transcription is initiated on both RNAs. To determine whether a single virion is capable of initiating reverse transcription more than once, we constructed a murine leukemia virus-based vector containing a second primer-binding site (PBS) derived from spleen necrosis virus and inserted the green fluorescent protein gene (GFP) between the two PBSs. Initiation of reverse transcription at either PBS results in a provirus that expresses GFP. However, initiation at both PBSs can result in the deletion of GFP, which can be detected by flow cytometry and Southern blotting analysis. Approximately 22-29% of the proviruses formed deleted the GFP in a single replication cycle, indicating the minimum proportion of virions that initiated reverse transcription on both PBSs. These results show that a significant proportion of MLV-based vectors containing two PBSs have the capacity to initiate reverse transcription more than once

  3. Scala for Real-Time Systems?

    DEFF Research Database (Denmark)

    Schoeberl, Martin

    2015-01-01

    Java served well as a general-purpose language. However, during its two decades of constant change it has gotten some weight and legacy in the language syntax and the libraries. Furthermore, Java's success for real-time systems is mediocre. Scala is a modern object-oriented and functional language...... with interesting new features. Although a new language, it executes on a Java virtual machine, reusing that technology. This paper explores Scala as language for future real-time systems....

  4. Real time monitoring of electron processors

    International Nuclear Information System (INIS)

    Nablo, S.V.; Kneeland, D.R.; McLaughlin, W.L.

    1995-01-01

    A real time radiation monitor (RTRM) has been developed for monitoring the dose rate (current density) of electron beam processors. The system provides continuous monitoring of processor output, electron beam uniformity, and an independent measure of operating voltage or electron energy. In view of the device's ability to replace labor-intensive dosimetry in verification of machine performance on a real-time basis, its application to providing archival performance data for in-line processing is discussed. (author)

  5. Identification of valid reference genes for gene expression studies of human stomach cancer by reverse transcription-qPCR

    International Nuclear Information System (INIS)

    Rho, Hyun-Wook; Lee, Byoung-Chan; Choi, Eun-Seok; Choi, Il-Ju; Lee, Yeon-Su; Goh, Sung-Ho

    2010-01-01

    Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for the analysis of gene expression. Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample. However, much effort has not been expended thus far in the search for reference genes suitable for the study of stomach cancer using RT-qPCR, although selection of optimal reference genes is critical for interpretation of results. We assessed the suitability of six possible reference genes, beta-actin (ACTB), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase 1 (HPRT1), beta-2-microglobulin (B2M), ribosomal subunit L29 (RPL29) and 18S ribosomal RNA (18S rRNA) in 20 normal and tumor stomach tissue pairs of stomach cancer patients and 6 stomach cancer cell lines, by RT-qPCR. Employing expression stability analyses using NormFinder and geNorm algorithms we determined the order of performance of these reference genes and their variation values. This RT-qPCR study showed that there are statistically significant (p < 0.05) differences in the expression levels of HPRT1 and 18S rRNA in 'normal-' versus 'tumor stomach tissues'. The stability analyses by geNorm suggest B2M-GAPDH, as best reference gene combination for 'stomach cancer cell lines'; RPL29-HPRT1, for 'all stomach tissues'; and ACTB-18S rRNA, for 'all stomach cell lines and tissues'. NormFinder also identified B2M as the best reference gene for 'stomach cancer cell lines', RPL29-B2M for 'all stomach tissues', and 18S rRNA-ACTB for 'all stomach cell lines and tissues'. The comparisons of normalized expression of the target gene, GPNMB, showed different interpretation of target gene expression depend on best single reference gene or combination. This study validated RPL29 and RPL29-B2M as the best single reference

  6. Benefits of real-time gas management

    International Nuclear Information System (INIS)

    Nolty, R.; Dolezalek, D. Jr.

    1994-01-01

    In today's competitive gas gathering, processing, storage and transportation business environment, the requirements to do business are continually changing. These changes arise from government regulations such as the amendments to the Clean Air Act concerning the environment and FERC Order 636 concerning business practices. Other changes are due to advances in technology such as electronic flow measurement (EFM) and real-time communications capabilities within the gas industry. Gas gathering, processing, storage and transportation companies must be flexible in adapting to these changes to remain competitive. These dynamic requirements can be met with an open, real-time gas management computer information system. Such a system provides flexible services with a variety of software applications. Allocations, nominations management and gas dispatching are examples of applications that are provided on a real-time basis. By providing real-time services, the gas management system enables operations personnel to make timely adjustments within the current accounting period. Benefits realized from implementing a real-time gas management system include reduced unaccountable gas, reduced imbalance penalties, reduced regulatory violations, improved facility operations and better service to customers. These benefits give a company the competitive edge. This article discusses the applications provided, the benefits from implementing a real-time gas management system, and the definition of such a system

  7. Typing of Poultry Influenza Virus (H5 and H7 by Reverse Transcription- Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Cesare Bonacina

    2010-01-01

    Full Text Available The ability of the influenza Orthomixovirus to undergo to continually antigenically changes that can affect its pathogenicity and its diffusion, explains the growing seriousness of this disease and the recent epizoozies in various parts of the world. There have been 15 HA and 9 NA type A sub-types of the influenza virus identified all of which are present in birds. Until now the very virulent avian influenza viruses identified were all included to the H5 and H7 sub-types. We here show that is possible to identify the H5 and H7 sub-types with reverse transcription-polymerase chain reaction (RT-PCR by using a set of specific primers for each HA sub-type. The RT-PCR is a quick and sensitive method of identifying the HA sub-types of the influenza virus directly from homogenised organs.

  8. Application of anti-listerial bacteriocins: monitoring enterocin expression by multiplex relative reverse transcription-PCR.

    Science.gov (United States)

    Williams, D Ross; Chanos, Panagiotis

    2012-12-01

    Listeriosis is a deadly food-borne disease, and its incidence may be limited through the biotechnological exploitation of a number of anti-listerial biocontrol agents. The most widely used of these agents are bacteriocins and the Class II enterocins are characterized by their activity against Listeria. Enterocins are primarily produced by enterococci, particularly Enterococcus faecium and many strains have been described, often encoding multiple bacteriocins. The use of these strains in food will require that they are free of virulence functions and that they exhibit a high level expression of anti-listerial enterocins in fermentation conditions. Multiplex relative RT (reverse transcription)-PCR is a technique that is useful in the discovery of advantageous expression characteristics among enterocin-producing strains. It allows the levels of individual enterocin gene expression to be monitored and determination of how expression is altered under different growth conditions.

  9. New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination

    OpenAIRE

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

    2014-01-01

    Loop mediated isothermal amplification (LAMP) assay, a promising diagnostic test, has been developed for detection of different pathogens of human as well as animals. Various positive points support its use as a field level test but the major problem is product cross contamination leading to false positive results. Different methods were adopted by various researchers to control this false positive amplification due to cross contamination but all have their own advantages and disadvantages. A...

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

    OpenAIRE

    Kong, Qing-Ming; Lu, Shao-Hong; Tong, Qun-Bo; Lou, Di; Chen, Rui; Zheng, Bin; Kumagai, Takashi; Wen, Li-Yong; Ohta, Nobuo; Zhou, Xiao-Nong

    2012-01-01

    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 perf...

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

    Directory of Open Access Journals (Sweden)

    Berit Aydin-Schmidt

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

  12. Real-time PCR in virology.

    Science.gov (United States)

    Mackay, Ian M; Arden, Katherine E; Nitsche, Andreas

    2002-03-15

    The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.

  13. REAL TIME SYSTEM OPERATIONS 2006-2007

    Energy Technology Data Exchange (ETDEWEB)

    Eto, Joseph H.; Parashar, Manu; Lewis, Nancy Jo

    2008-08-15

    The Real Time System Operations (RTSO) 2006-2007 project focused on two parallel technical tasks: (1) Real-Time Applications of Phasors for Monitoring, Alarming and Control; and (2) Real-Time Voltage Security Assessment (RTVSA) Prototype Tool. The overall goal of the phasor applications project was to accelerate adoption and foster greater use of new, more accurate, time-synchronized phasor measurements by conducting research and prototyping applications on California ISO's phasor platform - Real-Time Dynamics Monitoring System (RTDMS) -- that provide previously unavailable information on the dynamic stability of the grid. Feasibility assessment studies were conducted on potential application of this technology for small-signal stability monitoring, validating/improving existing stability nomograms, conducting frequency response analysis, and obtaining real-time sensitivity information on key metrics to assess grid stress. Based on study findings, prototype applications for real-time visualization and alarming, small-signal stability monitoring, measurement based sensitivity analysis and frequency response assessment were developed, factory- and field-tested at the California ISO and at BPA. The goal of the RTVSA project was to provide California ISO with a prototype voltage security assessment tool that runs in real time within California ISO?s new reliability and congestion management system. CERTS conducted a technical assessment of appropriate algorithms, developed a prototype incorporating state-of-art algorithms (such as the continuation power flow, direct method, boundary orbiting method, and hyperplanes) into a framework most suitable for an operations environment. Based on study findings, a functional specification was prepared, which the California ISO has since used to procure a production-quality tool that is now a part of a suite of advanced computational tools that is used by California ISO for reliability and congestion management.

  14. Loop-Mediated Isothermal Amplification for Detection of Endogenous Sad1 Gene in Cotton: An Internal Control for Rapid Onsite GMO Testing.

    Science.gov (United States)

    Singh, Monika; Bhoge, Rajesh K; Randhawa, Gurinderjit

    2018-04-20

    Background : Confirming the integrity of seed samples in powdered form is important priorto conducting a genetically modified organism (GMO) test. Rapid onsite methods may provide a technological solution to check for genetically modified (GM) events at ports of entry. In India, Bt cotton is the commercialized GM crop with four approved GM events; however, 59 GM events have been approved globally. GMO screening is required to test for authorized GM events. The identity and amplifiability of test samples could be ensured first by employing endogenous genes as an internal control. Objective : A rapid onsite detection method was developed for an endogenous reference gene, stearoyl acyl carrier protein desaturase ( Sad1 ) of cotton, employing visual and real-time loop-mediated isothermal amplification (LAMP). Methods : The assays were performed at a constant temperature of 63°C for 30 min for visual LAMP and 62ºC for 40 min for real-time LAMP. Positive amplification was visualized as a change in color from orange to green on addition of SYBR ® Green or detected as real-time amplification curves. Results : Specificity of LAMP assays was confirmed using a set of 10 samples. LOD for visual LAMP was up to 0.1%, detecting 40 target copies, and for real-time LAMP up to 0.05%, detecting 20 target copies. Conclusions : The developed methods could be utilized to confirm the integrity of seed powder prior to conducting a GMO test for specific GM events of cotton. Highlights : LAMP assays for the endogenous Sad1 gene of cotton have been developed to be used as an internal control for onsite GMO testing in cotton.

  15. Testing of real-time-software

    International Nuclear Information System (INIS)

    Friesland, G.; Ovenhausen, H.

    1975-05-01

    The situation in the area of testing real-time-software is unsatisfactory. During the first phase of the project PROMOTE (prozessorientiertes Modul- und Gesamttestsystem) an analysis of the momentary situation took place, results of which are summarized in the following study about some user interviews and an analysis of relevant literature. 22 users (industry, software-houses, hardware-manufacturers, and institutes) have been interviewed. Discussions were held about reliability of real-time software with special interest to error avoidance, testing, and debugging. Main aims of the analysis of the literature were elaboration of standard terms, comparison of existing test methods and -systems, and the definition of boundaries to related areas. During the further steps of this project some means and techniques will be worked out to systematically test real-time software. (orig.) [de

  16. Failure analysis of real-time systems

    International Nuclear Information System (INIS)

    Jalashgar, A.; Stoelen, K.

    1998-01-01

    This paper highlights essential aspects of real-time software systems that are strongly related to the failures and their course of propagation. The significant influence of means-oriented and goal-oriented system views in the description, understanding and analysing of those aspects is elaborated. The importance of performing failure analysis prior to reliability analysis of real-time systems is equally addressed. Problems of software reliability growth models taking the properties of such systems into account are discussed. Finally, the paper presents a preliminary study of a goal-oriented approach to model the static and dynamic characteristics of real-time systems, so that the corresponding analysis can be based on a more descriptive and informative picture of failures, their effects and the possibility of their occurrence. (author)

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

    Directory of Open Access Journals (Sweden)

    Owen Higgins

    2018-02-01

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

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

    Science.gov (United States)

    Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert

    2018-01-01

    Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology. PMID:29425124

  19. Real Time Linux - The RTOS for Astronomy?

    Science.gov (United States)

    Daly, P. N.

    The BoF was attended by about 30 participants and a free CD of real time Linux-based upon RedHat 5.2-was available. There was a detailed presentation on the nature of real time Linux and the variants for hard real time: New Mexico Tech's RTL and DIAPM's RTAI. Comparison tables between standard Linux and real time Linux responses to time interval generation and interrupt response latency were presented (see elsewhere in these proceedings). The present recommendations are to use RTL for UP machines running the 2.0.x kernels and RTAI for SMP machines running the 2.2.x kernel. Support, both academically and commercially, is available. Some known limitations were presented and the solutions reported e.g., debugging and hardware support. The features of RTAI (scheduler, fifos, shared memory, semaphores, message queues and RPCs) were described. Typical performance statistics were presented: Pentium-based oneshot tasks running > 30kHz, 486-based oneshot tasks running at ~ 10 kHz, periodic timer tasks running in excess of 90 kHz with average zero jitter peaking to ~ 13 mus (UP) and ~ 30 mus (SMP). Some detail on kernel module programming, including coding examples, were presented showing a typical data acquisition system generating simulated (random) data writing to a shared memory buffer and a fifo buffer to communicate between real time Linux and user space. All coding examples were complete and tested under RTAI v0.6 and the 2.2.12 kernel. Finally, arguments were raised in support of real time Linux: it's open source, free under GPL, enables rapid prototyping, has good support and the ability to have a fully functioning workstation capable of co-existing hard real time performance. The counter weight-the negatives-of lack of platforms (x86 and PowerPC only at present), lack of board support, promiscuous root access and the danger of ignorance of real time programming issues were also discussed. See ftp://orion.tuc.noao.edu/pub/pnd/rtlbof.tgz for the StarOffice overheads

  20. Axial Tomography from Digitized Real Time Radiography

    Science.gov (United States)

    Zolnay, A. S.; McDonald, W. M.; Doupont, P. A.; McKinney, R. L.; Lee, M. M.

    1985-01-18

    Axial tomography from digitized real time radiographs provides a useful tool for industrial radiography and tomography. The components of this system are: x-ray source, image intensifier, video camera, video line extractor and digitizer, data storage and reconstruction computers. With this system it is possible to view a two dimensional x-ray image in real time at each angle of rotation and select the tomography plane of interest by choosing which video line to digitize. The digitization of a video line requires less than a second making data acquisition relatively short. Further improvements on this system are planned and initial results are reported.

  1. Replacing OSE with Real Time capable Linux

    OpenAIRE

    Boman, Simon; Rutgersson, Olof

    2009-01-01

    For many years OSE has been a common used operating system, with real time extensions enhancements, in embed-ded systems. But in the last decades, Linux has grown and became a competitor to common operating systems and, in recent years, even as an operating system with real time extensions. With this in mind, ÅF was interested in replacing the quite expensive OSE with some distribution of the open source based Linux on a PowerPC MPC8360. Therefore, our purpose with thesis is to implement Linu...

  2. SignalR real time application development

    CERN Document Server

    Ingebrigtsen, Einar

    2013-01-01

    This step-by-step guide gives you practical advice, tips, and tricks that will have you writing real-time apps quickly and easily.If you are a .NET developer who wants to be at the cutting edge of development, then this book is for you. Real-time application development is made simple in this guide, so as long as you have basic knowledge of .NET, a copy of Visual Studio, and NuGet installed, you are ready to go.

  3. Real-time systems scheduling 2 focuses

    CERN Document Server

    Chetto, Maryline

    2014-01-01

    Real-time systems are used in a wide range of applications, including control, sensing, multimedia, etc. Scheduling is a central problem for these computing/communication systems since it is responsible for software execution in a timely manner. This book, the second of two volumes on the subject, brings together knowledge on specific topics and discusses the recent advances for some of them.  It addresses foundations as well as the latest advances and findings in real-time scheduling, giving comprehensive references to important papers, but the chapters are short and not overloaded with co

  4. Real-time ISEE data system

    Science.gov (United States)

    Tsurutani, B. T.; Baker, D. N.

    1979-01-01

    A real-time ISEE data system directed toward predicting geomagnetic substorms and storms is discussed. Such a system may allow up to 60+ minutes advance warning of magnetospheric substorms and up to 30 minute warnings of geomagnetic storms (and other disturbances) induced by high-speed streams and solar flares. The proposed system utilizes existing capabilities of several agencies (NASA, NOAA, USAF), and thereby minimizes costs. This same concept may be applicable to data from other spacecraft, and other NASA centers; thus, each individual experimenter can receive quick-look data in real time at his or her base institution.

  5. Mobile waste inspection real time radiography system

    International Nuclear Information System (INIS)

    Vigil, J.; Taggart, D.; Betts, S.; Rael, C.; Martinez, F.; Mendez, J.

    1995-01-01

    The 450-KeV Mobile Real Time Radiography System was designed and purchased to inspect containers of radioactive waste produced at Los Alamos National Laboratory (LANL). The Mobile Real Time Radiography System has the capability of inspecting waste containers of various sizes from 5-gal. buckets to standard waste boxes (SWB, dimensions 54.5 in. x 71 in. x 37 in.). The fact that this unit is mobile makes it an attractive alternative to the costly road closures associated with moving waste from the waste generator to storage or disposal facilities

  6. Real-time systems scheduling fundamentals

    CERN Document Server

    Chetto, Maryline

    2014-01-01

    Real-time systems are used in a wide range of applications, including control, sensing, multimedia, etc.  Scheduling is a central problem for these computing/communication systems since responsible of software execution in a timely manner. This book provides state of knowledge in this domain with special emphasis on the key results obtained within the last decade. This book addresses foundations as well as the latest advances and findings in Real-Time Scheduling, giving all references to important papers. But nevertheless the chapters will be short and not overloaded with confusing details.

  7. Automated real-time software development

    Science.gov (United States)

    Jones, Denise R.; Walker, Carrie K.; Turkovich, John J.

    1993-01-01

    A Computer-Aided Software Engineering (CASE) system has been developed at the Charles Stark Draper Laboratory (CSDL) under the direction of the NASA Langley Research Center. The CSDL CASE tool provides an automated method of generating source code and hard copy documentation from functional application engineering specifications. The goal is to significantly reduce the cost of developing and maintaining real-time scientific and engineering software while increasing system reliability. This paper describes CSDL CASE and discusses demonstrations that used the tool to automatically generate real-time application code.

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

    Science.gov (United States)

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

    2014-10-01

    Plum pox virus (PPV) causes the most destructive viral disease known as plum pox or Sharka disease in stone fruit trees. As an important regulated pathogen, detection of PPV is thus of critical importance to quarantine and eradication of the spreading disease. In this study, the innovative development of two AmplifyRP(®) tests is reported for a rapid isothermal detection of PPV using reverse transcription-recombinase polymerase amplification. In an AmplifyRP(®) test, all specific recombination and amplification reactions occur at a constant temperature without thermal cycling and the test results are either recorded in real-time with a portable fluorescence reader or displayed using a lateral flow strip contained inside an amplicon detection chamber. The major improvement of this assay is that the entire test from sample preparation to result can be completed in as little as 20min and can be performed easily both in laboratories and in the field. The results from this study demonstrated the ability of the AmplifyRP(®) technique to detect all nine PPV strains (An, C, CR, D, EA, M, Rec, T, or W). Among the economic benefits to pathogen surveys is the higher sensitivity of the AmplifyRP(®) to detect PPV when compared to the conventional ELISA and ImmunoStrip(®) assays. This is the first report describing the use of such an innovative technique to detect rapidly plant viruses affecting perennial crops. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae using reverse-transcription quantitative PCR.

    Directory of Open Access Journals (Sweden)

    Miao Yuan

    Full Text Available The brown planthopper (BPH, Nilaparvata lugens (Hemiptera, Delphacidae, is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR. Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT, muscle actin (MACT, ribosomal protein S11 (RPS11, ribosomal protein S15e (RPS15, alpha 2-tubulin (TUB, elongation factor 1 delta (EF, 18S ribosomal RNA (18S, and arginine kinase (AK and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for

  10. Compiling models into real-time systems

    International Nuclear Information System (INIS)

    Dormoy, J.L.; Cherriaux, F.; Ancelin, J.

    1992-08-01

    This paper presents an architecture for building real-time systems from models, and model-compiling techniques. This has been applied for building a real-time model-based monitoring system for nuclear plants, called KSE, which is currently being used in two plants in France. We describe how we used various artificial intelligence techniques for building it: a model-based approach, a logical model of its operation, a declarative implementation of these models, and original knowledge-compiling techniques for automatically generating the real-time expert system from those models. Some of those techniques have just been borrowed from the literature, but we had to modify or invent other techniques which simply did not exist. We also discuss two important problems, which are often underestimated in the artificial intelligence literature: size, and errors. Our architecture, which could be used in other applications, combines the advantages of the model-based approach with the efficiency requirements of real-time applications, while in general model-based approaches present serious drawbacks on this point

  11. Real-Time Operating System/360

    Science.gov (United States)

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

    1969-01-01

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

  12. Advances in Real-Time Systems

    CERN Document Server

    Chakraborty, Samarjit

    2012-01-01

    This volume contains the lectures given in honor to Georg Farber as tribute to his contributions in the area of real-time and embedded systems. The chapters of many leading scientists cover a wide range of aspects, like robot or automotive vision systems or medical aspects.

  13. Refactoring Real-Time Java Profiles

    DEFF Research Database (Denmark)

    Søndergaard, Hans; Thomsen, Bent; Ravn, Anders Peter

    2011-01-01

    Just like other software, Java profiles benefits from refactoring when they have been used and have evolved for some time. This paper presents a refactoring of the Real-Time Specification for Java (RTSJ) and the Safety Critical Java (SCJ) profile (JSR-302). It highlights core concepts and makes...

  14. Collecting data in real time with postcards

    DEFF Research Database (Denmark)

    Yee, Kwang Chien; Kanstrup, Anne Marie; Bertelsen, Pernille

    2013-01-01

    Systems. These methods often involve cross-sectional, retrospective data collection. This paper describes the postcard method for prospective real-time data collection, both in paper format and electronic format. This paper then describes the results obtained using postcard techniques in Denmark...

  15. Studying Complex Interactions in Real Time

    DEFF Research Database (Denmark)

    Mønster, Dan

    2017-01-01

    The study of human behavior must take into account the social context, and real-time, networked experiments with multiple participants is one increasingly popular way to achieve this. In this paper a framework based on Python and XMPP is presented that aims to make it easy to develop...

  16. Feedback as Real-Time Constructions

    Science.gov (United States)

    Keiding, Tina Bering; Qvortrup, Ane

    2014-01-01

    This article offers a re-description of feedback and the significance of time in feedback constructions based on systems theory. It describes feedback as internal, real-time constructions in a learning system. From this perspective, feedback is neither immediate nor delayed, but occurs in the very moment it takes place. This article argues for a…

  17. Interactive Real-time Magnetic Resonance Imaging

    DEFF Research Database (Denmark)

    Brix, Lau

    seeks to implement and assess existing reconstruction algorithms using multi-processors of modern graphics cards and many-core computer processors and to cover some of the potential clinical applications which might benefit from using an interactive real-time MRI system. First an off...

  18. Real-time PCR gene expression profiling

    Czech Academy of Sciences Publication Activity Database

    Kubista, Mikael; Sjögreen, B.; Forootan, A.; Šindelka, Radek; Jonák, Jiří; Andrade, J.M.

    2007-01-01

    Roč. 1, - (2007), s. 56-60 ISSN 1360-8606 R&D Projects: GA AV ČR KJB500520601 Institutional research plan: CEZ:AV0Z50520514 Keywords : real - time PCR, * expression profiling * statistical analysis Subject RIV: EB - Genetics ; Molecular Biology

  19. Real Time Grid Reliability Management 2005

    Energy Technology Data Exchange (ETDEWEB)

    Eto, Joe; Eto, Joe; Lesieutre, Bernard; Lewis, Nancy Jo; Parashar, Manu

    2008-07-07

    The increased need to manage California?s electricity grid in real time is a result of the ongoing transition from a system operated by vertically-integrated utilities serving native loads to one operated by an independent system operator supporting competitive energy markets. During this transition period, the traditional approach to reliability management -- construction of new transmission lines -- has not been pursued due to unresolved issues related to the financing and recovery of transmission project costs. In the absence of investments in new transmission infrastructure, the best strategy for managing reliability is to equip system operators with better real-time information about actual operating margins so that they can better understand and manage the risk of operating closer to the edge. A companion strategy is to address known deficiencies in offline modeling tools that are needed to ground the use of improved real-time tools. This project: (1) developed and conducted first-ever demonstrations of two prototype real-time software tools for voltage security assessment and phasor monitoring; and (2) prepared a scoping study on improving load and generator response models. Additional funding through two separate subsequent work authorizations has already been provided to build upon the work initiated in this project.

  20. Model Checking Real-Time Systems

    DEFF Research Database (Denmark)

    Bouyer, Patricia; Fahrenberg, Uli; Larsen, Kim Guldstrand

    2018-01-01

    This chapter surveys timed automata as a formalism for model checking real-time systems. We begin with introducing the model, as an extension of finite-state automata with real-valued variables for measuring time. We then present the main model-checking results in this framework, and give a hint...

  1. Real-time systems design and analysis

    CERN Document Server

    Laplante, Phillip A

    2004-01-01

    "Real-Time Systems Design and Analysis, Third Edition is essential for students and practicing software engineers who want improved designs, faster computation, and ultimate cost savings. Chapters discuss hardware considerations and software requirements, software systems design, the software production process, performance estimation and optimization, and engineering considerations."--Jacket.

  2. Testing Real-Time Systems Using UPPAAL

    DEFF Research Database (Denmark)

    Hessel, Anders; Larsen, Kim Guldstrand; Mikucionis, Marius

    2008-01-01

    This chapter presents principles and techniques for model-based black-box conformance testing of real-time systems using the Uppaal model-checking tool-suite. The basis for testing is given as a network of concurrent timed automata specified by the test engineer. Relativized input...

  3. Temporal logics and real time expert systems

    NARCIS (Netherlands)

    Blom, J.A.

    1996-01-01

    This paper introduces temporal logics. Due to the eternal compromise between expressive adequacy and reasoning efficiency that must decided upon in any application, full (first order logic or modal logic based) temporal logics are frequently not suitable. This is especially true in real time expert

  4. Ray Tracing for Real-time Games

    NARCIS (Netherlands)

    Bikker, J.

    2012-01-01

    This thesis describes efficient rendering algorithms based on ray tracing, and the application of these algorithms to real-time games. Compared to rasterizationbased approaches, rendering based on ray tracing allows elegant and correct simulation of important global effects, such as shadows,

  5. Compiling models into real-time systems

    International Nuclear Information System (INIS)

    Dormoy, J.L.; Cherriaux, F.; Ancelin, J.

    1992-08-01

    This paper presents an architecture for building real-time systems from models, and model-compiling techniques. This has been applied for building a real-time model-base monitoring system for nuclear plants, called KSE, which is currently being used in two plants in France. We describe how we used various artificial intelligence techniques for building it: a model-based approach, a logical model of its operation, a declarative implementation of these models, and original knowledge-compiling techniques for automatically generating the real-time expert system from those models. Some of those techniques have just been borrowed from the literature, but we had to modify or invent other techniques which simply did not exist. We also discuss two important problems, which are often underestimated in the artificial intelligence literature: size, and errors. Our architecture, which could be used in other applications, combines the advantages of the model-based approach with the efficiency requirements of real-time applications, while in general model-based approaches present serious drawbacks on this point

  6. Scene independent real-time indirect illumination

    DEFF Research Database (Denmark)

    Frisvad, Jeppe Revall; Christensen, Niels Jørgen; Falster, Peter

    2005-01-01

    A novel method for real-time simulation of indirect illumination is presented in this paper. The method, which we call Direct Radiance Mapping (DRM), is based on basal radiance calculations and does not impose any restrictions on scene geometry or dynamics. This makes the method tractable for rea...

  7. Composing Synchronisation and Real-Time Constraints

    NARCIS (Netherlands)

    Bergmans, Lodewijk; Aksit, Mehmet

    There have been a number of publications illustrating the successes of object-oriented techniques in creating highly reusable software systems. Several concurrent languages have been proposed for specifying reusable synchronization specifications. Recently, a number of real-time object-oriented

  8. In situ reverse transcription-PCR for monitoring gene expression in individual Methanosarcina mazei S-6 cells

    DEFF Research Database (Denmark)

    Lange, Marianne; Tolker-Nielsen, Tim; Molin, Søren

    2000-01-01

    An in situ reverse transcription-PCR protocol for detecting specific mRNA in Methanosarcina mazei S-6 is described. This method allowed us to detect heat shock-induced increases in the intracellular levels of the transcript of the universal stress gene dnaK. The cell walls of paraformaldehyde...

  9. Concepts of real time and semi-real time material control

    International Nuclear Information System (INIS)

    Lovett, J.E.

    1975-01-01

    After a brief consideration of the traditional material balance accounting on an MBA basis, this paper explores the basic concepts of real time and semi-real time material control, together with some of the major problems to be solved. Three types of short-term material control are discussed: storage, batch processing, and continuous processing. (DLC)

  10. 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.

  11. Reverse transcription and polymerase chain reaction: principles and applications in dentistry.

    Science.gov (United States)

    Santos, Carlos Ferreira Dos; Sakai, Vivien Thiemy; Machado, Maria Aparecida de Andrade Moreira; Schippers, Daniela Nicole; Greene, Andrew Seth

    2004-03-01

    Various molecular biology techniques have become available in the last few years. One of the most revolutionary of these techniques regarding nucleic acid analysis is the polymerase chain reaction (PCR), which was first described in 1985. This method relies on the exponential amplification of specific DNA fragments, resulting in millions of copies that can serve as templates for different kinds of analyses. PCR can be preceded by a reverse transcription (RT) reaction in order to produce cDNA from RNA (RT-PCR). RT-PCR provides the possibility to assess gene transcription in cells or tissues. PCR and RT-PCR techniques have been instrumental in dental research, and show potential to be used for diagnosis as well as for treatment and prevention of many diseases (dental caries, periodontal disease, endodontic infections and oral cancer). Compared to other traditional methodologies, PCR and RT-PCR show many advantages including high specificity, sensitivity, and speed. Since PCR and RT-PCR are relatively new techniques and are not available to most students and professionals involved with dentistry, the aim of this work is to present the details of these techniques as well as dental literature reports in which they were used.

  12. Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex.

    Science.gov (United States)

    Grigorov, Boyan; Bocquin, Anne; Gabus, Caroline; Avilov, Sergey; Mély, Yves; Agopian, Audrey; Divita, Gilles; Gottikh, Marina; Witvrouw, Myriam; Darlix, Jean-Luc

    2011-07-01

    Upon HIV-1 infection of a target cell, the viral reverse transcriptase (RT) copies the genomic RNA to synthesize the viral DNA. The genomic RNA is within the incoming HIV-1 core where it is coated by molecules of nucleocapsid (NC) protein that chaperones the reverse transcription process. Indeed, the RT chaperoning properties of NC extend from the initiation of cDNA synthesis to completion of the viral DNA. New and effective drugs against HIV-1 continue to be required, which prompted us to search for compounds aimed at inhibiting NC protein. Here, we report that the NC chaperoning activity is extensively inhibited in vitro by small methylated oligoribonucleotides (mODN). These mODNs were delivered intracellularly using a cell-penetrating-peptide and found to impede HIV-1 replication in primary human cells at nanomolar concentrations. Extensive analysis showed that viral cDNA synthesis was severely impaired by mODNs. Partially resistant viruses with mutations in NC and RT emerged after months of passaging in cell culture. A HIV-1 molecular clone (NL4.3) bearing these mutations was found to replicate at high concentrations of mODN, albeit with a reduced fitness. Small, methylated ODNs such as mODN-11 appear to be a new type of highly potent inhibitor of HIV-1.

  13. Detection of canine cytokine gene expression by reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Pinelli, E; van der Kaaij, S Y; Slappendel, R; Fragio, C; Ruitenberg, E J; Bernadina, W; Rutten, V P

    1999-08-02

    Further characterization of the canine immune system will greatly benefit from the availability of tools to detect canine cytokines. Our interest concerns the study on the role of cytokines in canine visceral leishmaniasis. For this purpose, we have designed specific primers using previously published sequences for the detection of canine IL-2, IFN-gamma and IL10 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). For IL-4, we have cloned and sequenced this cytokine gene, and developed canine-specific primers. To control for sample-to-sample variation in the quantity of mRNA and variation in the RT and PCR reactions, the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a housekeeping gene, were determined in parallel. Primers to amplify G3PDH were designed from consensus sequences obtained from the Genbank database. The mRNA levels of the cytokines mentioned here were detected from ConA-stimulated peripheral mononuclear cells derived from Leishmania-infected dogs. A different pattern of cytokine production among infected animals was found.

  14. Detection of HCV-RNA by Reverse Transcription Polymerase Chain Reaction Using Biotinylated and Radioiodinated Primers

    International Nuclear Information System (INIS)

    Ryu, Jin Sook; Moon, Dae Hyuk; Cheon, Jun Hong; Chung, Yoon Young; Park, Hung Dong; Chung, Young Hwa; Lee, Young Sang

    1994-01-01

    This study was performed to evaluate the clinical applicability of the reverse transcription polymerase chain reaction (RT-PCR) kit of HCV-RNA using biotinylated and radioiodinated primers. Study subjects were 118 patients with positive anti-HCV. HCV-RNA in patients serum was extracted by guanidium thiocyanate method. After first amplification, the product was reamplified by primers labelled with biotin and I-125. The final amplification product was detected by counting the radioactivity after incubation in avidin coated tubes. In 51 samples, the test was repeated for evaluation of reproducibility. This new method was also compared with conventional RT-PCR methods in 34 samples from patients with chronic liver disease. The results were as follows, 1) HCV-RNA was positive in 85(97%)of 88 patients with chronic liver disease, and in 23 (73%) of 30 patients with normal liver function. 2) In comparison with conventional method, HCV-RNA was detected in 32(94%) of 34 patients with new method, whereas in 27(79% ) of the same group with conventional method 3) Repeated test with new method in 52 samples demonstrated 82% of concordant result. In conclusion, new method with biotinylated and radioiodinated primers was more sensitive than conventional method. However, great care must be taken for quality control because there were considerable interassay variation and possibility of false positivity and false negativity.

  15. Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR.

    Science.gov (United States)

    Kembou Tsofack, Japhette Esther; Zamostiano, Rachel; Watted, Salsabeel; Berkowitz, Asaf; Rosenbluth, Ezra; Mishra, Nischay; Briese, Thomas; Lipkin, W Ian; Kabuusu, Richard M; Ferguson, Hugh; Del Pozo, Jorge; Eldar, Avi; Bacharach, Eran

    2017-03-01

    Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen. Copyright © 2017 American Society for Microbiology.

  16. Detection of HCV-RNA by Reverse Transcription Polymerase Chain Reaction Using Biotinylated and Radioiodinated Primers

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, Jin Sook; Moon, Dae Hyuk; Cheon, Jun Hong; Chung, Yoon Young; Park, Hung Dong; Chung, Young Hwa; Lee, Young Sang [Asan Medical Center, University of Ulsan, Seoul (Korea, Republic of)

    1994-07-15

    This study was performed to evaluate the clinical applicability of the reverse transcription polymerase chain reaction (RT-PCR) kit of HCV-RNA using biotinylated and radioiodinated primers. Study subjects were 118 patients with positive anti-HCV. HCV-RNA in patients serum was extracted by guanidium thiocyanate method. After first amplification, the product was reamplified by primers labelled with biotin and I-125. The final amplification product was detected by counting the radioactivity after incubation in avidin coated tubes. In 51 samples, the test was repeated for evaluation of reproducibility. This new method was also compared with conventional RT-PCR methods in 34 samples from patients with chronic liver disease. The results were as follows, 1) HCV-RNA was positive in 85(97%)of 88 patients with chronic liver disease, and in 23 (73%) of 30 patients with normal liver function. 2) In comparison with conventional method, HCV-RNA was detected in 32(94%) of 34 patients with new method, whereas in 27(79% ) of the same group with conventional method 3) Repeated test with new method in 52 samples demonstrated 82% of concordant result. In conclusion, new method with biotinylated and radioiodinated primers was more sensitive than conventional method. However, great care must be taken for quality control because there were considerable interassay variation and possibility of false positivity and false negativity.

  17. Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR

    Science.gov (United States)

    Kembou Tsofack, Japhette Esther; Zamostiano, Rachel; Watted, Salsabeel; Berkowitz, Asaf; Rosenbluth, Ezra; Mishra, Nischay; Briese, Thomas; Lipkin, W. Ian; Kabuusu, Richard M.; Ferguson, Hugh; del Pozo, Jorge

    2016-01-01

    ABSTRACT Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen. PMID:27974544

  18. Development of Loop-Mediated Isothermal Amplification Assay for Detection of Entamoeba histolytica▿

    Science.gov (United States)

    Liang, Shih-Yu; Chan, Yun-Hsien; Hsia, Kan-Tai; Lee, Jing-Lun; Kuo, Ming-Chu; Hwa, Kuo-Yuan; Chan, Chi-Wen; Chiang, Ting-Yi; Chen, Jung-Sheng; Wu, Fang-Tzy; Ji, Dar-Der

    2009-01-01

    A novel one-step, closed-tube, loop-mediated isothermal amplification (LAMP) assay for detecting Entamoeba histolytica, one of the leading causes of morbidity in developing countries, was developed. The sensitivity of the LAMP assay is 1 parasite per reaction. A total of 130 clinical samples were analyzed, and the results compared with those of conventional nested PCR to validate the practicability of this assay. No DNA was amplified from other diarrheal pathogens, such as other Entamoeba species, bacteria, and viruses. These results indicate that LAMP is a rapid, simple, and valuable diagnostic tool for epidemiological studies of amebiasis. PMID:19321720

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

    Science.gov (United States)

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

    2018-02-01

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

  20. Real-time PCR (qPCR) primer design using free online software.

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software. Copyright © 2010 Wiley Periodicals, Inc.

  1. Real-time ISEE data system

    International Nuclear Information System (INIS)

    Tsurutani, B.T.; Baker, D.N.

    1979-01-01

    Prediction of geomagnetic substorms and storms would be of great scientific and commercial interest. A real-time ISEE data system directed toward this purpose is discussed in detail. Such a system may allow up to 60+ minutes advance warning of magnetospheric substorms and up to 30 minute warnings of geomagnetic storms (and other disturbances) induced by high-speed streams and solar flares. The proposed system utilizes existing capabilities of several agencies (NASA, NOAA, USAF), and thereby minimizes costs. This same concept may be applicable to data from other spacecraft, and other NASA centers; thus, each individual experimenter can receive quick-look data in real time at his or her base institution. 6 figures, 1 table

  2. Real time animation of space plasma phenomena

    International Nuclear Information System (INIS)

    Jordan, K.F.; Greenstadt, E.W.

    1987-01-01

    In pursuit of real time animation of computer simulated space plasma phenomena, the code was rewritten for the Massively Parallel Processor (MPP). The program creates a dynamic representation of the global bowshock which is based on actual spacecraft data and designed for three dimensional graphic output. This output consists of time slice sequences which make up the frames of the animation. With the MPP, 16384, 512 or 4 frames can be calculated simultaneously depending upon which characteristic is being computed. The run time was greatly reduced which promotes the rapid sequence of images and makes real time animation a foreseeable goal. The addition of more complex phenomenology in the constructed computer images is now possible and work proceeds to generate these images

  3. Real Time Radiation Monitoring Using Nanotechnology

    Science.gov (United States)

    Li, Jing (Inventor); Hanratty, James J. (Inventor); Wilkins, Richard T. (Inventor); Lu, Yijiang (Inventor)

    2016-01-01

    System and method for monitoring receipt and estimating flux value, in real time, of incident radiation, using two or more nanostructures (NSs) and associated terminals to provide closed electrical paths and to measure one or more electrical property change values .DELTA.EPV, associated with irradiated NSs, during a sequence of irradiation time intervals. Effects of irradiation, without healing and with healing, of the NSs, are separately modeled for first order and second order healing. Change values.DELTA.EPV are related to flux, to cumulative dose received by NSs, and to radiation and healing effectivity parameters and/or.mu., associated with the NS material and to the flux. Flux and/or dose are estimated in real time, based on EPV change values, using measured .DELTA.EPV values. Threshold dose for specified changes of biological origin (usually undesired) can be estimated. Effects of time-dependent radiation flux are analyzed in pre-healing and healing regimes.

  4. Real time gamma-ray signature identifier

    Science.gov (United States)

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

    2012-05-15

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

  5. Real-time sonography in obstetrics.

    Science.gov (United States)

    Anderson, S G

    1978-03-01

    Three hundred fifty real-time scans were performed on pregnant women for various indications. Placental localization was satisfactorily obtained in 173 of 174 studies. Estimates of fetal gestation from directly measured biparietal diameter were +/-2 weeks of actual gestation in 153 of 172 (88.9%) measurements. The presence or absence of fetal motion and cardiac activity established a diagnosis of fetal viability or fetal death in 32 patients after the first trimester. Accurate diagnosis was made in 52 of 57 patients with threatened abortions, and two of these errors occurred in scans performed before completion of the eighth postmenstrual week. Because of the ability to demonstrate fetal motion, real-time sonography should have many applications in obstetrics.

  6. Real time processor for array speckle interferometry

    International Nuclear Information System (INIS)

    Chin, G.; Florez, J.; Borelli, R.; Fong, W.; Miko, J.; Trujillo, C.

    1989-01-01

    With the construction of several new large aperture telescopes and the development of large format array detectors in the near IR, the ability to obtain diffraction limited seeing via IR array speckle interferometry offers a powerful tool. We are constructing a real-time processor to acquire image frames, perform array flat-fielding, execute a 64 x 64 element 2D complex FFT, and to average the power spectrum all within the 25 msec coherence time for speckles at near IR wavelength. The processor is a compact unit controlled by a PC with real time display and data storage capability. It provides the ability to optimize observations and obtain results on the telescope rather than waiting several weeks before the data can be analyzed and viewed with off-line methods

  7. Real time processor for array speckle interferometry

    Science.gov (United States)

    Chin, Gordon; Florez, Jose; Borelli, Renan; Fong, Wai; Miko, Joseph; Trujillo, Carlos

    1989-02-01

    The authors are constructing a real-time processor to acquire image frames, perform array flat-fielding, execute a 64 x 64 element two-dimensional complex FFT (fast Fourier transform) and average the power spectrum, all within the 25 ms coherence time for speckles at near-IR (infrared) wavelength. The processor will be a compact unit controlled by a PC with real-time display and data storage capability. This will provide the ability to optimize observations and obtain results on the telescope rather than waiting several weeks before the data can be analyzed and viewed with offline methods. The image acquisition and processing, design criteria, and processor architecture are described.

  8. Real Time Structured Light and Applications

    DEFF Research Database (Denmark)

    Wilm, Jakob

    Structured light scanning is a versatile method for 3D shape acquisition. While much faster than most competing measurement techniques, most high-end structured light scans still take in the order of seconds to complete. Low-cost sensors such as Microsoft Kinect and time of flight cameras have made......, increased processing power, and methods presented in this thesis, it is possible to perform structured light scans in real time with 20 depth measurements per second. This offers new opportunities for studying dynamic scenes, quality control, human-computer interaction and more. This thesis discusses...... several aspects of real time structured light systems and presents contributions within calibration, scene coding and motion correction aspects. The problem of reliable and fast calibration of such systems is addressed with a novel calibration scheme utilising radial basis functions [Contribution B...

  9. Real time automatic discriminating of ultrasonic flaws

    International Nuclear Information System (INIS)

    Suhairy Sani; Mohd Hanif Md Saad; Marzuki Mustafa; Mohd Redzwan Rosli

    2009-01-01

    This paper is concerned with the real time automatic discriminating of flaws from two categories; i. cracks (planar defect) and ii. Non-cracks (volumetric defect such as cluster porosity and slag) using pulse-echo ultrasound. The raw ultrasonic flaws signal were collected from a computerized robotic plane scanning system over the whole of each reflector as the primary source of data. The signal is then filtered and the analysis in both time and frequency domain were executed to obtain the selected feature. The real time feature analysis techniques measured the number of peaks, maximum index, pulse duration, rise time and fall time. The obtained features could be used to distinguish between quantitatively classified flaws by using various tools in artificial intelligence such as neural networks. The proposed algorithm and complete system were implemented in a computer software developed using Microsoft Visual BASIC 6.0 (author)

  10. Real-time imaging of quantum entanglement.

    Science.gov (United States)

    Fickler, Robert; Krenn, Mario; Lapkiewicz, Radek; Ramelow, Sven; Zeilinger, Anton

    2013-01-01

    Quantum Entanglement is widely regarded as one of the most prominent features of quantum mechanics and quantum information science. Although, photonic entanglement is routinely studied in many experiments nowadays, its signature has been out of the grasp for real-time imaging. Here we show that modern technology, namely triggered intensified charge coupled device (ICCD) cameras are fast and sensitive enough to image in real-time the effect of the measurement of one photon on its entangled partner. To quantitatively verify the non-classicality of the measurements we determine the detected photon number and error margin from the registered intensity image within a certain region. Additionally, the use of the ICCD camera allows us to demonstrate the high flexibility of the setup in creating any desired spatial-mode entanglement, which suggests as well that visual imaging in quantum optics not only provides a better intuitive understanding of entanglement but will improve applications of quantum science.

  11. Real-time interactive treatment planning

    International Nuclear Information System (INIS)

    Otto, Karl

    2014-01-01

    The goal of this work is to develop an interactive treatment planning platform that permits real-time manipulation of dose distributions including DVHs and other dose metrics. The hypothesis underlying the approach proposed here is that the process of evaluating potential dose distribution options and deciding on the best clinical trade-offs may be separated from the derivation of the actual delivery parameters used for the patient’s treatment. For this purpose a novel algorithm for deriving an Achievable Dose Estimate (ADE) was developed. The ADE algorithm is computationally efficient so as to update dose distributions in effectively real-time while accurately incorporating the limits of what can be achieved in practice. The resulting system is a software environment for interactive real-time manipulation of dose that permits the clinician to rapidly develop a fully customized 3D dose distribution. Graphical navigation of dose distributions is achieved by a sophisticated method of identifying contributing fluence elements, modifying those elements and re-computing the entire dose distribution. 3D dose distributions are calculated in ∼2–20 ms. Including graphics processing overhead, clinicians may visually interact with the dose distribution (e.g. ‘drag’ a DVH) and display updates of the dose distribution at a rate of more than 20 times per second. Preliminary testing on various sites shows that interactive planning may be completed in ∼1–5 min, depending on the complexity of the case (number of targets and OARs). Final DVHs are derived through a separate plan optimization step using a conventional VMAT planning system and were shown to be achievable within 2% and 4% in high and low dose regions respectively. With real-time interactive planning trade-offs between Target(s) and OARs may be evaluated efficiently providing a better understanding of the dosimetric options available to each patient in static or adaptive RT. (paper)

  12. Real time simulator for material testing reactor

    Energy Technology Data Exchange (ETDEWEB)

    Takemoto, Noriyuki; Imaizumi, Tomomi; Izumo, Hironobu; Hori, Naohiko; Suzuki, Masahide [Japan Atomic Energy Agency, Oarai Research and Development Center, Oarai, Ibaraki (Japan); Ishitsuka, Tatsuo; Tamura, Kazuo [ITOCHU Techno-Solutions Corp., Tokyo (Japan)

    2012-03-15

    Japan Atomic Energy Agency (JAEA) is now developing a real time simulator for a material testing reactor based on Japan Materials Testing Reactor (JMTR). The simulator treats reactor core system, primary and secondary cooling system, electricity system and irradiation facility systems. Possible simulations are normal reactor operation, unusual transient operation and accidental operation. The developed simulator also contains tool to revise/add facility in it for the future development. (author)

  13. Probabilistic real-time contingency ranking method

    International Nuclear Information System (INIS)

    Mijuskovic, N.A.; Stojnic, D.

    2000-01-01

    This paper describes a real-time contingency method based on a probabilistic index-expected energy not supplied. This way it is possible to take into account the stochastic nature of the electric power system equipment outages. This approach enables more comprehensive ranking of contingencies and it is possible to form reliability cost values that can form the basis for hourly spot price calculations. The electric power system of Serbia is used as an example for the method proposed. (author)

  14. Advanced real time radioscopy and computed tomography

    International Nuclear Information System (INIS)

    Sauerwein, Ch.; Nuding, W.; Grimm, R.; Wiacker, H.

    1996-01-01

    The paper describes three x-ray inspection systems. One radioscopic system is designed for the inspection of castings. The next integrates a radioscopic and a tomographic mode. The radioscopy has a high resolution camera and real time image processor. Radiation sources are a 450 kV industrial and a 200 kV microfocus tube. The third system is a tomographic system with 30 scintillation detectors for the inspection of nuclear waste containers. (author)

  15. Real time computer controlled weld skate

    Science.gov (United States)

    Wall, W. A., Jr.

    1977-01-01

    A real time, adaptive control, automatic welding system was developed. This system utilizes the general case geometrical relationships between a weldment and a weld skate to precisely maintain constant weld speed and torch angle along a contoured workplace. The system is compatible with the gas tungsten arc weld process or can be adapted to other weld processes. Heli-arc cutting and machine tool routing operations are possible applications.

  16. Real-time controller for hydrostatic transmission

    OpenAIRE

    2014-01-01

    M. Ing. (Electrical and Electronic Engineering) This dissertation describes the development of a modular real-time controller implemented on a personal computer for a hydrostatically driven vehicle. In such a vehicle the conventional mechanical transmission is replaced with a hydrostatic pump and two hydrostatic motors, making use of the secondary control principle. The infinitely variable transmission and wheel pair controller gives the vehicle superior traction and mobility over conventi...

  17. Telepositional portable real time radiation monitoring system

    International Nuclear Information System (INIS)

    Talpalariu, Jeni; Matei, Corina; Popescu, Oana

    2010-01-01

    Technology development for complex portable networks is on going to meet the area dosimetry challenge, improving the basic design using new telepositional GPS satellite methods and GSM terrestrial civil radio transmission networks. The system and devices proposed overcome the limitations of fixed and portable dosimeters, providing wireless real time radiations data and geospatial information's means, using many portable dosimeter stations and a mobile dosimeter computerised central console. (authors)

  18. Real time simulator for material testing reactor

    International Nuclear Information System (INIS)

    Takemoto, Noriyuki; Imaizumi, Tomomi; Izumo, Hironobu; Hori, Naohiko; Suzuki, Masahide; Ishitsuka, Tatsuo; Tamura, Kazuo

    2012-01-01

    Japan Atomic Energy Agency (JAEA) is now developing a real time simulator for a material testing reactor based on Japan Materials Testing Reactor (JMTR). The simulator treats reactor core system, primary and secondary cooling system, electricity system and irradiation facility systems. Possible simulations are normal reactor operation, unusual transient operation and accidental operation. The developed simulator also contains tool to revise/add facility in it for the future development. (author)

  19. Boundary Correct Real-Time Soft Shadows

    DEFF Research Database (Denmark)

    Jacobsen, Bjarke; Christensen, Niels Jørgen; Larsen, Bent Dalgaard

    2004-01-01

    This paper describes a method to determine correct shadow boundaries from an area light source using umbra and penumbra volumes. The light source is approximated by a circular disk as this gives a fast way to extrude the volumes. The method also gives a crude estimate of the visibility of the are...... for implementation on most programmable hardware. Though some crude approximations are used in the visibility function, the method can be used to produce soft shadows with correct boundaries in real time....

  20. Real-time Astrometry Using Phase Congruency

    Science.gov (United States)

    Lambert, A.; Polo, M.; Tang, Y.

    Phase congruency is a computer vision technique that proves to perform well for determining the tracks of optical objects (Flewelling, AMOS 2014). We report on a real-time implementation of this using an FPGA and CMOS Image Sensor, with on-sky data. The lightweight instrument can provide tracking update signals to the mount of the telescope, as well as determine abnormal objects in the scene.

  1. Real-time multiple image manipulations

    International Nuclear Information System (INIS)

    Arenson, J.S.; Shalev, S.; Legris, J.; Goertzen, Y.

    1984-01-01

    There are many situations in which it is desired to manipulate two or more images under real-time operator control. The authors have investigated a number of such cases in order to determine their value and applicability in clinical medicine and laboratory research. Several examples are presented in detail. The DICOM-8 video image computer system was used due to its capability of storing two 512 x 512 x 8 bit images and operating on them, and/or an incoming video frame, with any of a number of real time operations including addition, subtraction, inversion, averaging, logical AND, NAND, OR, NOR, NOT, XOR and XNOR, as well as combinations of these. Some applications involve manipulations of or among the stored images. In others, a stored image is used as a mask or template for positioning or adjusting a second image to be grabbed via a video camera. The accuracy of radiotherapy treatment is verified by comparing port films with the original radiographic planning film, which is previously digitized and stored. Moving the port film on the light box while viewing the real-time subtraction image allows for adjustments of zoom, translation and rotation, together with contrast and edge enhancement

  2. Real time ultrasonography in obstructive jaundice

    International Nuclear Information System (INIS)

    Cho, Kyung Sik; Kim, Ho Kyun; Sung, Nak Kwan; Kim, Soon Yong

    1982-01-01

    Ultrasonography is a predominantly accurate, relatively simple unique diagnostic method of obstructive jaundice. The ultrasonographic findings of obstructive jaundice are dilated intra- and extrahepatic duct with intraluminal hyper reflective echo or mass in and/ or around the bile duct. The superiority of high resolution real time ultrasonography for the diagnosis of obstructive jaundice is bases on the easy detectability of extra- and intrahepatic bile ducts by its multiple sectional images in a short time, the flexibility of probe and small crystal size. Author evaluated real time sonographic findings 46 obstructive jaundice patients confirmed by surgery or radiographical examinations. The results were: 1. Diameter of extrahepatic duct in obstructive jaundice were varied from normal to 4.0 Cm, mostly 8 to 10 mm in diameter (26%). Degree of dilatation of biliary duct appeared more prominent in cancer patients than other causes of obstruction. 2. The site of obstruction was detected in 85% (39/46) and its common site was common bile duct in 63% (29/46). 3. The diagnostic accuracy of choledocholithiasis and cancer was 82% (22/27) and 44% (4/9), respectively. Diagnostic accuracy of the real time ultrasonography in obstructive jaundice was over all 75% (34/46)

  3. Real-time video quality monitoring

    Science.gov (United States)

    Liu, Tao; Narvekar, Niranjan; Wang, Beibei; Ding, Ran; Zou, Dekun; Cash, Glenn; Bhagavathy, Sitaram; Bloom, Jeffrey

    2011-12-01

    The ITU-T Recommendation G.1070 is a standardized opinion model for video telephony applications that uses video bitrate, frame rate, and packet-loss rate to measure the video quality. However, this model was original designed as an offline quality planning tool. It cannot be directly used for quality monitoring since the above three input parameters are not readily available within a network or at the decoder. And there is a great room for the performance improvement of this quality metric. In this article, we present a real-time video quality monitoring solution based on this Recommendation. We first propose a scheme to efficiently estimate the three parameters from video bitstreams, so that it can be used as a real-time video quality monitoring tool. Furthermore, an enhanced algorithm based on the G.1070 model that provides more accurate quality prediction is proposed. Finally, to use this metric in real-world applications, we present an example emerging application of real-time quality measurement to the management of transmitted videos, especially those delivered to mobile devices.

  4. Real-time earthquake data feasible

    Science.gov (United States)

    Bush, Susan

    Scientists agree that early warning devices and monitoring of both Hurricane Hugo and the Mt. Pinatubo volcanic eruption saved thousands of lives. What would it take to develop this sort of early warning and monitoring system for earthquake activity?Not all that much, claims a panel assigned to study the feasibility, costs, and technology needed to establish a real-time earthquake monitoring (RTEM) system. The panel, drafted by the National Academy of Science's Committee on Seismology, has presented its findings in Real-Time Earthquake Monitoring. The recently released report states that “present technology is entirely capable of recording and processing data so as to provide real-time information, enabling people to mitigate somewhat the earthquake disaster.” RTEM systems would consist of two parts—an early warning system that would give a few seconds warning before severe shaking, and immediate postquake information within minutes of the quake that would give actual measurements of the magnitude. At this time, however, this type of warning system has not been addressed at the national level for the United States and is not included in the National Earthquake Hazard Reduction Program, according to the report.

  5. The Raptor Real-Time Processing Architecture

    Science.gov (United States)

    Galassi, M.; Starr, D.; Wozniak, P.; Brozdin, K.

    The primary goal of Raptor is ambitious: to identify interesting optical transients from very wide field of view telescopes in real time, and then to quickly point the higher resolution Raptor ``fovea'' cameras and spectrometer to the location of the optical transient. The most interesting of Raptor's many applications is the real-time search for orphan optical counterparts of Gamma Ray Bursts. The sequence of steps (data acquisition, basic calibration, source extraction, astrometry, relative photometry, the smarts of transient identification and elimination of false positives, telescope pointing feedback, etc.) is implemented with a ``component'' approach. All basic elements of the pipeline functionality have been written from scratch or adapted (as in the case of SExtractor for source extraction) to form a consistent modern API operating on memory resident images and source lists. The result is a pipeline which meets our real-time requirements and which can easily operate as a monolithic or distributed processing system. Finally, the Raptor architecture is entirely based on free software (sometimes referred to as ``open source'' software). In this paper we also discuss the interplay between various free software technologies in this type of astronomical problem.

  6. Tuning Linux to meet real time requirements

    Science.gov (United States)

    Herbel, Richard S.; Le, Dang N.

    2007-04-01

    There is a desire to use Linux in military systems. Customers are requesting contractors to use open source to the maximal possible extent in contracts. Linux is probably the best operating system of choice to meet this need. It is widely used. It is free. It is royalty free, and, best of all, it is completely open source. However, there is a problem. Linux was not originally built to be a real time operating system. There are many places where interrupts can and will be blocked for an indeterminate amount of time. There have been several attempts to bridge this gap. One of them is from RTLinux, which attempts to build a microkernel underneath Linux. The microkernel will handle all interrupts and then pass it up to the Linux operating system. This does insure good interrupt latency; however, it is not free [1]. Another is RTAI, which provides a similar typed interface; however, the PowerPC platform, which is used widely in real time embedded community, was stated as "recovering" [2]. Thus this is not suited for military usage. This paper provides a method for tuning a standard Linux kernel so it can meet the real time requirement of an embedded system.

  7. Real-time RT-PCR, a necessary tool to support the diagnosis and surveillance of rotavirus in Mexico.

    Science.gov (United States)

    De La Cruz Hernández, Sergio Isaac; Anaya Molina, Yazmin; Gómez Santiago, Fabián; Terán Vega, Heidi Lizbeth; Monroy Leyva, Elda; Méndez Pérez, Héctor; García Lozano, Herlinda

    2018-04-01

    Rotavirus produces diarrhea in children under 5 years old. Most of those conventional methods such as polyacrylamide gel electrophoresis (PAGE) and reverse transcription-polymerase chain reaction (RT-PCR) have been used for rotavirus detection. However, these techniques need a multi-step process to get the results. In comparison with conventional methods, the real-time RT-PCR is a highly sensitive method, which allows getting the results in only one day. In this study a real-time RT-PCR assay was tested using a panel of 440 samples from patients with acute gastroenteritis, and characterized by PAGE and RT-PCR. The results show that the real-time RT-PCR detected rotavirus from 73% of rotavirus-negative samples analyzed by PAGE and RT-PCR; thus, the percentage of rotavirus-positive samples increased to 81%. The results indicate that this real-time RT-PCR should be part of a routine analysis, and as a support of the diagnosis of rotavirus in Mexico. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification

    Directory of Open Access Journals (Sweden)

    Luiz Tadeu M Figueiredo

    1997-05-01

    Full Text Available We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37oC for RT followed by a variable amount of cycles of two-step PCR amplification (92oC for 60 sec, 53oC for 60 sec with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 102.8 TCID50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique

  9. Reverse Transcription Errors and RNA-DNA Differences at Short Tandem Repeats.

    Science.gov (United States)

    Fungtammasan, Arkarachai; Tomaszkiewicz, Marta; Campos-Sánchez, Rebeca; Eckert, Kristin A; DeGiorgio, Michael; Makova, Kateryna D

    2016-10-01

    Transcript variation has important implications for organismal function in health and disease. Most transcriptome studies focus on assessing variation in gene expression levels and isoform representation. Variation at the level of transcript sequence is caused by RNA editing and transcription errors, and leads to nongenetically encoded transcript variants, or RNA-DNA differences (RDDs). Such variation has been understudied, in part because its detection is obscured by reverse transcription (RT) and sequencing errors. It has only been evaluated for intertranscript base substitution differences. Here, we investigated transcript sequence variation for short tandem repeats (STRs). We developed the first maximum-likelihood estimator (MLE) to infer RT error and RDD rates, taking next generation sequencing error rates into account. Using the MLE, we empirically evaluated RT error and RDD rates for STRs in a large-scale DNA and RNA replicated sequencing experiment conducted in a primate species. The RT error rates increased exponentially with STR length and were biased toward expansions. The RDD rates were approximately 1 order of magnitude lower than the RT error rates. The RT error rates estimated with the MLE from a primate data set were concordant with those estimated with an independent method, barcoded RNA sequencing, from a Caenorhabditis elegans data set. Our results have important implications for medical genomics, as STR allelic variation is associated with >40 diseases. STR nonallelic transcript variation can also contribute to disease phenotype. The MLE and empirical rates presented here can be used to evaluate the probability of disease-associated transcripts arising due to RDD. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  10. Comparison of multiplex RT-PCR and real-time HybProbe assay for serotyping of dengue virus using reference strains and clinical samples from India

    Directory of Open Access Journals (Sweden)

    Anita Chakravarti

    2016-01-01

    Full Text Available Background: Dengue virus serotyping is crucial from clinical management and epidemiological point of view. Aims: To compare efficacy of two molecular detection and typing methods, namely, multiplex reverse transcription polymerase chain reaction (RT-PCR and real-time Hybprobe assay using a panel of known dilution of four reference Dengue virus strains and a panel of sera collected from clinically suspected dengue patients. Settings: This study was conducted at a tertiary-care teaching hospital in Delhi, India. Materials and Methods: Dengue serotype specific virus strains were used as prototypes for serotyping assays. Viral load was quantified by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR. Acute phase serum samples were collected from 79 patients with clinically suspected Dengue fever on their first day of presentation during September-October 2012. Viral RNA from serum and cell culture supernatant was extracted. Reverse transcription was carried out. Quantitative detection of DENV RNA from reference strain culture supernatants and each of the 79 patient samples by real-time PCR was performed using light cycler Taqman master mix kit. Serotyping was done by multiplex RT-PCR assay and Hybprobe assay. Results: The multiplex RT-PCR assay, though found to be 100% specific, couldn't serotype either patient or reference strains with viral load less than 1000 RNA copies/ml. The Hybprobe assay was found to have 100% specificity and had a lower limit of serotype detection of merely 3.54 RNA copies/ml. Conclusions: HybProbe assay has an important role especially in situations where serotyping is to be performed in clinical samples with low viral load.

  11. Exploring Earthquakes in Real-Time

    Science.gov (United States)

    Bravo, T. K.; Kafka, A. L.; Coleman, B.; Taber, J. J.

    2013-12-01

    Earthquakes capture the attention of students and inspire them to explore the Earth. Adding the ability to view and explore recordings of significant and newsworthy earthquakes in real-time makes the subject even more compelling. To address this opportunity, the Incorporated Research Institutions for Seismology (IRIS), in collaboration with Moravian College, developed ';jAmaSeis', a cross-platform application that enables students to access real-time earthquake waveform data. Students can watch as the seismic waves are recorded on their computer, and can be among the first to analyze the data from an earthquake. jAmaSeis facilitates student centered investigations of seismological concepts using either a low-cost educational seismograph or streamed data from other educational seismographs or from any seismic station that sends data to the IRIS Data Management System. After an earthquake, students can analyze the seismograms to determine characteristics of earthquakes such as time of occurrence, distance from the epicenter to the station, magnitude, and location. The software has been designed to provide graphical clues to guide students in the analysis and assist in their interpretations. Since jAmaSeis can simultaneously record up to three stations from anywhere on the planet, there are numerous opportunities for student driven investigations. For example, students can explore differences in the seismograms from different distances from an earthquake and compare waveforms from different azimuthal directions. Students can simultaneously monitor seismicity at a tectonic plate boundary and in the middle of the plate regardless of their school location. This can help students discover for themselves the ideas underlying seismic wave propagation, regional earthquake hazards, magnitude-frequency relationships, and the details of plate tectonics. The real-time nature of the data keeps the investigations dynamic, and offers students countless opportunities to explore.

  12. Simultaneous real-time data collection methods

    Science.gov (United States)

    Klincsek, Thomas

    1992-01-01

    This paper describes the development of electronic test equipment which executes, supervises, and reports on various tests. This validation process uses computers to analyze test results and report conclusions. The test equipment consists of an electronics component and the data collection and reporting unit. The PC software, display screens, and real-time data-base are described. Pass-fail procedures and data replay are discussed. The OS2 operating system and Presentation Manager user interface system were used to create a highly interactive automated system. The system outputs are hardcopy printouts and MS DOS format files which may be used as input for other PC programs.

  13. Temporal logics and real time expert systems.

    Science.gov (United States)

    Blom, J A

    1996-10-01

    This paper introduces temporal logics. Due to the eternal compromise between expressive adequacy and reasoning efficiency that must decided upon in any application, full (first order logic or modal logic based) temporal logics are frequently not suitable. This is especially true in real time expert systems, where a fixed (and usually small) response time must be guaranteed. One such expert system, Fagan's VM, is reviewed, and a delineation is given of how to formally describe and reason with time in medical protocols. It is shown that Petri net theory is a useful tool to check the correctness of formalised protocols.

  14. CUDA-based real time surgery simulation.

    Science.gov (United States)

    Liu, Youquan; De, Suvranu

    2008-01-01

    In this paper we present a general software platform that enables real time surgery simulation on the newly available compute unified device architecture (CUDA)from NVIDIA. CUDA-enabled GPUs harness the power of 128 processors which allow data parallel computations. Compared to the previous GPGPU, it is significantly more flexible with a C language interface. We report implementation of both collision detection and consequent deformation computation algorithms. Our test results indicate that the CUDA enables a twenty times speedup for collision detection and about fifteen times speedup for deformation computation on an Intel Core 2 Quad 2.66 GHz machine with GeForce 8800 GTX.

  15. Real-time PCR in virology

    OpenAIRE

    Mackay, Ian M.; Arden, Katherine E.; Nitsche, Andreas

    2002-01-01

    The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of P...

  16. Systems Analyze Water Quality in Real Time

    Science.gov (United States)

    2010-01-01

    A water analyzer developed under Small Business Innovation Research (SBIR) contracts with Kennedy Space Center now monitors treatment processes at water and wastewater facilities around the world. Originally designed to provide real-time detection of nutrient levels in hydroponic solutions for growing plants in space, the ChemScan analyzer, produced by ASA Analytics Inc., of Waukesha, Wisconsin, utilizes spectrometry and chemometric algorithms to automatically analyze multiple parameters in the water treatment process with little need for maintenance, calibration, or operator intervention. The company has experienced a compound annual growth rate of 40 percent over its 15-year history as a direct result of the technology's success.

  17. Real-Time Thevenin Impedance Computation

    DEFF Research Database (Denmark)

    Sommer, Stefan Horst; Jóhannsson, Hjörtur

    2013-01-01

    operating state, and strict time constraints are difficult to adhere to as the complexity of the grid increases. Several suggested approaches for real-time stability assessment require Thevenin impedances to be determined for the observed system conditions. By combining matrix factorization, graph reduction......, and parallelization, we develop an algorithm for computing Thevenin impedances an order of magnitude faster than previous approaches. We test the factor-and-solve algorithm with data from several power grids of varying complexity, and we show how the algorithm allows realtime stability assessment of complex power...

  18. Real-time modeling of heat distributions

    Science.gov (United States)

    Hamann, Hendrik F.; Li, Hongfei; Yarlanki, Srinivas

    2018-01-02

    Techniques for real-time modeling temperature distributions based on streaming sensor data are provided. In one aspect, a method for creating a three-dimensional temperature distribution model for a room having a floor and a ceiling is provided. The method includes the following steps. A ceiling temperature distribution in the room is determined. A floor temperature distribution in the room is determined. An interpolation between the ceiling temperature distribution and the floor temperature distribution is used to obtain the three-dimensional temperature distribution model for the room.

  19. Linear Regression Based Real-Time Filtering

    Directory of Open Access Journals (Sweden)

    Misel Batmend

    2013-01-01

    Full Text Available This paper introduces real time filtering method based on linear least squares fitted line. Method can be used in case that a filtered signal is linear. This constraint narrows a band of potential applications. Advantage over Kalman filter is that it is computationally less expensive. The paper further deals with application of introduced method on filtering data used to evaluate a position of engraved material with respect to engraving machine. The filter was implemented to the CNC engraving machine control system. Experiments showing its performance are included.

  20. A real-time Global Warming Index.

    Science.gov (United States)

    Haustein, K; Allen, M R; Forster, P M; Otto, F E L; Mitchell, D M; Matthews, H D; Frame, D J

    2017-11-13

    We propose a simple real-time index of global human-induced warming and assess its robustness to uncertainties in climate forcing and short-term climate fluctuations. This index provides improved scientific context for temperature stabilisation targets and has the potential to decrease the volatility of climate policy. We quantify uncertainties arising from temperature observations, climate radiative forcings, internal variability and the model response. Our index and the associated rate of human-induced warming is compatible with a range of other more sophisticated methods to estimate the human contribution to observed global temperature change.

  1. General purpose computers in real time

    International Nuclear Information System (INIS)

    Biel, J.R.

    1989-01-01

    I see three main trends in the use of general purpose computers in real time. The first is more processing power. The second is the use of higher speed interconnects between computers (allowing more data to be delivered to the processors). The third is the use of larger programs running in the computers. Although there is still work that needs to be done, I believe that all indications are that the online need for general purpose computers should be available for the SCC and LHC machines. 2 figs

  2. Detection of Brazilian hantavirus by reverse transcription polymerase chain reaction amplification of N gene in patients with hantavirus cardiopulmonary syndrome

    OpenAIRE

    Marcos Lázaro Moreli; Ricardo Luiz Moro de Sousa; Luiz Tadeu Moraes Figueiredo

    2004-01-01

    We report a nested reverse transcription-polymerase chain reaction (RT-PCR) assay for hantavirus using primers selected to match high homology regions of hantavirus genomes detected from the whole blood of hantavirus cardiopulmonary syndrome (HCPS) patients from Brazil, also including the N gene nucleotide sequence of Araraquara virus. Hantavirus genomes were detected in eight out of nine blood samples from the HCPS patients by RT-PCR (88.9% positivity) and in all 9 blood samples (100% positi...

  3. Selection of suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using quantitative real-time polymerase chain reaction

    DEFF Research Database (Denmark)

    Zornhagen, K. W.; Kristensen, A. T.; Hansen, Anders Elias

    2015-01-01

    Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a sensitive technique for quantifying gene expression. Stably expressed reference genes are necessary for normalization of RT-qPCR data. Only a few articles have been published on reference genes in canine tumours....... The objective of this study was to demonstrate how to identify suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using RT-qPCR. Primer pairs for 17 potential reference genes were designed and tested in archival tumour biopsies from six dogs. The geNorm algorithm...

  4. Software Design Methods for Real-Time Systems

    Science.gov (United States)

    1989-12-01

    This module describes the concepts and methods used in the software design of real time systems . It outlines the characteristics of real time systems , describes...the role of software design in real time system development, surveys and compares some software design methods for real - time systems , and

  5. Real-time inextensible surgical thread simulation.

    Science.gov (United States)

    Xu, Lang; Liu, Qian

    2018-03-27

    This paper discusses a real-time simulation method of inextensible surgical thread based on the Cosserat rod theory using position-based dynamics (PBD). The method realizes stable twining and knotting of surgical thread while including inextensibility, bending, twisting and coupling effects. The Cosserat rod theory is used to model the nonlinear elastic behavior of surgical thread. The surgical thread model is solved with PBD to achieve a real-time, extremely stable simulation. Due to the one-dimensional linear structure of surgical thread, the direct solution of the distance constraint based on tridiagonal matrix algorithm is used to enhance stretching resistance in every constraint projection iteration. In addition, continuous collision detection and collision response guarantee a large time step and high performance. Furthermore, friction is integrated into the constraint projection process to stabilize the twining of multiple threads and complex contact situations. Through comparisons with existing methods, the surgical thread maintains constant length under large deformation after applying the direct distance constraint in our method. The twining and knotting of multiple threads correspond to stable solutions to contact and friction forces. A surgical suture scene is also modeled to demonstrate the practicality and simplicity of our method. Our method achieves stable and fast simulation of inextensible surgical thread. Benefiting from the unified particle framework, the rigid body, elastic rod, and soft body can be simultaneously simulated. The method is appropriate for applications in virtual surgery that require multiple dynamic bodies.

  6. Real-time applications of neural nets

    International Nuclear Information System (INIS)

    Spencer, J.E.

    1989-05-01

    Producing, accelerating and colliding very high power, low emittance beams for long periods is a formidable problem in real-time control. As energy has grown exponentially in time so has the complexity of the machines and their control systems. Similar growth rates have occurred in many areas, e.g., improved integrated circuits have been paid for with comparable increases in complexity. However, in this case, reliability, capability and cost have improved due to reduced size, high production and increased integration which allow various kinds of feedback. In contrast, most large complex systems (LCS) are perceived to lack such possibilities because only one copy is made. Neural nets, as a metaphor for LCS, suggest ways to circumvent such limitations. It is argued that they are logically equivalent to multi-loop feedback/forward control of faulty systems. While complimentary to AI, they mesh nicely with characteristics desired for real-time systems. Such issues are considered, examples given and possibilities discussed. 21 refs., 6 figs

  7. A real-time radiation mapping system

    International Nuclear Information System (INIS)

    Scoggins, W.A.; VanEtten, D.M.

    1988-01-01

    A prototype of a real-time radiation mapping system, Ranger, was developed to respond to an accident involving the release of plutonium for the Department of Energy's Accident Response Group. In 1987 Ranger demonstrated that it can provide an efficient method of monitoring large areas of land for radioactive contamination. With the experience gained from the operation of the prototype, the external computer and software are being upgraded in order to obtain a fully operational system. The new system uses the prototype's commercially available line-of-sight microwave system for determining position and the same radiation detection instruments. The data obtained from the radiation detection instrument(s) are linked back to the external computer along with the relative position of the measurement through the ranging system. The data are displayed on a gridded map as colored circles and permanently stored in real-time. The different colors represent different contamination levels. Contours can be drawn using the permanently stored data. 4 figs

  8. Real time water chemistry monitoring and diagnostics

    International Nuclear Information System (INIS)

    Gaudreau, T.M.; Choi, S.S.

    2002-01-01

    EPRI has produced a real time water chemistry monitoring and diagnostic system. This system is called SMART ChemWorks and is based on the EPRI ChemWorks codes. System models, chemistry parameter relationships and diagnostic approaches from these codes are integrated with real time data collection, an intelligence engine and Internet technologies to allow for automated analysis of system chemistry. Significant data management capabilities are also included which allow the user to evaluate data and create automated reporting. Additional features have been added to the system in recent years including tracking and evaluation of primary chemistry as well as the calculation and tracking of primary to secondary leakage in PWRs. This system performs virtual sensing, identifies normal and upset conditions, and evaluates the consistency of on-line monitor and grab sample readings. The system also makes use of virtual fingerprinting to identify the cause of any chemistry upsets. This technology employs plant-specific data and models to determine the chemical state of the steam cycle. (authors)

  9. Interfacing real-time information with OILMAP

    International Nuclear Information System (INIS)

    Howlett, E.; Jayko, K.; Spaulding, M.

    1993-01-01

    OILMAP is a state-of-the-art, microcomputer-based oil spill response system applicable to oil spill contingency planning and real-time response for any location in the world. OILMAP has a graphic user interface and was designed in a modular framework so that different spill models could be incorporated into the system, as well as a suite of sophisticated data management tools, without increasing the complexity of the user interface. The basic OILMAP configuration contains a surface trajectory model intended for rapid, first-order estimates of spill movement. A variety of additional models are available within the OILMAP shell to address issues such as weathering, cleanup activities, and probabilities of oiling. A simplified geographic information system (GIS) allows display and manipulation of point, line, and area data geographically referenced to the spill domain. The GIS can import raster data so that images collected by satellite and aerial photography may be displayed. Several new capabilities have been implemented for OILMAP that allow real-time data to be integrated. These features include linking with the OILTRACKER free-floating buoys via a global positioning system, linking of hydrodynamic data from the Ocean Data and Information Network, the Harvard ocean forecasting system, and SeaSonde radar, and the capability of importing spill observations from any remotely sensed data. A further link between OILMAP's GIS and spill models has been developed which allows model predictions to be corrected to observed oil locations while the model runs. 13 refs., 6 figs

  10. Real-time applications of neural nets

    Energy Technology Data Exchange (ETDEWEB)

    Spencer, J.E.

    1989-05-01

    Producing, accelerating and colliding very high power, low emittance beams for long periods is a formidable problem in real-time control. As energy has grown exponentially in time so has the complexity of the machines and their control systems. Similar growth rates have occurred in many areas, e.g., improved integrated circuits have been paid for with comparable increases in complexity. However, in this case, reliability, capability and cost have improved due to reduced size, high production and increased integration which allow various kinds of feedback. In contrast, most large complex systems (LCS) are perceived to lack such possibilities because only one copy is made. Neural nets, as a metaphor for LCS, suggest ways to circumvent such limitations. It is argued that they are logically equivalent to multi-loop feedback/forward control of faulty systems. While complimentary to AI, they mesh nicely with characteristics desired for real-time systems. Such issues are considered, examples given and possibilities discussed. 21 refs., 6 figs.

  11. Real-time applications of neural nets

    International Nuclear Information System (INIS)

    Spencer, J.E.

    1989-01-01

    Producing, accelerating and colliding very high power, low emittance beams for long periods is a formidable problem in real-time control. As energy has grown exponentially in time so has the complexity of the machines and their control systems. Similar growth rates have occurred in many areas e.g. improved integrated circuits have been paid for with comparable increases in complexity. However, in this case, reliability, capability and cost have improved due to reduced size, high production and increased integration which allow various kinds of feedback. In contrast, most large complex systems (LCS) are perceived to lack such possibilities because only one copy is made. Neural nets, as a metaphor for LCS, suggest ways to circumvent such limitations. It is argued that they are logically equivalent to multi-loop feedback/forward control of faulty systems. While complimentary to AI, they mesh nicely with characteristics desired for real-time systems. In this paper, such issues are considered, examples given and possibilities discussed

  12. Detection of mRNA by reverse transcription PCR as an indicator of viability in Phytophthora ramorum

    Science.gov (United States)

    Antonio Chimento; Santa Olga Cacciola; Matteo Garbelotto

    2008-01-01

    Real-Time PCR technologies offer increasing opportunities to detect and study phytopathogenic fungi. They combine the sensitivity of conventional PCR with the generation of a specific fluorescent signal providing both real-time analysis of the reaction kinetics and quantification of specific DNA targets. Before the development of Real-Time PCR and...

  13. Memory controllers for real-time embedded systems predictable and composable real-time systems

    CERN Document Server

    Akesson, Benny

    2012-01-01

      Verification of real-time requirements in systems-on-chip becomes more complex as more applications are integrated. Predictable and composable systems can manage the increasing complexity using formal verification and simulation.  This book explains the concepts of predictability and composability and shows how to apply them to the design and analysis of a memory controller, which is a key component in any real-time system. This book is generally intended for readers interested in Systems-on-Chips with real-time applications.   It is especially well-suited for readers looking to use SDRAM memories in systems with hard or firm real-time requirements. There is a strong focus on real-time concepts, such as predictability and composability, as well as a brief discussion about memory controller architectures for high-performance computing. Readers will learn step-by-step how to go from an unpredictable SDRAM memory, offering highly variable bandwidth and latency, to a predictable and composable shared memory...

  14. Evaluation of Reference Genes for Reverse Transcription Quantitative PCR Studies of Physiological Responses in the Ghost Moth, Thitarodes armoricanus (Lepidoptera, Hepialidae.

    Directory of Open Access Journals (Sweden)

    Guiqing Liu

    Full Text Available Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR is the sensitive method to quantify the expression levels of target genes on the basis of endogenous control. An appropriate reference gene set for normalization is essential for reliable results. The ghost moth, Thitarodes armoricanus, a host species of a medicinal fungus, Ophiocordyceps sinensis, is an economically important member of the Lepidoptera. Recent studies have focused on the mechanism of adaptation of this species to its high-altitude environment and host immune response to O. sinensis infection and RT-qPCR is commonly used in these studies to decipher the genetic basis of physiological functions. However, a thorough assessment of candidate reference genes in the genus Thitarodes is lacking. Here, the expression levels of eight candidate reference genes (ACT, EF, EIF4A, GAPDH, G6PDH, RPL13A, TUB and 18S in T. armoricanus at different developmental stages and in different body parts of the seventh instar larvae were analyzed, along with larvae kept under low temperatures, larvae exposed to two fungal infections and larvae fed different diets. Three established software programs-Bestkeeper, geNorm and NormFinder-were employed to calculate variation among the treatments. The results revealed that the best-suited reference genes differed across the treatments, with EF, EIF4A and GAPDH found to be the best suited for the different developmental stages and larvae body parts; EF, EIF4A and RPL13A found to be the best suited for low-temperature challenge; and EF, EIF4A and TUB found to be the best suited for the fungal infections and dietary treatments. This study thus further contributes to the establishment of an accurate method for normalizing RT-qPCR results for T. armoricanus and serves as a reference for gene expression studies of related insect species.

  15. A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay.

    Science.gov (United States)

    Babu, Binoy; Washburn, Brian K; Ertek, Tülin Sarigül; Miller, Steven H; Riddle, Charles B; Knox, Gary W; Ochoa-Corona, Francisco M; Olson, Jennifer; Katırcıoğlu, Yakup Zekai; Paret, Mathews L

    2017-09-01

    Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early detection and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and are inconsistent in detecting the virus from symptomatic plants. Real-time RT-qPCR assay is highly sensitive for detection of RRV, but it is expensive and requires well-equipped laboratories. Both the RT-PCR and RT-qPCR cannot be used in a field-based testing for RRV. Hence a novel probe based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, using primer/probe designed based on the nucleocapsid gene of the RRV has been developed. The assay is highly specific and did not give a positive reaction to other viruses infecting roses belonging to both inclusive and exclusive genus. Dilution assays using the in vitro transcript showed that the primer/probe set is highly sensitive, with a detection limit of 1 fg/μl. In addition, a rapid technique for the extraction of viral RNA (rose varieties, collected from different states in the U.S. The entire process, including the extraction can be completed in 25min, with less sophisticated equipments. The developed assay can be used with high efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscapes. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Reverse-transcriptional gene expression of anammox and ammonia-oxidizing archaea and bacteria in soybean and rice paddy soils of Northeast China.

    Science.gov (United States)

    Wang, Jing; Dong, Hailiang; Wang, Weidong; Gu, Ji-Dong

    2014-03-01

    The relative gene expression of hydrazine oxidoreductase encoding gene (hzo) for anaerobic ammonium oxidizing bacteria (anammox) and ammonia monooxygenase encoding gene (amoA) for both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in Sanjiang Plain soybean and rice paddy soils of Northeast China was investigated by using real-time reverse-transcriptional quantitative PCR. Metabolically active populations of anammox, AOA, and AOB in rice paddy soils were evident by the presence and successful quantification of hzo mRNA and amoA mRNA genes. The expression ratio of amoA gene for both AOA and AOB varied between soybean soils and different rice paddy soils while the expression of hzo gene for anammox was detectable only in rice paddy soils by showing a diverse relative expression ratio in each soil sample. Gene expression of both archaeal and bacterial amoA genes in rice paddy soils differed among the three sampling depths, but that of hzo was not. Both archaeal and bacterial amoA genes showed an increase trend of expression level with continuation of rice paddy cultivation, but the low expression ratio of hzo gene indicated a relatively small contribution of anammox in overall removal of inorganic nitrogen through N2 even under anoxic and high nitrogen input in agriculture. Bacterial amoA gene from two soybean fields and three rice paddy fields were also analyzed for community composition by denaturing gradient gel electrophoresis fingerprint. Community shift was observed between soybean and paddy fields and within each of them. The consistent occurrence of three bands 5, 6, and 7 in all samples showed their high adaptability for both arid cultivation and continuous rice paddy cultivation. Our data suggest that AOA and AOB are playing a more important role in nitrogen transformation in agricultural soils in oxic or anoxic environment and anammox bacteria may also contribute but in a less extent to N transformation in these agricultural soils

  17. Development of a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction assay for the differential diagnosis of Feline leukemia virus vaccine and wild strains.

    Science.gov (United States)

    Ho, Chia-Fang; Chan, Kun-Wei; Yang, Wei-Cheng; Chiang, Yu-Chung; Chung, Yang-Tsung; Kuo, James; Wang, Chi-Young

    2014-07-01

    A multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) was developed for the differential diagnosis of Feline leukemia virus (FeLV) vaccine and wild-type strains based on a point mutation between the vaccine strain (S) and the wild-type strain (T) located in the p27 gene. This system was further upgraded to obtain a real-time ARMS RT-PCR (ARMS qRT-PCR) with a high-resolution melt analysis (HRMA) platform. The genotyping of various strains of FeLV was determined by comparing the HRMA curves with the defined wild-type FeLV (strain TW1), and the results were expressed as a percentage confidence. The detection limits of ARMS RT-PCR and ARMS qRT-PCR combined with HRMA were 100 and 1 copies of transcribed FeLV RNA per 0.5 ml of sample, respectively. No false-positive results were obtained with 6 unrelated pathogens and 1 feline cell line. Twelve FeLV Taiwan strains were correctly identified using ARMS qRT-PCR combined with HRMA. The genotypes of the strains matched the defined FeLV wild-type strain genotype with at least 91.17% confidence. A higher degree of sequence polymorphism was found throughout the p27 gene compared with the long terminal repeat region. In conclusion, the current study describes the phylogenetic relationship of the FeLV Taiwan strains and demonstrates that the developed ARMS RT-PCR assay is able to be used to detect the replication of a vaccine strain that has not been properly inactivated, thus acting as a safety check for the quality of FeLV vaccines.

  18. Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus.

    Science.gov (United States)

    Ambagala, A; Fisher, M; Goolia, M; Nfon, C; Furukawa-Stoffer, T; Ortega Polo, R; Lung, O

    2017-10-01

    Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, which can decimate the livestock industry and economy of countries previously free of this disease. Rapid detection of foot-and-mouth disease virus (FMDV) is critical to containing an FMD outbreak. Availability of a rapid, highly sensitive and specific, yet simple and field-deployable assay would support local decision-making during an FMDV outbreak. Here we report validation of a novel reverse transcription-insulated isothermal PCR (RT-iiPCR) assay that can be performed on a commercially available, compact and portable POCKIT ™ analyser that automatically analyses data and displays '+' or '-' results. The FMDV RT-iiPCR assay targets the 3D region of the FMDV genome and was capable of detecting 9 copies of in vitro-transcribed RNA standard with 95% confidence. It accurately identified 63 FMDV strains belonging to all seven serotypes and showed no cross-reactivity with viruses causing similar clinical diseases in cloven-hoofed animals. The assay was able to identify FMDV RNA in multiple sample types including oral, nasal and lesion swabs, epithelial tissue suspensions, vesicular and oral fluid samples, even before the appearance of clinical signs. Clinical sensitivity of the assay was comparable or slightly higher than the laboratory-based real-time RT-PCR assay in use. The assay was able to detect FMDV RNA in vesicular fluid samples without nucleic acid extraction. For RNA extraction from more complex sample types, a commercially available taco ™ mini transportable magnetic bead-based, automated extraction system was used. This assay provides a potentially useful field-deployable diagnostic tool for rapid detection of FMDV in an outbreak in FMD-free countries or for routine diagnostics in endemic countries with less structured laboratory systems. © 2016 Her Majesty the Queen in Right of Canada.

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

    Science.gov (United States)

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

    2015-05-04

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

  20. A novel method of multiple nucleic acid detection: Real-time RT-PCR coupled with probe-melting curve analysis.

    Science.gov (United States)

    Han, Yang; Hou, Shao-Yang; Ji, Shang-Zhi; Cheng, Juan; Zhang, Meng-Yue; He, Li-Juan; Ye, Xiang-Zhong; Li, Yi-Min; Zhang, Yi-Xuan

    2017-11-15

    A novel method, real-time reverse transcription PCR (real-time RT-PCR) coupled with probe-melting curve analysis, has been established to detect two kinds of samples within one fluorescence channel. Besides a conventional TaqMan probe, this method employs another specially designed melting-probe with a 5' terminus modification which meets the same label with the same fluorescent group. By using an asymmetric PCR method, the melting-probe is able to detect an extra sample in the melting stage effectively while it almost has little influence on the amplification detection. Thus, this method allows the availability of united employment of both amplification stage and melting stage for detecting samples in one reaction. The further demonstration by simultaneous detection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in one channel as a model system is presented in this essay. The sensitivity of detection by real-time RT-PCR coupled with probe-melting analysis was proved to be equal to that detected by conventional real-time RT-PCR. Because real-time RT-PCR coupled with probe-melting analysis can double the detection throughputs within one fluorescence channel, it is expected to be a good solution for the problem of low-throughput in current real-time PCR. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Standardization and application of real-time polymerase chain reaction for rapid detection of bluetongue virus

    Directory of Open Access Journals (Sweden)

    I. Karthika Lakshmi

    2018-04-01

    Full Text Available Aim: The present study was designed to standardize real-time polymerase chain reaction (PCR for detecting the bluetongue virus from blood samples of sheep collected during outbreaks of bluetongue disease in the year 2014 in Andhra Pradesh and Telangana states of India. Materials and Methods: A 10-fold serial dilution of Plasmid PUC59 with bluetongue virus (BTV NS3 insert was used to plot the standard curve. BHK-21 and KC cells were used for in vitro propagation of virus BTV-9 at a TCID50/ml of 105 ml and RNA was isolated by the Trizol method. Both reverse transcription -PCR and real-time PCR using TaqMan probe were carried out with RNA extracted from virus-spiked culture medium and blood to compare the sensitivity by means of finding out the limit of detection (LoD. The results were verified by inoculating the detected and undetected dilutions onto cell cultures with further cytological (cytopathic effect and molecular confirmation (by BTV-NS1 group-specific PCR. The standardized technique was then applied to field samples (blood for detecting BTV. Results: The slope of the standard curve obtained was -3.23, and the efficiency was 103%. The LoD with RT-PCR was 8.269Ex103 number of copies of plasmid, whereas it was 13 with real-time PCR for plasmid dilutions. Similarly, LoD was determined for virus-spiked culture medium, and blood with both the types of PCR and the values were 103 TCID 50/ml and 104 TCID 50/ml with RT-PCR and 10° TCID 50/ml and 102 TCID 50/ml with real-time PCR, respectively. The standardized technique was applied to blood samples collected from BTV suspected animals; 10 among 20 samples were found positive with Cq values ranging from 27 to 39. The Cq value exhibiting samples were further processed in cell cultures and were confirmed to be BT positive. Likewise, Cq undetected samples on processing in cell cultures turned out to be BTV negative. Conclusion: Real-time PCR was found to be a very sensitive as well as reliable method

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

    Directory of Open Access Journals (Sweden)

    Pravas Ranjan Sahoo

    2016-05-01

    Full Text Available 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.

  3. 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.

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

    Science.gov (United States)

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

    2013-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Gardner Shea N

    2011-06-01

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

  6. Urine sample used for detection of toxoplasma gondii infection by loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Hu, Xin; Pan, Chang-Wang; Li, Ya-Fei; Wang, Han; Tan, Feng

    2012-02-01

    In this study, a loop-mediated isothermal amplification (LAMP) assay was established to detect Toxoplasma gondii DNA in mice infected with T. gondii PRU strain. This LAMP assay was based on the sequence of highly repetitive B1 gene. The detection limit of T. gondii LAMP assay was 1 pg of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. The LAMP assay was also highly specific for T. gondii and able to detect T. gondii DNA in urine of mice treated with dexamethasone at 90 day post infection (p.i.), although this assay could not detect the DNA in mice urine 2-6 days p.i. These results demonstrated that LAMP is effective for evaluation of therapy effectiveness for T. gondii infection. The established LAMP assay may represent a useful and practical tool for the routine diagnosis and therapeutic evaluation of human toxoplasmosis.

  7. Loop-mediated isothermal amplification assay for rapid detection of Streptococcus agalactiae (group B streptococcus) in vaginal swabs - a proof of concept study.

    Science.gov (United States)

    McKenna, James Patrick; Cox, Ciara; Fairley, Derek John; Burke, Rachael; Shields, Michael D; Watt, Alison; Coyle, Peter Valentine

    2017-03-01

    Neonatal sepsis caused by Streptococcus agalactiae [group B streptococcus (GBS)] is a life-threatening condition, which is preventable if colonized mothers are identified and given antibiotic prophylaxis during labour. Conventional culture is time consuming and unreliable, and many available non-culture diagnostics are too complex to implement routinely at point of care. Loop-mediated isothermal amplification (LAMP) is a method that, enables the rapid and specific detection of target nucleic acid sequences in clinical materials without the requirement for extensive sample preparation. A prototype LAMP assay targeting GBS sip gene is described. The assay was 100 % specific for GBS, with a limit of detection of 14 genome copies per reaction. The clinical utility of the LAMP assay for rapid direct molecular detection of GBS was determined by testing a total of 157 vaginal swabs with minimal sample processing using a rapid lysis solution. Compared to a reference quantitative real-time PCR assay, the direct LAMP protocol had a sensitivity and specificity of 95.4 and 100 %, respectively, with positive and negative predictive values of 100 and 98.3 %, respectively. Positive and negative likelihood ratios were infinity and 0.05, respectively. The direct LAMP method required a mean time of 45 min from the receipt of a swab to generation of a confirmed result, compared to 2 h 30 min for the reference quantitative real-time PCR test. The direct LAMP protocol described is easy to perform, facilitating rapid and accurate detection of GBS in vaginal swabs. This test has a potential for use at point of care.

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

    Directory of Open Access Journals (Sweden)

    Ponce Camille

    2017-01-01

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

  9. Robust synthesis for real-time systems

    DEFF Research Database (Denmark)

    Larsen, Kim Guldstrand; Legay, Axel; Traonouez, Luois-Marie

    2014-01-01

    Specification theories for real-time systems allow reasoning about interfaces and their implementation models, using a set of operators that includes satisfaction, refinement, logical and parallel composition. To make such theories applicable throughout the entire design process from an abstract...... of introducing small perturbations into formal models. We address this problem of robust implementations in timed specification theories. We first consider a fixed perturbation and study the robustness of timed specifications with respect to the operators of the theory. To this end we synthesize robust...... specification to an implementation, we need to reason about the possibility to effectively implement the theoretical specifications on physical systems, despite their limited precision. In the literature, this implementation problem has been linked to the robustness problem that analyzes the consequences...

  10. Real time speech formant analyzer and display

    Science.gov (United States)

    Holland, George E.; Struve, Walter S.; Homer, John F.

    1987-01-01

    A speech analyzer for interpretation of sound includes a sound input which converts the sound into a signal representing the sound. The signal is passed through a plurality of frequency pass filters to derive a plurality of frequency formants. These formants are converted to voltage signals by frequency-to-voltage converters and then are prepared for visual display in continuous real time. Parameters from the inputted sound are also derived and displayed. The display may then be interpreted by the user. The preferred embodiment includes a microprocessor which is interfaced with a television set for displaying of the sound formants. The microprocessor software enables the sound analyzer to present a variety of display modes for interpretive and therapeutic used by the user.

  11. CONSIDERATIONS ON REAL TIME DATA WAREHOUSING (RTDW

    Directory of Open Access Journals (Sweden)

    Marius Bogdan DINU

    2014-05-01

    Full Text Available The RTDW concept originated in the early 2000s. By that time, computing power had increased to a level that was allowing extraction of data collections for reporting purposes. Such collections were used almost in real time and at speeds nearly comparable to what an operation system was capable to deliver. The main idea will be to eliminate some of the components of the classic extraction process which is basically the most costly factor less time - consuming. We anticipate that the following factors will be decisive: elimination of batch-type processes [1], data compression techniques, data capture techniques, ability to keep in cache a large volume of data, parallel processing, and data mining algorithms that can adapt to such applications.

  12. Real-Time Optical Antimicrobial Susceptibility Testing

    DEFF Research Database (Denmark)

    Fredborg, Marlene; Andersen, Klaus R; Jørgensen, Erik

    2013-01-01

    Rapid antibiotic susceptibility testing is in highly demand in health-care fields as antimicrobial resistant bacterial strains emerge and spread. Here we describe an optical screening system (oCelloScope), which based on time-lapse imaging of 96 bacteria-antibiotic combinations at a time......, introduces real-time detection of bacterial growth and antimicrobial susceptibility, with imaging material to support the automatically generated graphs. Automated antibiotic susceptibility tests of a monoculture showed statistically significant antibiotic effect within 6 minutes and within 30 minutes...... from multidrug-resistant pathogenic bacteria. The oCelloScope system can be employed for a broad range of applications within bacteriology and may present new vistas as a point-of-care instrument in both clinical and veterinarian settings....

  13. Operational and real-time Business Intelligence

    Directory of Open Access Journals (Sweden)

    Daniela Ioana SANDU

    2008-01-01

    Full Text Available A key component of a company’s IT framework is a business intelligence (BI system. BI enables business users to report on, analyze and optimize business operations to reduce costs and increase revenues. Organizations use BI for strategic and tactical decision making where the decision-making cycle may span a time period of several weeks (e.g., campaign management or months (e.g., improving customer satisfaction.Competitive pressures coming from a very dynamic business environment are forcing companies to react faster to changing business conditions and customer requirements. As a result, there is now a need to use BI to help drive and optimize business operations on a daily basis, and, in some cases, even for intraday decision making. This type of BI is usually called operational business intelligence and real-time business intelligence.

  14. Performance evaluation of real time radiographic systems

    International Nuclear Information System (INIS)

    Venkatraman, B.; Saravanan, S.; Jayakumar, T.; Kalyanasundaram, P.; Baldev Raj

    1996-01-01

    The Real Time Radiography (RTR) system can be studied completely by knowing the modulation transfer function (MTF) of the whole system. The MTF curve is a special form of contrast/detail-size diagram in which the image contrast is plotted against the spatial frequency of a test object measured in line-pairs per millimetre (lp/mm). MTF curves are widely used to measure the characteristics of optical equipment, particularly for assessing the contribution of individual items in a complex imaging transfer system. Codes of practice indicate that the image intensifier systems should be checked periodically to assess its performance through the use of MTF curves and step wedges for contrast ratio. Authors, instead, suggest the use of performance curves which are simple to obtain and can be easily interpreted by radiographers. (author)

  15. Real time ray tracing based on shader

    Science.gov (United States)

    Gui, JiangHeng; Li, Min

    2017-07-01

    Ray tracing is a rendering algorithm for generating an image through tracing lights into an image plane, it can simulate complicate optical phenomenon like refraction, depth of field and motion blur. Compared with rasterization, ray tracing can achieve more realistic rendering result, however with greater computational cost, simple scene rendering can consume tons of time. With the GPU's performance improvement and the advent of programmable rendering pipeline, complicated algorithm can also be implemented directly on shader. So, this paper proposes a new method that implement ray tracing directly on fragment shader, mainly include: surface intersection, importance sampling and progressive rendering. With the help of GPU's powerful throughput capability, it can implement real time rendering of simple scene.

  16. Kalman Filtering with Real-Time Applications

    CERN Document Server

    Chui, Charles K

    2009-01-01

    Kalman Filtering with Real-Time Applications presents a thorough discussion of the mathematical theory and computational schemes of Kalman filtering. The filtering algorithms are derived via different approaches, including a direct method consisting of a series of elementary steps, and an indirect method based on innovation projection. Other topics include Kalman filtering for systems with correlated noise or colored noise, limiting Kalman filtering for time-invariant systems, extended Kalman filtering for nonlinear systems, interval Kalman filtering for uncertain systems, and wavelet Kalman filtering for multiresolution analysis of random signals. Most filtering algorithms are illustrated by using simplified radar tracking examples. The style of the book is informal, and the mathematics is elementary but rigorous. The text is self-contained, suitable for self-study, and accessible to all readers with a minimum knowledge of linear algebra, probability theory, and system engineering.

  17. Optimal, real-time control--colliders

    International Nuclear Information System (INIS)

    Spencer, J.E.

    1991-05-01

    With reasonable definitions, optimal control is possible for both classical and quantal systems with new approaches called PISC(Parallel) and NISC(Neural) from analogy with RISC (Reduced Instruction Set Computing). If control equals interaction, observation and comparison to some figure of merit with interaction via external fields, then optimization comes from varying these fields to give design or operating goals. Structural stability can then give us tolerance and design constraints. But simulations use simplified models, are not in real-time and assume fixed or stationary conditions, so optimal control goes far beyond convergence rates of algorithms. It is inseparable from design and this has many implications for colliders. 12 refs., 3 figs

  18. Real-time rockmass response from microseismics

    Energy Technology Data Exchange (ETDEWEB)

    Andrew King; Michael Lofgren; Matt van de Werken [CSIRO Exploration and Mining (Australia)

    2009-06-15

    The primary objective of this project was to develop a prototype real-time microseismic monitoring system for strata control management and forewarning of geotechnical hazards. Power and communications problems have been addressed by developing a wirelessly connected network of solar-powered acquisition nodes, one at the top of each instrumented borehole. The open-source 'earthworm' earthquake acquisition software, which can run on different hardware platforms and use different acquisition cards, was modified for use in a coal environment by developing special new arrival-picking and event-location procedures. The system was field-trialled at Moranbah North mine. The acquisition software performed well, as did wireless communications and solar power. There were issues with the acquisition hardware selected, including problems with timing synchronisation, which is essential for seismic event location. Although these were fixed during the test, different hardware is likely to be used in future installations.

  19. Analyzer of neutron flux in real time

    International Nuclear Information System (INIS)

    Rojas S, A.S.; Carrillo M, R.A.; Balderas, E.G.

    1999-01-01

    With base in the study of the real signals of neutron flux of instability events occurred in the Laguna Verde nuclear power plant where the nucleus oscillation phenomena of the reactor are in the 0 to 2.5 Hz range, it has been seen the possibility about the development a surveillance and diagnostic equipment capable to analyze in real time the behavior of nucleus in this frequencies range. An important method for surveillance the stability of the reactor nucleus is the use of the Power spectral density which allows to determine the frequencies and amplitudes contained in the signals. It is used an instrument carried out by LabVIEW graphic programming with a data acquisition card of 16 channels which works at Windows 95/98 environment. (Author)

  20. Development of the real time monitor system

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Katsumi [Research Organization for Information Science and Technology, Tokai, Ibaraki (Japan); Watanabe, Tadashi; Kaburaki, Hideo

    1996-10-01

    Large-scale simulation technique is studied at the Center for Promotion of Computational Science and Engineering (CCSE) for the computational science research in nuclear fields. Visualization and animation processing technique are studied and developed for efficient understanding of simulation results. The real time monitor system, in which on-going simulation results are transferred from a supercomputer or workstation to a graphic workstation and are visualized and recorded, is described in this report. This system is composed of the graphic workstation and the video equipment connected to the network. The control shell programs are the job-execution shell for simulations on supercomputers, the file-transfer shell for output files for visualization, and the shell for starting visualization tools. Special image processing technique and hardware are not necessary in this system and the standard visualization tool AVS and the UNIX commands are used, so that this system can be implemented and applied in various computer environments. (author)

  1. Embedded and real-time operating systems

    CERN Document Server

    Wang, K C

    2017-01-01

    This book covers the basic concepts and principles of operating systems, showing how to apply them to the design and implementation of complete operating systems for embedded and real-time systems. It includes all the foundational and background information on ARM architecture, ARM instructions and programming, toolchain for developing programs, virtual machines for software implementation and testing, program execution image, function call conventions, run-time stack usage and link C programs with assembly code. It describes the design and implementation of a complete OS for embedded systems in incremental steps, explaining the design principles and implementation techniques. For Symmetric Multiprocessing (SMP) embedded systems, the author examines the ARM MPcore processors, which include the SCU and GIC for interrupts routing and interprocessor communication and synchronization by Software Generated Interrupts (SGIs). Throughout the book, complete working sample systems demonstrate the design principles and...

  2. Real-time Human Activity Recognition

    Science.gov (United States)

    Albukhary, N.; Mustafah, Y. M.

    2017-11-01

    The traditional Closed-circuit Television (CCTV) system requires human to monitor the CCTV for 24/7 which is inefficient and costly. Therefore, there’s a need for a system which can recognize human activity effectively in real-time. This paper concentrates on recognizing simple activity such as walking, running, sitting, standing and landing by using image processing techniques. Firstly, object detection is done by using background subtraction to detect moving object. Then, object tracking and object classification are constructed so that different person can be differentiated by using feature detection. Geometrical attributes of tracked object, which are centroid and aspect ratio of identified tracked are manipulated so that simple activity can be detected.

  3. The RHIC real time data link system

    International Nuclear Information System (INIS)

    Hartmann, H.

    1997-01-01

    The RHIC Real Time Data Link (RTDL) System distributes to all locations around the RHIC ring machine parameters of general interest to accelerator systems and users. The system, along with supporting host interface, is centrally located. The RTDL System is comprised of two module types: the Encoder Module (V105) and the Input Module (V106). There is only one V105 module, but many (up to 128) Input Modules. Multiple buffered outputs are provided for use locally or for retransmission to other RHIC equipment locations. Machine parameters are generated from the V115 Waveform Generator Module (WFG) or from machine hardware and coupled directly through a fiber optic serial link to one of the V106 input channels

  4. REAL TIME DATA FOR REMEDIATION ACTIVITIES (11505)

    International Nuclear Information System (INIS)

    Brock, C.T.

    2011-01-01

    Health physicists from the CH2M HILL Plateau Remediation Company collaborated with Berkeley Nucleonics Corporation to modify the SAM 940 isotope identifier instrument to be used for nuclear waste remediation. These modifications coupled with existing capabilities of the SAM 940 have proven to be invaluable during remediation activities, reducing disposal costs by allowing swift remediation of targeted areas that have been identified as having isotopes of concern (IOC), and eliminating multiple visits to sites by declaring an excavation site clear of IOCs before demobilizing from the site. These advantages are enabled by accumulating spectral data for specific isotopes that is nearly 100 percent free of false positives, which are filtered out in 'real time.'

  5. A distributed real-time operating system

    International Nuclear Information System (INIS)

    Tuynman, F.; Hertzberger, L.O.

    1984-07-01

    A distributed real-time operating system, Fados, has been developed for an embedded multi-processor system. The operating system is based on a host target approach and provides for communication between arbitrary processes on host and target machine. The facilities offered are, apart from process communication, access to the file system on the host by programs on the target machine and monitoring and debugging of programs on the target machine from the host. The process communication has been designed in such a way that the possibilities are the same as those offered by the Ada programming language. The operating system is implemented on a MC 68000 based multiprocessor system in combination with a Unix host. (orig.)

  6. REAL TIME SPEED ESTIMATION FROM MONOCULAR VIDEO

    Directory of Open Access Journals (Sweden)

    M. S. Temiz

    2012-07-01

    Full Text Available In this paper, detailed studies have been performed for developing a real time system to be used for surveillance of the traffic flow by using monocular video cameras to find speeds of the vehicles for secure travelling are presented. We assume that the studied road segment is planar and straight, the camera is tilted downward a bridge and the length of one line segment in the image is known. In order to estimate the speed of a moving vehicle from a video camera, rectification of video images is performed to eliminate the perspective effects and then the interest region namely the ROI is determined for tracking the vehicles. Velocity vectors of a sufficient number of reference points are identified on the image of the vehicle from each video frame. For this purpose sufficient number of points from the vehicle is selected, and these points must be accurately tracked on at least two successive video frames. In the second step, by using the displacement vectors of the tracked points and passed time, the velocity vectors of those points are computed. Computed velocity vectors are defined in the video image coordinate system and displacement vectors are measured by the means of pixel units. Then the magnitudes of the computed vectors in the image space are transformed to the object space to find the absolute values of these magnitudes. The accuracy of the estimated speed is approximately ±1 – 2 km/h. In order to solve the real time speed estimation problem, the authors have written a software system in C++ programming language. This software system has been used for all of the computations and test applications.

  7. Real-time visualization of joint cavitation.

    Directory of Open Access Journals (Sweden)

    Gregory N Kawchuk

    Full Text Available Cracking sounds emitted from human synovial joints have been attributed historically to the sudden collapse of a cavitation bubble formed as articular surfaces are separated. Unfortunately, bubble collapse as the source of joint cracking is inconsistent with many physical phenomena that define the joint cracking phenomenon. Here we present direct evidence from real-time magnetic resonance imaging that the mechanism of joint cracking is related to cavity formation rather than bubble collapse. In this study, ten metacarpophalangeal joints were studied by inserting the finger of interest into a flexible tube tightened around a length of cable used to provide long-axis traction. Before and after traction, static 3D T1-weighted magnetic resonance images were acquired. During traction, rapid cine magnetic resonance images were obtained from the joint midline at a rate of 3.2 frames per second until the cracking event occurred. As traction forces increased, real-time cine magnetic resonance imaging demonstrated rapid cavity inception at the time of joint separation and sound production after which the resulting cavity remained visible. Our results offer direct experimental evidence that joint cracking is associated with cavity inception rather than collapse of a pre-existing bubble. These observations are consistent with tribonucleation, a known process where opposing surfaces resist separation until a critical point where they then separate rapidly creating sustained gas cavities. Observed previously in vitro, this is the first in-vivo macroscopic demonstration of tribonucleation and as such, provides a new theoretical framework to investigate health outcomes associated with joint cracking.

  8. Real Time Monitor of Grid job executions

    International Nuclear Information System (INIS)

    Colling, D J; Martyniak, J; McGough, A S; Krenek, A; Sitera, J; Mulac, M; Dvorak, F

    2010-01-01

    In this paper we describe the architecture and operation of the Real Time Monitor (RTM), developed by the Grid team in the HEP group at Imperial College London. This is arguably the most popular dissemination tool within the EGEE [1] Grid. Having been used, on many occasions including GridFest and LHC inauguration events held at CERN in October 2008. The RTM gathers information from EGEE sites hosting Logging and Bookkeeping (LB) services. Information is cached locally at a dedicated server at Imperial College London and made available for clients to use in near real time. The system consists of three main components: the RTM server, enquirer and an apache Web Server which is queried by clients. The RTM server queries the LB servers at fixed time intervals, collecting job related information and storing this in a local database. Job related data includes not only job state (i.e. Scheduled, Waiting, Running or Done) along with timing information but also other attributes such as Virtual Organization and Computing Element (CE) queue - if known. The job data stored in the RTM database is read by the enquirer every minute and converted to an XML format which is stored on a Web Server. This decouples the RTM server database from the client removing the bottleneck problem caused by many clients simultaneously accessing the database. This information can be visualized through either a 2D or 3D Java based client with live job data either being overlaid on to a 2 dimensional map of the world or rendered in 3 dimensions over a globe map using OpenGL.

  9. Development of a primer–probe energy transfer based real-time PCR for the detection of Swine influenza virus

    DEFF Research Database (Denmark)

    Kowalczyk, Andrzej; Markowska-Daniel, Iwona; Rasmussen, Thomas Bruun

    2013-01-01

    Swine influenza virus (SIV) causes a contagious and requiring official notification disease of pigs and humans. In this study, a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay based on primer–probe energy transfer (PriProET) for the detection of SIV RNA was developed...... of the specific product amplification. The assay is specific for influenza virus with a sensitivity of detection limit of approximately 10 copies of RNA by PCR. Based on serial dilutions of SIV, the detection limit of the assay was approximately 0.003 TCID50/ml for H1N1 A/Swine/Poland/KPR9/2004 virus. The Pri...

  10. Development of a Real-Time Resistance Measurement for Vibrio parahaemolyticus Detection by the Lecithin-Dependent Hemolysin Gene

    Science.gov (United States)

    Xiang, Guiming; Pu, Xiaoyun; Jiang, Dongneng; Liu, Linlin; Liu, Chang; Liu, Xiaobo

    2013-01-01

    The marine bacterium Vibrio parahaemolyticus (V. parahaemolyticus) causes gastroenteritis in humans via the ingestion of raw or undercooked contaminated seafood, and early diagnosis and prompt treatment are important for the prevention of V. parahaemolyticus-related diseases. In this study, a real-time resistance measurement based on loop-mediated isothermal amplification (LAMP), electrochemical ion bonding (Crystal violet and Mg2+), real-time monitoring, and derivative analysis was developed. V. parahaemolyticus DNA was first amplified by LAMP, and the products (DNA and pyrophosphate) represented two types of negative ions that could combine with a positive dye (Crystal violet) and positive ions (Mg2+) to increase the resistance of the reaction liquid. This resistance was measured in real-time using a specially designed resistance electrode, thus permitting the quantitative detection of V. parahaemolyticus. The results were obtained in 1–2 hours, with a minimum bacterial density of 10 CFU.mL−1 and high levels of accuracy (97%), sensitivity (96.08%), and specificity (97.96%) when compared to cultivation methods. Therefore, this simple and rapid method has a potential application in the detection of V. parahaemolyticus on a gene chip or in point-of-care testing. PMID:23991096

  11. Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics.

    Science.gov (United States)

    Zambenedetti, Miriam Ribas; Pavoni, Daniela Parada; Dallabona, Andreia Cristine; Dominguez, Alejandro Correa; Poersch, Celina de Oliveira; Fragoso, Stenio Perdigão; Krieger, Marco Aurélio

    2017-05-01

    Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

  12. Approaching near real-time biosensing: microfluidic microsphere based biosensor for real-time analyte detection.

    Science.gov (United States)

    Cohen, Noa; Sabhachandani, Pooja; Golberg, Alexander; Konry, Tania

    2015-04-15

    In this study we describe a simple lab-on-a-chip (LOC) biosensor approach utilizing well mixed microfluidic device and a microsphere-based assay capable of performing near real-time diagnostics of clinically relevant analytes such cytokines and antibodies. We were able to overcome the adsorption kinetics reaction rate-limiting mechanism, which is diffusion-controlled in standard immunoassays, by introducing the microsphere-based assay into well-mixed yet simple microfluidic device with turbulent flow profiles in the reaction regions. The integrated microsphere-based LOC device performs dynamic detection of the analyte in minimal amount of biological specimen by continuously sampling micro-liter volumes of sample per minute to detect dynamic changes in target analyte concentration. Furthermore we developed a mathematical model for the well-mixed reaction to describe the near real time detection mechanism observed in the developed LOC method. To demonstrate the specificity and sensitivity of the developed real time monitoring LOC approach, we applied the device for clinically relevant analytes: Tumor Necrosis Factor (TNF)-α cytokine and its clinically used inhibitor, anti-TNF-α antibody. Based on the reported results herein, the developed LOC device provides continuous sensitive and specific near real-time monitoring method for analytes such as cytokines and antibodies, reduces reagent volumes by nearly three orders of magnitude as well as eliminates the washing steps required by standard immunoassays. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Application of XML in real-time data warehouse

    Science.gov (United States)

    Zhao, Yanhong; Wang, Beizhan; Liu, Lizhao; Ye, Su

    2009-07-01

    At present, XML is one of the most widely-used technologies of data-describing and data-exchanging, and the needs for real-time data make real-time data warehouse a popular area in the research of data warehouse. What effects can we have if we apply XML technology to the research of real-time data warehouse? XML technology solves many technologic problems which are impossible to be addressed in traditional real-time data warehouse, and realize the integration of OLAP (On-line Analytical Processing) and OLTP (Online transaction processing) environment. Then real-time data warehouse can truly be called "real time".

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

    Science.gov (United States)

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

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

    NARCIS (Netherlands)

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

    2017-01-01

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

  16. Real Time Seismic Prediction while Drilling

    Science.gov (United States)

    Schilling, F. R.; Bohlen, T.; Edelmann, T.; Kassel, A.; Heim, A.; Gehring, M.; Lüth, S.; Giese, R.; Jaksch, K.; Rechlin, A.; Kopf, M.; Stahlmann, J.; Gattermann, J.; Bruns, B.

    2009-12-01

    Efficient and safe drilling is a prerequisite to enhance the mobility of people and goods, to improve the traffic as well as utility infrastructure of growing megacities, and to ensure the growing energy demand while building geothermal and in hydroelectric power plants. Construction within the underground is often building within the unknown. An enhanced risk potential for people and the underground building may arise if drilling enters fracture zones, karsts, brittle rocks, mixed solid and soft rocks, caves, or anthropogenic obstacles. Knowing about the material behavior ahead of the drilling allows reducing the risk during drilling and construction operation. In drilling operations direct observations from boreholes can be complemented with geophysical investigations. In this presentation we focus on “real time” seismic prediction while drilling which is seen as a prerequisite while using geophysical methods in modern drilling operations. In solid rocks P- and S-wave velocity, refraction and reflection as well as seismic wave attenuation can be used for the interpretation of structures ahead of the drilling. An Integrated Seismic Imaging System (ISIS) for exploration ahead of a construction is used, where a pneumatic hammer or a magnetostrictive vibration source generate repetitive signals behind the tunneling machine. Tube waves are generated which travel along the tunnel to the working face. There the tube waves are converted to mainly S- but also P-Waves which interact with the formation ahead of the heading face. The reflected or refracted waves travel back to the working front are converted back to tube waves and recorded using three-component geophones which are fit into the tips of anchor rods. In near real time, the ISIS software allows for an integrated 3D imaging and interpretation of the observed data, geological and geotechnical parameters. Fracture zones, heterogeneities, and variations in the rock properties can be revealed during the drilling

  17. Real Time Earthquake Information System in Japan

    Science.gov (United States)

    Doi, K.; Kato, T.

    2003-12-01

    An early earthquake notification system in Japan had been developed by the Japan Meteorological Agency (JMA) as a governmental organization responsible for issuing earthquake information and tsunami forecasts. The system was primarily developed for prompt provision of a tsunami forecast to the public with locating an earthquake and estimating its magnitude as quickly as possible. Years after, a system for a prompt provision of seismic intensity information as indices of degrees of disasters caused by strong ground motion was also developed so that concerned governmental organizations can decide whether it was necessary for them to launch emergency response or not. At present, JMA issues the following kinds of information successively when a large earthquake occurs. 1) Prompt report of occurrence of a large earthquake and major seismic intensities caused by the earthquake in about two minutes after the earthquake occurrence. 2) Tsunami forecast in around three minutes. 3) Information on expected arrival times and maximum heights of tsunami waves in around five minutes. 4) Information on a hypocenter and a magnitude of the earthquake, the seismic intensity at each observation station, the times of high tides in addition to the expected tsunami arrival times in 5-7 minutes. To issue information above, JMA has established; - An advanced nationwide seismic network with about 180 stations for seismic wave observation and about 3,400 stations for instrumental seismic intensity observation including about 2,800 seismic intensity stations maintained by local governments, - Data telemetry networks via landlines and partly via a satellite communication link, - Real-time data processing techniques, for example, the automatic calculation of earthquake location and magnitude, the database driven method for quantitative tsunami estimation, and - Dissemination networks, via computer-to-computer communications and facsimile through dedicated telephone lines. JMA operationally

  18. The INGV Real Time Strong Motion Database

    Science.gov (United States)

    Massa, Marco; D'Alema, Ezio; Mascandola, Claudia; Lovati, Sara; Scafidi, Davide; Gomez, Antonio; Carannante, Simona; Franceschina, Gianlorenzo; Mirenna, Santi; Augliera, Paolo

    2017-04-01

    The INGV real time strong motion data sharing is assured by the INGV Strong Motion Database. ISMD (http://ismd.mi.ingv.it) was designed in the last months of 2011 in cooperation among different INGV departments, with the aim to organize the distribution of the INGV strong-motion data using standard procedures for data acquisition and processing. The first version of the web portal was published soon after the occurrence of the 2012 Emilia (Northern Italy), Mw 6.1, seismic sequence. At that time ISMD was the first European real time web portal devoted to the engineering seismology community. After four years of successfully operation, the thousands of accelerometric waveforms collected in the archive need necessary a technological improvement of the system in order to better organize the new data archiving and to make more efficient the answer to the user requests. ISMD 2.0 was based on PostgreSQL (www.postgresql.org), an open source object- relational database. The main purpose of the web portal is to distribute few minutes after the origin time the accelerometric waveforms and related metadata of the Italian earthquakes with ML≥3.0. Data are provided both in raw SAC (counts) and automatically corrected ASCII (gal) formats. The web portal also provide, for each event, a detailed description of the ground motion parameters (i.e. Peak Ground Acceleration, Velocity and Displacement, Arias and Housner Intensities) data converted in velocity and displacement, response spectra up to 10.0 s and general maps concerning the recent and the historical seismicity of the area together with information about its seismic hazard. The focal parameters of the events are provided by the INGV National Earthquake Center (CNT, http://cnt.rm.ingv.it). Moreover, the database provides a detailed site characterization section for each strong motion station, based on geological, geomorphological and geophysical information. At present (i.e. January 2017), ISMD includes 987 (121

  19. RTMOD: Real-Time MODel evaluation

    International Nuclear Information System (INIS)

    Graziani, G; Galmarini, S.; Mikkelsen, T.

    2000-01-01

    The 1998 - 1999 RTMOD project is a system based on an automated statistical evaluation for the inter-comparison of real-time forecasts produced by long-range atmospheric dispersion models for national nuclear emergency predictions of cross-boundary consequences. The background of RTMOD was the 1994 ETEX project that involved about 50 models run in several Institutes around the world to simulate two real tracer releases involving a large part of the European territory. In the preliminary phase of ETEX, three dry runs (i.e. simulations in real-time of fictitious releases) were carried out. At that time, the World Wide Web was not available to all the exercise participants, and plume predictions were therefore submitted to JRC-Ispra by fax and regular mail for subsequent processing. The rapid development of the World Wide Web in the second half of the nineties, together with the experience gained during the ETEX exercises suggested the development of this project. RTMOD featured a web-based user-friendly interface for data submission and an interactive program module for displaying, intercomparison and analysis of the forecasts. RTMOD has focussed on model intercomparison of concentration predictions at the nodes of a regular grid with 0.5 degrees of resolution both in latitude and in longitude, the domain grid extending from 5W to 40E and 40N to 65N. Hypothetical releases were notified around the world to the 28 model forecasters via the web on a one-day warning in advance. They then accessed the RTMOD web page for detailed information on the actual release, and as soon as possible they then uploaded their predictions to the RTMOD server and could soon after start their inter-comparison analysis with other modelers. When additional forecast data arrived, already existing statistical results would be recalculated to include the influence by all available predictions. The new web-based RTMOD concept has proven useful as a practical decision-making tool for realtime

  20. Business Hypervisors for Real-time Applications

    Directory of Open Access Journals (Sweden)

    L. Perneel

    2015-08-01

    Full Text Available System virtualization is one of the hottest trends in information technology today. It is not just another nice to use technology but has become fundamental across the business world. It is successfully used with many business application classes where cloud computing is the most visual one. Recently, it started to be used for soft Real-Time (RT applications such as IP telephony, media servers, audio and video streaming servers, automotive and communication systems in general. Running these applications on a traditional system (Hardware + Operating System guarantee their Quality of Service (QoS; virtualizing them means inserting a new layer between the hardware and the (virtual Operating System (OS, and thus adding extra overhead. Although these applications’ areas do not always demand hard time guarantees, they require the underlying virtualization layer supports low latency and provide adequate computational resources for completion within a reasonable or predictable timeframe. These aspects are intimately intertwined with the logic of the hypervisor scheduler. In this paper, a series of tests are conducted on three hypervisors (VMware ESXi, Hyper-V server and Xen to provide a benchmark of the latencies added to the applications running on top of them. These tests are conducted for different scenarios (use cases to take into consideration all the parameters and configurations of the hypervisors’ schedulers. Finally, this benchmark can be used as a reference for choosing the best hypervisor-application combination.

  1. Real time biometric surveillance with gait recognition

    Science.gov (United States)

    Mohapatra, Subasish; Swain, Anisha; Das, Manaswini; Mohanty, Subhadarshini

    2018-04-01

    Bio metric surveillance has become indispensable for every system in the recent years. The contribution of bio metric authentication, identification, and screening purposes are widely used in various domains for preventing unauthorized access. A large amount of data needs to be updated, segregated and safeguarded from malicious software and misuse. Bio metrics is the intrinsic characteristics of each individual. Recently fingerprints, iris, passwords, unique keys, and cards are commonly used for authentication purposes. These methods have various issues related to security and confidentiality. These systems are not yet automated to provide the safety and security. The gait recognition system is the alternative for overcoming the drawbacks of the recent bio metric based authentication systems. Gait recognition is newer as it hasn't been implemented in the real-world scenario so far. This is an un-intrusive system that requires no knowledge or co-operation of the subject. Gait is a unique behavioral characteristic of every human being which is hard to imitate. The walking style of an individual teamed with the orientation of joints in the skeletal structure and inclinations between them imparts the unique characteristic. A person can alter one's own external appearance but not skeletal structure. These are real-time, automatic systems that can even process low-resolution images and video frames. In this paper, we have proposed a gait recognition system and compared the performance with conventional bio metric identification systems.

  2. The Colliderscope: a real-time show

    CERN Multimedia

    Francesco Poppi

    2010-01-01

    Ninety-six LED lights distributed over the facade of the Niels Bohr Institute (NBI) in Blegdamsvej (Denmark) reproduce the actual signals coming from the Transition Radiation Detector (TRT) in ATLAS. Thanks to the Colliderscope, when a collision occurs below the ground in Geneva, people passing by in Blegdamsvej will be aware of it almost in real-time.   Niels Bohr Institute facade lit up to reflect the latest data from ATLAS-TRT . The pattern, intensity and duration of the Colliderscope’s flashes of light depend on the physical parameters of particles crossing the ATLAS TRT detector. “At the Colliderscope very little happens randomly”, explains Troels Petersen, a physicist at NBI and one of the people who conceived it. “Particularly interesting events, such as electrons, are shown by a bright light that remains on the facade for several seconds”. The Niels Bohr Institute has participated in the development of the TRT detector, and this is why t...

  3. Real-Time 3D Visualization

    Science.gov (United States)

    1997-01-01

    Butler Hine, former director of the Intelligent Mechanism Group (IMG) at Ames Research Center, and five others partnered to start Fourth Planet, Inc., a visualization company that specializes in the intuitive visual representation of dynamic, real-time data over the Internet and Intranet. Over a five-year period, the then NASA researchers performed ten robotic field missions in harsh climes to mimic the end- to-end operations of automated vehicles trekking across another world under control from Earth. The core software technology for these missions was the Virtual Environment Vehicle Interface (VEVI). Fourth Planet has released VEVI4, the fourth generation of the VEVI software, and NetVision. VEVI4 is a cutting-edge computer graphics simulation and remote control applications tool. The NetVision package allows large companies to view and analyze in virtual 3D space such things as the health or performance of their computer network or locate a trouble spot on an electric power grid. Other products are forthcoming. Fourth Planet is currently part of the NASA/Ames Technology Commercialization Center, a business incubator for start-up companies.

  4. Real-time control of fusion reactors

    International Nuclear Information System (INIS)

    Goncalves, B.; Sousa, J.; Varandas, C.A.F.

    2010-01-01

    The next generation fusion experiments, e.g. ITER, will be highly complex and raise new challenges in the field of control and data acquisition systems. The more advanced operation scenarios have to be capable of sustaining long pulse steady-state plasma and to suppress plasma instabilities almost completely. Such scenarios will heavily rely on Multiple-Input-Multiple-Output (MIMO) fast control systems. To ensure safety for the operation these systems have to be robust and resilient to faults while ensuring high availability. Mindful of the importance of such features for future fusion experiments ATCA based systems have been successfully used in fusion experiment as MIMO fast controller. This is the most promising architecture to substantially enhance the performance and capability of existing standard systems delivering well high throughput as well as high availability. The real-time control needs of a fusion experiment, the rational for the presently pursued solutions, the existing problems and the broad scientific and technical questions that need to be addressed on the path to a fusion power plant will be discussed.

  5. Real-time petroleum spill detection system

    International Nuclear Information System (INIS)

    Dakin, D.T.

    2001-01-01

    A real-time autonomous oil and fuel spill detection system has been developed to rapidly detect of a wide range of petroleum products floating on, or suspended in water. The system consists of an array of spill detection buoys distributed within the area to be monitored. The buoys are composed of a float and a multispectral fluorometer, which looks up through the top 5 cm of water to detect floating and suspended petroleum products. The buoys communicate to a base station computer that controls the sampling of the buoys and analyses the data from each buoy to determine if a spill has occurred. If statistically significant background petroleum levels are detected, the system raises an oil spill alarm. The system is useful because early detection of a marine oil spill allows for faster containment, thereby minimizing the contaminated area and reducing cleanup costs. This paper also provided test results for biofouling, various petroleum product detection, water turbidity and wave tolerance. The technology has been successfully demonstrated. The UV light source keeps the optic window free from biofouling, and the electronics are fully submerged so there is no risk that the unit could ignite the vapours of a potential oil spill. The system can also tolerate moderately turbid waters and can therefore be used in many rivers, harbours, water intakes and sumps. The system can detect petroleum products with an average thickness of less than 3 micrometers floating on the water surface. 3 refs., 15 figs

  6. Real-time ultrasonic weld evaluation system

    Science.gov (United States)

    Katragadda, Gopichand; Nair, Satish; Liu, Harry; Brown, Lawrence M.

    1996-11-01

    Ultrasonic testing techniques are currently used as an alternative to radiography for detecting, classifying,and sizing weld defects, and for evaluating weld quality. Typically, ultrasonic weld inspections are performed manually, which require significant operator expertise and time. Thus, in recent years, the emphasis is to develop automated methods to aid or replace operators in critical weld inspections where inspection time, reliability, and operator safety are major issues. During this period, significant advances wee made in the areas of weld defect classification and sizing. Very few of these methods, however have found their way into the market, largely due to the lack of an integrated approach enabling real-time implementation. Also, not much research effort was directed in improving weld acceptance criteria. This paper presents an integrated system utilizing state-of-the-art techniques for a complete automation of the weld inspection procedure. The modules discussed include transducer tracking, classification, sizing, and weld acceptance criteria. Transducer tracking was studied by experimentally evaluating sonic and optical position tracking techniques. Details for this evaluation are presented. Classification is obtained using a multi-layer perceptron. Results from different feature extraction schemes, including a new method based on a combination of time and frequency-domain signal representations are given. Algorithms developed to automate defect registration and sizing are discussed. A fuzzy-logic acceptance criteria for weld acceptance is presented describing how this scheme provides improved robustness compared to the traditional flow-diagram standards.

  7. Real-Time Accumulative Computation Motion Detectors

    Directory of Open Access Journals (Sweden)

    Saturnino Maldonado-Bascón

    2009-12-01

    Full Text Available The neurally inspired accumulative computation (AC method and its application to motion detection have been introduced in the past years. This paper revisits the fact that many researchers have explored the relationship between neural networks and finite state machines. Indeed, finite state machines constitute the best characterized computational model, whereas artificial neural networks have become a very successful tool for modeling and problem solving. The article shows how to reach real-time performance after using a model described as a finite state machine. This paper introduces two steps towards that direction: (a A simplification of the general AC method is performed by formally transforming it into a finite state machine. (b A hardware implementation in FPGA of such a designed AC module, as well as an 8-AC motion detector, providing promising performance results. We also offer two case studies of the use of AC motion detectors in surveillance applications, namely infrared-based people segmentation and color-based people tracking, respectively.

  8. Mobility and language change in real time

    DEFF Research Database (Denmark)

    Monka, Malene

    Diachronic studies of the interrelationship between mobility and language change leave us with some unanswered questions of causation. The most important question is whether language change is caused by mobility, or if mobile informants mark themselves linguistically different than their non-mobi...... mobile and non-mobile informants. I also suggest a human geographic approach to place to explain the differences between the language change of the mobile informants (e.g. Britain 2009; Johnstone 2004).......Diachronic studies of the interrelationship between mobility and language change leave us with some unanswered questions of causation. The most important question is whether language change is caused by mobility, or if mobile informants mark themselves linguistically different than their non......-mobile peers prior to being geographically and socially mobile (e.g. Andersson & Thelander 1994). In the presentation I discuss this question by presenting a real time panel-study of language change in 23 speakers from three municipalities in distinct dialect areas in Denmark. The language change of six mobile...

  9. The Fast Tracker Real Time Processor

    CERN Document Server

    Annovi, A; The ATLAS collaboration

    2011-01-01

    As the LHC luminosity is ramped up to the SLHC Phase I level and beyond, the high rates, multiplicities, and energies of particles seen by the detectors will pose a unique challenge. Only a tiny fraction of the produced collisions can be stored on tape and immense real-time data reduction is needed. An effective trigger system must maintain high trigger efficiencies for the physics we are most interested in, and at the same time suppress the enormous QCD backgrounds. This requires massive computing power to minimize the online execution time of complex algorithms. A multi-level trigger is an effective solution for an otherwise impossible problem. The Fast Tracker (FTK)[1], is a proposed upgrade to the current ATLAS trigger system that will operate at full Level-1 output rates and provide high quality tracks reconstructed over the entire detector by the start of processing in Level-2. FTK solves the combinatorial challenge inherent to tracking by exploiting massive parallelism of associative memories [2] that ...

  10. Near Real Time Ship Detection Experiments

    Science.gov (United States)

    Brusch, S.; Lehner, S.; Schwarz, E.; Fritz, T.

    2010-04-01

    A new Near Real Time (NRT) ship detection processor SAINT (SAR AIS Integrated Toolbox) was developed in the framework of the ESA project MARISS. Data are received at DLRs ground segment DLR-BN (Neustrelitz, Germany). Results of the ship detection are available on ftp server within 30 min after the acquisition started. The detectability of ships on Synthetic Aperture Radar (SAR) ERS-2, ENVISAT ASAR and TerraSAR-X (TS-X) images is validated by coastal (live) AIS and space AIS. The monitoring areas chosen for surveillance are the North-, Baltic Sea, and Cape Town. The detectability in respect to environmental parameters like wind field, sea state, currents and changing coastlines due to tidal effects is investigated. In the South Atlantic a tracking experiment of the German research vessel Polarstern has been performed. Issues of piracy in particular in respect to ships hijacked at the Somali coast are discussed. Some examples using high resolution images from TerraSAR-X are given.

  11. Real-time scheduling of software tasks

    International Nuclear Information System (INIS)

    Hoff, L.T.

    1995-01-01

    When designing real-time systems, it is often desirable to schedule execution of software tasks based on the occurrence of events. The events may be clock ticks, interrupts from a hardware device, or software signals from other software tasks. If the nature of the events, is well understood, this scheduling is normally a static part of the system design. If the nature of the events is not completely understood, or is expected to change over time, it may be necessary to provide a mechanism for adjusting the scheduling of the software tasks. RHIC front-end computers (FECs) provide such a mechanism. The goals in designing this mechanism were to be as independent as possible of the underlying operating system, to allow for future expansion of the mechanism to handle new types of events, and to allow easy configuration. Some considerations which steered the design were programming paradigm (object oriented vs. procedural), programming language, and whether events are merely interesting moments in time, or whether they intrinsically have data associated with them. The design also needed to address performance and robustness tradeoffs involving shared task contexts, task priorities, and use of interrupt service routine (ISR) contexts vs. task contexts. This paper will explore these considerations and tradeoffs

  12. An improved real time superresolution FPGA system

    Science.gov (United States)

    Lakshmi Narasimha, Pramod; Mudigoudar, Basavaraj; Yue, Zhanfeng; Topiwala, Pankaj

    2009-05-01

    In numerous computer vision applications, enhancing the quality and resolution of captured video can be critical. Acquired video is often grainy and low quality due to motion, transmission bottlenecks, etc. Postprocessing can enhance it. Superresolution greatly decreases camera jitter to deliver a smooth, stabilized, high quality video. In this paper, we extend previous work on a real-time superresolution application implemented in ASIC/FPGA hardware. A gradient based technique is used to register the frames at the sub-pixel level. Once we get the high resolution grid, we use an improved regularization technique in which the image is iteratively modified by applying back-projection to get a sharp and undistorted image. The algorithm was first tested in software and migrated to hardware, to achieve 320x240 -> 1280x960, about 30 fps, a stunning superresolution by 16X in total pixels. Various input parameters, such as size of input image, enlarging factor and the number of nearest neighbors, can be tuned conveniently by the user. We use a maximum word size of 32 bits to implement the algorithm in Matlab Simulink as well as in FPGA hardware, which gives us a fine balance between the number of bits and performance. The proposed system is robust and highly efficient. We have shown the performance improvement of the hardware superresolution over the software version (C code).

  13. Optimizing near real time accountability for reprocessing

    International Nuclear Information System (INIS)

    Cipiti, Benjamin B.

    2010-01-01

    Near Real Time Accountability (NRTA) of actinides at high precision in reprocessing plants has been a long sought-after goal in the safeguards community. Achieving this goal is hampered by the difficulty of making precision measurements in the reprocessing environment, equipment cost, and impact to plant operations. Thus the design of future reprocessing plants requires an optimization of different approaches. The Separations and Safeguards Performance Model, developed at Sandia National Laboratories, was used to evaluate a number of NRTA strategies in a UREX+ reprocessing plant. Strategies examined include the incorporation of additional actinide measurements of internal plant vessels, more use of process monitoring data, and the option of periodic draining of inventory to key tanks. Preliminary results show that the addition of measurement technologies can increase the overall measurement uncertainty due to additional error propagation, so care must be taken when designing an advanced system. Initial results also show that relying on a combination of different NRTA techniques will likely be the best option. The model provides a platform for integrating all the data. The modeling results for the different NRTA options under various material loss conditions will be presented.

  14. Improved Detection of Lassa Virus by Reverse Transcription-PCR Targeting the 5′ Region of S RNA▿

    OpenAIRE

    Ölschläger, Stephan; Lelke, Michaela; Emmerich, Petra; Panning, Marcus; Drosten, Christian; Hass, Meike; Asogun, Danny; Ehichioya, Deborah; Omilabu, Sunday; Günther, Stephan

    2010-01-01

    The method of choice for the detection of Lassa virus is reverse transcription (RT)-PCR. However, the high degree of genetic variability of the virus poses a problem with the design of RT-PCR assays that will reliably detect all strains. Recently, we encountered difficulties in detecting some strains from Liberia and Nigeria in a commonly used glycoprotein precursor (GPC) gene-specific RT-PCR assay (A. H. Demby, J. Chamberlain, D. W. Brown, and C. S. Clegg, J. Clin. Microbiol. 32:2898-2903, 1...

  15. Microdroplet sandwich real-time rt-PCR for detection of pandemic and seasonal influenza subtypes.

    Directory of Open Access Journals (Sweden)

    Stephanie L Angione

    Full Text Available As demonstrated by the recent 2012/2013 flu epidemic, the continual emergence of new viral strains highlights the need for accurate medical diagnostics in multiple community settings. If rapid, robust, and sensitive diagnostics for influenza subtyping were available, it would help identify epidemics, facilitate appropriate antiviral usage, decrease inappropriate antibiotic usage, and eliminate the extra cost of unnecessary laboratory testing and treatment. Here, we describe a droplet sandwich platform that can detect influenza subtypes using real-time reverse-transcription polymerase chain reaction (rtRT-PCR. Using clinical samples collected during the 2010/11 season, we effectively differentiate between H1N1p (swine pandemic, H1N1s (seasonal, and H3N2 with an overall assay sensitivity was 96%, with 100% specificity for each subtype. Additionally, we demonstrate the ability to detect viral loads as low as 10(4 copies/mL, which is two orders of magnitude lower than viral loads in typical infected patients. This platform performs diagnostics in a miniaturized format without sacrificing any sensitivity, and can thus be easily developed into devices which are ideal for small clinics and pharmacies.

  16. Identification of Reference Genes for Normalizing Quantitative Real-Time PCR in Urechis unicinctus

    Science.gov (United States)

    Bai, Yajiao; Zhou, Di; Wei, Maokai; Xie, Yueyang; Gao, Beibei; Qin, Zhenkui; Zhang, Zhifeng

    2018-06-01

    The reverse transcription quantitative real-time PCR (RT-qPCR) has become one of the most important techniques of studying gene expression. A set of valid reference genes are essential for the accurate normalization of data. In this study, five candidate genes were analyzed with geNorm, NormFinder, BestKeeper and ΔCt methods to identify the genes stably expressed in echiuran Urechis unicinctus, an important commercial marine benthic worm, under abiotic (sulfide stress) and normal (adult tissues, embryos and larvae at different development stages) conditions. The comprehensive results indicated that the expression of TBP was the most stable at sulfide stress and in developmental process, while the expression of EF- 1- α was the most stable at sulfide stress and in various tissues. TBP and EF- 1- α were recommended as a suitable reference gene combination to accurately normalize the expression of target genes at sulfide stress; and EF- 1- α, TBP and TUB were considered as a potential reference gene combination for normalizing the expression of target genes in different tissues. No suitable gene combination was obtained among these five candidate genes for normalizing the expression of target genes for developmental process of U. unicinctus. Our results provided a valuable support for quantifying gene expression using RT-qPCR in U. unicinctus.

  17. Automated Real-Time Clearance Analyzer (ARCA), Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — The Automated Real-Time Clearance Analyzer (ARCA) addresses the future safety need for Real-Time System-Wide Safety Assurance (RSSA) in aviation and progressively...

  18. Specification and Automated Verification of Real-Time Behaviour

    DEFF Research Database (Denmark)

    Kristensen, C.H.; Andersen, J.H.; Skou, A.

    1995-01-01

    In this paper we sketch a method for specification and automatic verification of real-time software properties.......In this paper we sketch a method for specification and automatic verification of real-time software properties....

  19. Specification and Automated Verification of Real-Time Behaviour

    DEFF Research Database (Denmark)

    Andersen, J.H.; Kristensen, C.H.; Skou, A.

    1996-01-01

    In this paper we sketch a method for specification and automatic verification of real-time software properties.......In this paper we sketch a method for specification and automatic verification of real-time software properties....

  20. Internet-accessible real-time weather information system

    Digital Repository Service at National Institute of Oceanography (India)

    Desai, R.G.P.; Joseph, A.; Desa, E.; Mehra, P.; Desa, E.; Gouveia, A.D.

    An internet-accessible real-time weather information system has been developed. This system provides real-time accessibility to weather information from a multitude of spatially distributed weather stations. The Internet connectivity also offers...

  1. An integrated technique for developing real-time systems

    NARCIS (Netherlands)

    Hooman, J.J.M.; Vain, J.

    1995-01-01

    The integration of conceptual modeling techniques, formal specification, and compositional verification is considered for real time systems within the knowledge engineering context. We define constructive transformations from a conceptual meta model to a real time specification language and give

  2. Virtual timers in hierarchical real-time systems

    NARCIS (Netherlands)

    Heuvel, van den M.M.H.P.; Holenderski, M.J.; Cools, W.A.; Bril, R.J.; Lukkien, J.J.; Zhu, D.

    2009-01-01

    Hierarchical scheduling frameworks (HSFs) provide means for composing complex real-time systems from welldefined subsystems. This paper describes an approach to provide hierarchically scheduled real-time applications with virtual event timers, motivated by the need for integrating priority

  3. Spying on real-time computers to improve performance

    International Nuclear Information System (INIS)

    Taff, L.M.

    1975-01-01

    The sampled program-counter histogram, an established technique for shortening the execution times of programs, is described for a real-time computer. The use of a real-time clock allows particularly easy implementation. (Auth.)

  4. Real-time subway information for improving transit ridership.

    Science.gov (United States)

    2016-08-01

    In recent years, the standardization of transit schedule information has yielded a dramatic increase in the accessibility of computerized transit schedules and given rise to real-time service schedules. Two such real-time service schedules are the Ge...

  5. Improving Timeliness in Real-Time Secure Database Systems

    National Research Council Canada - National Science Library

    Son, Sang H; David, Rasikan; Thuraisingham, Bhavani

    2006-01-01

    .... In addition to real-time requirements, security is usually required in many applications. Multilevel security requirements introduce a new dimension to transaction processing in real-time database systems...

  6. On Real-Time Systems Using Local Area Networks.

    Science.gov (United States)

    1987-07-01

    87-35 July, 1987 CS-TR-1892 On Real - Time Systems Using Local Area Networks*I VShem-Tov Levi Department of Computer Science Satish K. Tripathit...1892 On Real - Time Systems Using Local Area Networks* Shem-Tov Levi Department of Computer Science Satish K. Tripathit Department of Computer Science...constraints and the clock systems that feed the time to real - time systems . A model for real-time system based on LAN communication is presented in

  7. Energy efficient approach for transient fault recovery in real time ...

    African Journals Online (AJOL)

    DR OKE

    Keywords: DVS, Fault tolerance, Real Time System, Transient Fault. ... in which missing the deadline may cause a failure and soft real time system, ..... Pillai, P., Shin, K., Real-time dynamic voltage scaling for low-power embedded operating ...

  8. ClockWork: a Real-Time Feasibility Analysis Tool

    NARCIS (Netherlands)

    Jansen, P.G.; Hanssen, F.T.Y.; Mullender, Sape J.

    ClockWork shows that we can improve the flexibility and efficiency of real-time kernels. We do this by proposing methods for scheduling based on so-called Real-Time Transactions. ClockWork uses Real-Time Transactions which allow scheduling decisions to be taken by the system. A programmer does not

  9. The real-time price elasticity of electricity

    NARCIS (Netherlands)

    Lijesen, M.G.

    2007-01-01

    The real-time price elasticity of electricity contains important information on the demand response of consumers to the volatility of peak prices. Despite the importance, empirical estimates of the real-time elasticity are hardly available. This paper provides a quantification of the real-time

  10. Temporal Specification and Verification of Real-Time Systems.

    Science.gov (United States)

    1991-08-30

    of concrete real - time systems can be modeled adequately. Specification: We present two conservative extensions of temporal logic that allow for the...logic. We present both model-checking algorithms for the automatic verification of finite-state real - time systems and proof methods for the deductive verification of real - time systems .

  11. Real-time volumetric scintillation dosimetry

    International Nuclear Information System (INIS)

    Beddar, S

    2015-01-01

    The goal of this brief review is to review the current status of real-time 3D scintillation dosimetry and what has been done so far in this area. The basic concept is to use a large volume of a scintillator material (liquid or solid) to measure or image the dose distributions from external radiation therapy (RT) beams in three dimensions. In this configuration, the scintillator material fulfills the dual role of being the detector and the phantom material in which the measurements are being performed. In this case, dose perturbations caused by the introduction of a detector within a phantom will not be at issue. All the detector configurations that have been conceived to date used a Charge-Coupled Device (CCD) camera to measure the light produced within the scintillator. In order to accurately measure the scintillation light, one must correct for various optical artefacts that arise as the light propagates from the scintillating centers through the optical chain to the CCD chip. Quenching, defined in its simplest form as a nonlinear response to high-linear energy transfer (LET) charged particles, is one of the disadvantages when such systems are used to measure the absorbed dose from high-LET particles such protons. However, correction methods that restore the linear dose response through the whole proton range have been proven to be effective for both liquid and plastic scintillators. Volumetric scintillation dosimetry has the potential to provide fast, high-resolution and accurate 3D imaging of RT dose distributions. Further research is warranted to optimize the necessary image reconstruction methods and optical corrections needed to achieve its full potential

  12. Real time PV manufacturing diagnostic system

    Energy Technology Data Exchange (ETDEWEB)

    Kochergin, Vladimir [MicroXact Inc., Blacksburg, VA (United States); Crawford, Michael A. [MicroXact Inc., Blacksburg, VA (United States)

    2015-09-01

    The main obstacle Photovoltaic (PV) industry is facing at present is the higher cost of PV energy compared to that of fossil energy. While solar cell efficiencies continue to make incremental gains these improvements are so far insufficient to drive PV costs down to match that of fossil energy. Improved in-line diagnostics however, has the potential to significantly increase the productivity and reduce cost by improving the yield of the process. On this Phase I/Phase II SBIR project MicroXact developed and demonstrated at CIGS pilot manufacturing line a high-throughput in-line PV manufacturing diagnostic system, which was verified to provide fast and accurate data on the spatial uniformity of thickness, an composition of the thin films comprising the solar cell as the solar cell is processed reel-to-reel. In Phase II project MicroXact developed a stand-alone system prototype and demonstrated the following technical characteristics: 1) ability of real time defect/composition inconsistency detection over 60cm wide web at web speeds up to 3m/minute; 2) Better than 1mm spatial resolution on 60cm wide web; 3) an average better than 20nm spectral resolution resulting in more than sufficient sensitivity to composition imperfections (copper-rich and copper-poor regions were detected). The system was verified to be high vacuum compatible. Phase II results completely validated both technical and economic feasibility of the proposed concept. MicroXact’s solution is an enabling technique for in-line PV manufacturing diagnostics to increase the productivity of PV manufacturing lines and reduce the cost of solar energy, thus reducing the US dependency on foreign oil while simultaneously reducing emission of greenhouse gasses.

  13. Novel functions of prototype foamy virus Gag glycine- arginine-rich boxes in reverse transcription and particle morphogenesis.

    Science.gov (United States)

    Müllers, Erik; Uhlig, Tobias; Stirnnagel, Kristin; Fiebig, Uwe; Zentgraf, Hanswalter; Lindemann, Dirk

    2011-02-01

    Prototype foamy virus (PFV) Gag lacks the characteristic orthoretroviral Cys-His motifs that are essential for various steps of the orthoretroviral replication cycle, such as RNA packaging, reverse transcription, infectivity, integration, and viral assembly. Instead, it contains three glycine-arginine-rich boxes (GR boxes) in its C terminus that putatively represent a functional equivalent. We used a four-plasmid replication-deficient PFV vector system, with uncoupled RNA genome packaging and structural protein translation, to analyze the effects of deletion and various substitution mutations within each GR box on particle release, particle-associated protein composition, RNA packaging, DNA content, infectivity, particle morphology, and intracellular localization. The degree of viral particle release by all mutants was similar to that of the wild type. Only minimal effects on Pol encapsidation, exogenous reverse transcriptase (RT) activity, and genomic viral RNA packaging were observed. In contrast, particle-associated DNA content and infectivity were drastically reduced for all deletion mutants and were undetectable for all alanine substitution mutants. Furthermore, GR box I mutants had significant changes in particle morphology, and GR box II mutants lacked the typical nuclear localization pattern of PFV Gag. Finally, it could be shown that GR boxes I and III, but not GR box II, can functionally complement each other. It therefore appears that, similar to the orthoretroviral Cys-His motifs, the PFV Gag GR boxes are important for RNA encapsidation, genome reverse transcription, and virion infectivity as well as for particle morphogenesis.

  14. Mixed - mode Operating System for Real - time Performance

    OpenAIRE

    Hasan M. M.; Sultana S.; Foo C.K.

    2017-01-01

    The purpose of the mixed-mode system research is to handle devices with the accuracy of real-time systems and at the same time, having all the benefits and facilities of a matured Graphic User Interface(GUI)operating system which is typicallynon-real-time. This mixed-mode operating system comprising of a real-time portion and a non-real-time portion was studied and implemented to identify the feasibilities and performances in practical applications (in the context of scheduled the real-time e...

  15. 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.

  16. New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination.

    Science.gov (United States)

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

    2014-01-01

    Loop mediated isothermal amplification (LAMP) assay, a promising diagnostic test, has been developed for detection of different pathogens of human as well as animals. Various positive points support its use as a field level test but the major problem is product cross contamination leading to false positive results. Different methods were adopted by various researchers to control this false positive amplification due to cross contamination but all have their own advantages and disadvantages. A new closed tube LAMP assay based on agar dye capsule was developed in the present study and this technique has some advantages over the other closed tube technique.•Agar at the concentration of 1.5% was used to sandwich SYBR green dye I with the aid of intradermal syringe. This agar dye capsule was placed over the LAMP reaction mixture before it was amplified.•To eliminate the hazardous nature of Ultra Violet (UV) light during result visualization of LAMP products, the present study demonstrates the use of Light Emitting Diode (LED) lights for result visualization.•LAMP was carried out for Brucella species detection using this modified techniques yielding good results without any cross contamination and LED showed similar fluorescence compared to UV.

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

    Science.gov (United States)

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

    2016-09-01

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

  18. Loop-mediated isothermal amplification for detection of Staphylococcus aureus in dairy cow suffering from mastitis.

    Science.gov (United States)

    Tie, Zhang; Chunguang, Wang; Xiaoyuan, Wei; Xinghua, Zhao; Xiuhui, Zhong

    2012-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Abhishek Mewara

    2017-01-01

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

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    Na Feng

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

  2. 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.

  3. 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.

  4. Immunocapture loop-mediated isothermal amplification assays for the detection of canine parvovirus.

    Science.gov (United States)

    Sun, Yu-Ling; Yen, Chon-Ho; Tu, Ching-Fu

    2017-11-01

    A loop-mediated isothermal amplification (LAMP) assay was used for rapid canine parvovirus (CPV) diagnosis. To reduce the time required and increase the sensitivity of the assay, an immunocapture (IC) technique was developed in this study to exclude the DNA extraction step in molecular diagnostic procedures for CPV. A polyclonal rabbit anti-CPV serum was produced against VP2-EpC that was cloned via DNA recombination. The polyclonal anti-VP2-EpC serum was used for virus capture to prepare microtubes. IC-LAMP was performed to amplify a specific CPV target gene sequence from the CPV viral particles that were captured on the microtubes, and the amplicons were analyzed using agarose electrophoresis or enzyme-linked immunosorbent assay (IC-LAMP-ELISA) and lateral-flow dipstick (IC-LAMP-LFD). The detection sensitivities of IC-LAMP, IC-LAMP-ELISA, and IC-LAMP-LFD were 10 -1 , 10 -1 , and 10 -1 TCID 50 /mL, respectively. Using the IC-LAMP-ELISA and IC-LAMP-LFD assays, the complete CPV diagnostic process can be achieved within 1.5h. Both of the developed IC-LAMP-based assays are simple, direct visual and efficient techniques that are applicable to the detection of CPV. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Detection of bovine mastitis pathogens by loop-mediated isothermal amplification and an electrochemical DNA chip.

    Science.gov (United States)

    Kawai, Kazuhiro; Inada, Mika; Ito, Keiko; Hashimoto, Koji; Nikaido, Masaru; Hata, Eiji; Katsuda, Ken; Kiku, Yoshio; Tagawa, Yuichi; Hayashi, Tomohito

    2017-12-22

    Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pathogenic microorganism. However, bacterial identification by culture has drawbacks in that the sensitivity may be low and the procedure can be complex. In this study, we developed a genetic detection method to identify mastitis pathogens using a simple and highly sensitive electrochemical DNA chip which can specifically detect bacterial DNA in milk specimens. First, we selected microorganisms belonging to 12 families and/or genera associated with mastitis for which testing should be performed. Next, we optimized the conditions for amplifying microorganism DNA by loop-mediated isothermal amplification (LAMP) using 32 primers and the use of a DNA chip capable of measuring all pathogens simultaneously. Sample detection could be completed in just a few hours using this method. Comparison of the results obtained with our DNA chip method and those obtained by bacterial culture verified that when the culture method was set to 100%, the total positive concordance rate of the DNA chip was 85.0% and the total negative concordance rate was 86.9%. Furthermore, the proposed method allows both rapid and highly sensitive detection of mastitis pathogens. We believe that this method will contribute to the development of an effective mastitis control program.

  6. Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Cannabis sativa.

    Science.gov (United States)

    Kitamura, Masashi; Aragane, Masako; Nakamura, Kou; Watanabe, Kazuhito; Sasaki, Yohei

    2016-07-01

    In many parts of the world, the possession and cultivation of Cannabis sativa L. are restricted by law. As chemical or morphological analyses cannot identify the plant in some cases, a simple yet accurate DNA-based method for identifying C. sativa is desired. We have developed a loop-mediated isothermal amplification (LAMP) assay for the rapid identification of C. sativa. By optimizing the conditions for the LAMP reaction that targets a highly conserved region of tetrahydrocannabinolic acid (THCA) synthase gene, C. sativa was identified within 50 min at 60-66°C. The detection limit was the same as or higher than that of conventional PCR. The LAMP assay detected all 21 specimens of C. sativa, showing high specificity. Using a simple protocol, the identification of C. sativa could be accomplished within 90 min from sample treatment to detection without use of special equipment. A rapid, sensitive, highly specific, and convenient method for detecting and identifying C. sativa has been developed and is applicable to forensic investigations and industrial quality control.

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

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    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.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2012-11-07

    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.

  10. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. 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.

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Science.gov (United States)

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

    2015-08-01

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

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

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    Esther A. Shiraho

    2016-01-01

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

  15. Loop-mediated isothermal amplification assays for screening of bacterial integrons

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    Guangchao Yu

    2014-01-01

    Full Text Available BACKGROUND: The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection. RESULTS: In this study, three loop-mediated isothermal amplification (LAMP assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets. Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains. According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 µL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%. CONCLUSIONS: The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.

  16. 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.

  17. 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.

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

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    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.

  19. Accuracy of loop-mediated isothermal amplification for diagnosis of human leptospirosis in Thailand.

    Science.gov (United States)

    Sonthayanon, Piengchan; Chierakul, Wirongrong; Wuthiekanun, Vanaporn; Thaipadungpanit, Janjira; Kalambaheti, Thareerat; Boonsilp, Siriphan; Amornchai, Premjit; Smythe, Lee D; Limmathurotsakul, Direk; Day, Nicholas P; Peacock, Sharon J

    2011-04-01

    There is a lack of diagnostic tests for leptospirosis in technology-restricted settings. We developed loop-mediated isothermal amplification (LAMP) specific for the 16S ribosomal RNA gene (rrs) of pathogenic and intermediate group Leptospira species. The lower limit of detection was 10 genomic equivalents/reaction, and analytical specificity was high; we observed positive reactions for pathogenic/intermediate groups and negative reactions for non-pathogenic Leptospira species and other bacterial species. We evaluated this assay in Thailand by using a case-control study of 133 patients with laboratory-proven leptospirosis and 133 patients with other febrile illnesses. Using admission blood, we found that the rrs LAMP showed positive results in 58 of 133 cases (diagnostic sensitivity = 43.6, 95% confidence interval [CI] = 35.0-52.5) and in 22 of 133 controls (diagnostic specificity = 83.5, 95% CI = 76.0-89.3). Sensitivity was high for 39 patients who were culture positive for Leptospira spp. (84.6, 95% CI = 69.5-94.1). The rrs LAMP can provide an admission diagnosis in approximately half of patients with leptospirosis, but its clinical utility is reduced by a lower specificity.

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

    Science.gov (United States)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2007-05-01

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

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

    Science.gov (United States)

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

    2017-11-22

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

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

    Science.gov (United States)

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

    2017-07-01

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

  4. Real Time Wide Area Radiation Surveillance System

    Science.gov (United States)

    Biafore, M.

    2012-04-01

    We present the REWARD project, financed within the FP7 programme, theme SEC-2011.1.5-1 (Development of detection capabilities of difficult to detect radioactive sources and nuclear materials - Capability Project). Within this project, we propose a novel mobile system for real time, wide area radiation surveillance. The system is based on the integration of new miniaturized solid-state radiation sensors: a CdZnTe detector for gamma radiation and a high efficiency neutron detector based on novel silicon technologies. The sensing unit will include a wireless communication interface to send the data remotely to a monitoring base station which also uses a GPS system to calculate the position of the tag. The system will also incorporate middleware and high level software to provide web-service interfaces for the exchange of information, and that will offer top level functionalities as management of users, mobile tags and environment data and alarms, database storage and management and a web-based graphical user interface. Effort will be spent to ensure that the software is modular and re-usable across as many architectural levels as possible. Finally, an expert system will continuously analyze the information from the radiation sensor and correlate it with historical data from the tag location in order to generate an alarm when an abnormal situation is detected. The system will be useful for many different scenarios, including such lost radioactive sources and radioactive contamination. It will be possible to deploy in emergency units and in general in any type of mobile or static equipment. The sensing units will be highly portable thanks to their low size and low energy consumption. The complete system will be scalable in terms of complexity and cost and will offer very high precision on both the measurement and the location of the radiation. The modularity and flexibility of the system will allow for a realistic introduction to the market. Authorities may start with a

  5. Real Time Seismic Loss Estimation in Italy

    Science.gov (United States)

    Goretti, A.; Sabetta, F.

    2009-04-01

    By more than 15 years the Seismic Risk Office is able to perform a real-time evaluation of the earthquake potential loss in any part of Italy. Once the epicentre and the magnitude of the earthquake are made available by the National Institute for Geophysiscs and Volca-nology, the model, based on the Italian Geographic Information Sys-tems, is able to evaluate the extent of the damaged area and the consequences on the built environment. In recent years the model has been significantly improved with new methodologies able to conditioning the uncertainties using observa-tions coming from the fields during the first days after the event. However it is reputed that the main challenges in loss analysis are related to the input data, more than to methodologies. Unlike the ur-ban scenario, where the missing data can be collected with enough accuracy, the country-wise analysis requires the use of existing data bases, often collected for other purposed than seismic scenario evaluation, and hence in some way lacking of completeness and homogeneity. Soil properties, building inventory and population dis-tribution are the main input data that are to be known in any site of the whole Italian territory. To this end the National Census on Popu-lation and Dwellings has provided information on the residential building types and the population that lives in that building types. The critical buildings, such as Hospital, Fire Brigade Stations, Schools, are not included in the inventory, since the national plan for seismic risk assessment of critical buildings is still under way. The choice of a proper soil motion parameter, its attenuation with distance and the building type fragility are important ingredients of the model as well. The presentation will focus on the above mentioned issues, highlight-ing the different data sets used and their accuracy, and comparing the model, input data and results when geographical areas with dif-ferent extent are considered: from the urban scenarios

  6. Instrumentation development for real time brainwave monitoring.

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Lawrence Frederick; Clough, Benjamin W.

    2005-12-01

    The human brain functions through a chemically-induced biological process which operates in a manner similar to electrical systems. The signal resulting from this biochemical process can actually be monitored and read using tools and having patterns similar to those found in electrical and electronics engineering. The primary signature of this electrical activity is the ''brain wave'', which looks remarkably similar to the output of many electrical systems. Likewise, the device currently used in medical arenas to read brain electrical activity is the electroencephalogram (EEG) which is synonymous with a multi-channel oscilloscope reading. Brain wave readings and recordings for medical purposes are traditionally taken in clinical settings such as hospitals, laboratories or diagnostic clinics. The signal is captured via externally applied scalp electrodes using semi-viscous gel to reduce impedance. The signal will be in the 10 to 100 microvolt range. In other instances, where surgeons are attempting to isolate particular types of minute brain signals, the electrodes may actually be temporarily implanted in the brain during a preliminary procedure. The current configurations of equipment required for EEGs involve large recording instruments, many electrodes, wires, and large amounts of hard disk space devoted to storing large files of brain wave data which are then eventually analyzed for patterns of concern. Advances in sensors, signal processing, data storage and microelectronics over the last decade would seem to have paved the way for the realization of devices capable of ''real time'' external monitoring, and possible assessment, of brain activity. A myriad of applications for such a capability are likewise presenting themselves, including the ability to assess brain functioning, level of functioning and malfunctioning. Our plan is to develop the sensors, signal processing, and portable instrumentation package which could

  7. Real-time Forensic Disaster Analysis

    Science.gov (United States)

    Wenzel, F.; Daniell, J.; Khazai, B.; Mühr, B.; Kunz-Plapp, T.; Markus, M.; Vervaeck, A.

    2012-04-01

    The Center for Disaster Management and Risk Reduction Technology (CEDIM, www.cedim.de) - an interdisciplinary research center founded by the German Research Centre for Geoscience (GFZ) and Karlsruhe Institute of Technology (KIT) - has embarked on a new style of disaster research known as Forensic Disaster Analysis. The notion has been coined by the Integrated Research on Disaster Risk initiative (IRDR, www.irdrinternational.org) launched by ICSU in 2010. It has been defined as an approach to studying natural disasters that aims at uncovering the root causes of disasters through in-depth investigations that go beyond the reconnaissance reports and case studies typically conducted after disasters. In adopting this comprehensive understanding of disasters CEDIM adds a real-time component to the assessment and evaluation process. By comprehensive we mean that most if not all relevant aspects of disasters are considered and jointly analysed. This includes the impact (human, economy, and infrastructure), comparisons with recent historic events, social vulnerability, reconstruction and long-term impacts on livelihood issues. The forensic disaster analysis research mode is thus best characterized as "event-based research" through systematic investigation of critical issues arising after a disaster across various inter-related areas. The forensic approach requires (a) availability of global data bases regarding previous earthquake losses, socio-economic parameters, building stock information, etc.; (b) leveraging platforms such as the EERI clearing house, relief-web, and the many sources of local and international sources where information is organized; and (c) rapid access to critical information (e.g., crowd sourcing techniques) to improve our understanding of the complex dynamics of disasters. The main scientific questions being addressed are: What are critical factors that control loss of life, of infrastructure, and for economy? What are the critical interactions

  8. Real-time {sup 90}Sr Counter

    Energy Technology Data Exchange (ETDEWEB)

    Kaneko, Naomi; Kawai, Hideyuki; Kodama, Satoshi; Kobayashi, Atsushi; Tabata, Makoto; Ito, Hiroshi [Graduate School of Science, Chiba University, Chiba, (Japan); Han, Soorim [Graduate School of Science, Chiba University, Chiba, (Japan); National Institute of Radiological Science, Chiba, (Japan)

    2015-07-01

    Radioisotopes have been emitted around Japan due to a nuclear accident at the Fukushima Daiichi nuclear power station in March 2011. A problem is the contaminated water including the atomic nucleus which relatively has a long half- life time and soluble such as {sup 90}Sr, {sup 137}Cs. Internal exposures by {sup 90}Sr are more dangerous than {sup 137}Cs's because Sr has effective half-life time of 18 years and property of accumulation in a born. We have developed real-time {sup 90}Sr counter which is sensitive beta-ray of maximum kinematic energy of 2.28 MeV from {sup 90}Sr and insensitive of beta-ray of maximum kinematic energy of 1.17 MeV and gamma-ray from {sup 90}Sr by Cherenkov detection. This counter composes of Cerenkov counter, trigger scintillation counter and veto counter. Silica aerogel for Cherenkov counter can obtain refractive index between 1.017 and 1.049 easily. And wavelength shifting fiber (WLSF) is used as a light guide for extending effective area and producing lower cost. A mechanism of the identification of {sup 90}Sr is explained in following. In case of {sup 90}Sr, when the trigger counter reacts on the beta-ray from {sup 90}Sr, aerogel emits the Cherenkov light and WLSF reacts and read the Cherenkov light. On the other hand, in case of {sup 137}Cs, the trigger counter reacts on the beta-ray, aerogel stops the beta- ray and Cherenkov light is not emitted. Therefore, aerogel has a function as a radiator and shielding material. the gamma-ray is not reacted on the lower density detector. Cosmic rays would be also reacted by the veto counter. A prototype counter whose the effective area is 30 cm x 10 cm was obtained (2.0±1.2){sup 3} of mis-identification as {sup 137}Cs/{sup 90}Sr. Detection limit in the surface contamination inspection depends on measurement time and effective area mainly. The sensitivity of wide range, 10{sup -2} - 10{sup 4} Bq/cm{sup 2}, is obtained by adjustment of detection level in circuit of this counter. A lower

  9. Mixed - mode Operating System for Real - time Performance

    Directory of Open Access Journals (Sweden)

    Hasan M. M.

    2017-11-01

    Full Text Available The purpose of the mixed-mode system research is to handle devices with the accuracy of real-time systems and at the same time, having all the benefits and facilities of a matured Graphic User Interface(GUIoperating system which is typicallynon-real-time. This mixed-mode operating system comprising of a real-time portion and a non-real-time portion was studied and implemented to identify the feasibilities and performances in practical applications (in the context of scheduled the real-time events. In this research an i8751 microcontroller-based hardware was used to measure the performance of the system in real-time-only as well as non-real-time-only configurations. The real-time portion is an 486DX-40 IBM PC system running under DOS-based real-time kernel and the non-real-time portion is a Pentium IIIbased system running under Windows NT. It was found that mixed-mode systems performed as good as a typical real-time system and in fact, gave many additional benefits such as simplified/modular programming and load tolerance.

  10. Real-time data access layer for MDSplus

    International Nuclear Information System (INIS)

    Manduchi, G.; Luchetta, A.; Taliercio, C.; Fredian, T.; Stillerman, J.

    2008-01-01

    Recent extensions to MDSplus allow data handling in long discharges and provide a real-time data access and communication layer. The real-time data access layer is an additional component of MDSplus: it is possible to use the traditional MDSplus API during normal operation, and to select a subset of data items to be used in real time. Real-time notification is provided by a communication layer using a publish-subscribe pattern. The notification covers processes sharing the same data items even running on different machines, thus allowing the implementation of distributed control systems. The real-time data access layer has been developed for Windows, Linux, and VxWorks; it is currently being ported to Linux RTAI. In order to quantify the fingerprint of the presented system, the performance of the real-time access layer approach is compared with that of an ad hoc, manually optimized program in a sample real-time application

  11. Integration of MDSplus in real-time systems

    International Nuclear Information System (INIS)

    Luchetta, A.; Manduchi, G.; Taliercio, C.

    2006-01-01

    RFX-mod makes extensive usage of real-time systems for feedback control and uses MDSplus to interface them to the main Data Acquisition system. For this purpose, the core of MDSplus has been ported to VxWorks, the operating system used for real-time control in RFX. Using this approach, it is possible to integrate real-time systems, but MDSplus is used only for non-real-time tasks, i.e. those tasks which are executed before and after the pulse and whose performance does not affect the system time constraints. More extensive use of MDSplus in real-time systems is foreseen, and a real-time layer for MDSplus is under development, which will provide access to memory-mapped pulse files, shared by the tasks running on the same CPU. Real-time communication will also be integrated in the MDSplus core to provide support for distributed memory-mapped pulse files

  12. Characterization of the gut microbiota of Papua New Guineans using reverse transcription quantitative PCR.

    Directory of Open Access Journals (Sweden)

    Andrew R Greenhill

    Full Text Available There has been considerable interest in composition of gut microbiota in recent years, leading to a better understanding of the role the gut microbiota plays in health and disease. Most studies have been limited in their geographical and socioeconomic diversity to high-income settings, and have been conducted using small sample sizes. To date, few analyses have been conducted in low-income settings, where a better understanding of the gut microbiome could lead to the greatest return in terms of health benefits. Here, we have used quantitative real-time polymerase chain reaction targeting dominant and sub-dominant groups of microorganisms associated with human gut microbiome in 115 people living a subsistence lifestyle in rural areas of Papua New Guinea. Quantification of Clostridium coccoides group, C. leptum subgroup, C. perfringens, Bacteroides fragilis group, Bifidobacterium, Atopobium cluster, Prevotella, Enterobacteriaceae, Enterococcus, Staphylococcus, and Lactobacillus spp. was conducted. Principle coordinates analysis (PCoA revealed two dimensions with Prevotella, clostridia, Atopobium, Enterobacteriaceae, Enterococcus and Staphylococcus grouping in one dimension, while B. fragilis, Bifidobacterium and Lactobacillus grouping in the second dimension. Highland people had higher numbers of most groups of bacteria detected, and this is likely a key factor for the differences revealed by PCoA between highland and lowland study participants. Age and sex were not major determinants in microbial population composition. The study demonstrates a gut microbial composition with some similarities to those observed in other low-income settings where traditional diets are consumed, which have previously been suggested to favor energy extraction from a carbohydrate rich diet.

  13. Characterization of the gut microbiota of Papua New Guineans using reverse transcription quantitative PCR.

    Science.gov (United States)

    Greenhill, Andrew R; Tsuji, Hirokazu; Ogata, Kiyohito; Natsuhara, Kazumi; Morita, Ayako; Soli, Kevin; Larkins, Jo-Ann; Tadokoro, Kiyoshi; Odani, Shingo; Baba, Jun; Naito, Yuichi; Tomitsuka, Eriko; Nomoto, Koji; Siba, Peter M; Horwood, Paul F; Umezaki, Masahiro

    2015-01-01

    There has been considerable interest in composition of gut microbiota in recent years, leading to a better understanding of the role the gut microbiota plays in health and disease. Most studies have been limited in their geographical and socioeconomic diversity to high-income settings, and have been conducted using small sample sizes. To date, few analyses have been conducted in low-income settings, where a better understanding of the gut microbiome could lead to the greatest return in terms of health benefits. Here, we have used quantitative real-time polymerase chain reaction targeting dominant and sub-dominant groups of microorganisms associated with human gut microbiome in 115 people living a subsistence lifestyle in rural areas of Papua New Guinea. Quantification of Clostridium coccoides group, C. leptum subgroup, C. perfringens, Bacteroides fragilis group, Bifidobacterium, Atopobium cluster, Prevotella, Enterobacteriaceae, Enterococcus, Staphylococcus, and Lactobacillus spp. was conducted. Principle coordinates analysis (PCoA) revealed two dimensions with Prevotella, clostridia, Atopobium, Enterobacteriaceae, Enterococcus and Staphylococcus grouping in one dimension, while B. fragilis, Bifidobacterium and Lactobacillus grouping in the second dimension. Highland people had higher numbers of most groups of bacteria detected, and this is likely a key factor for the differences revealed by PCoA between highland and lowland study participants. Age and sex were not major determinants in microbial population composition. The study demonstrates a gut microbial composition with some similarities to those observed in other low-income settings where traditional diets are consumed, which have previously been suggested to favor energy extraction from a carbohydrate rich diet.

  14. Real-Time Wireless Data Acquisition System

    Science.gov (United States)

    Valencia, Emilio J.; Perotti, Jose; Lucena, Angel; Mata, Carlos

    2007-01-01

    Current and future aerospace requirements demand the creation of a new breed of sensing devices, with emphasis on reduced weight, power consumption, and physical size. This new generation of sensors must possess a high degree of intelligence to provide critical data efficiently and in real-time. Intelligence will include self-calibration, self-health assessment, and pre-processing of raw data at the sensor level. Most of these features are already incorporated in the Wireless Sensors Network (SensorNet(TradeMark)), developed by the Instrumentation Group at Kennedy Space Center (KSC). A system based on the SensorNet(TradeMark) architecture consists of data collection point(s) called Central Stations (CS) and intelligent sensors called Remote Stations (RS) where one or more CSs can be accommodated depending on the specific application. The CS's major function is to establish communications with the Remote Stations and to poll each RS for data and health information. The CS also collects, stores and distributes these data to the appropriate systems requiring the information. The system has the ability to perform point-to-point, multi-point and relay mode communications with an autonomous self-diagnosis of each communications link. Upon detection of a communication failure, the system automatically reconfigures to establish new communication paths. These communication paths are automatically and autonomously selected as the best paths by the system based on the existing operating environment. The data acquisition system currently under development at KSC consists of the SensorNet(TradeMark) wireless sensors as the remote stations and the central station called the Radio Frequency Health Node (RFHN). The RFF1N is the central station which remotely communicates with the SensorNet(TradeMark) sensors to control them and to receive data. The system's salient feature is the ability to provide deterministic sensor data with accurate time stamps for both time critical and non

  15. Real time measurement of air radioactivity

    International Nuclear Information System (INIS)

    Galeriu, D.; Craciunescu, T.; Teles, S.

    1998-01-01

    commands to the logger according to the command protocol. - ODAS reads the configuration from logger. At initialization the software sends to the logger the desired configuration which is overlooked in the following operations. It calculates the averages of readings over 10 minute and other parameters. Following that the software records the data into a file (one line of data at every 10 min). The interrogation of logger and data collection is repeated at every 10 minute. - After every file recording, ODAS sends a code signal to the workstation. Transmission of data to RODOS workstation is done periodically (every 10 min) or when requested by users. Advantages of our Local Meteorological and Radiological Monitoring System are the following: - it is automate, able to acquire, calculate and transmit data in non-stop mode; - it is a real-time and on-line system; - it is easy to upgrade due to the fact that it was developed as modules working independently. (authors)

  16. 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.

  17. Loop-mediated isothermal amplification (LAMP) assay for the diagnosis of fasciolosis in sheep and its application under field conditions

    OpenAIRE

    Mart?nez-Valladares, Mar?a; Rojo-V?zquez, Francisco Antonio

    2016-01-01

    Background Loop-mediated isothermal amplification (LAMP) is a very specific, efficient, and rapid gene amplification procedure in which the reaction can run at a constant temperature. In the current study we have developed a LAMP assay to improve the diagnosis of Fasciola spp. in the faeces of sheep. Findings After the optimisation of the LAMP assay we have shown similar results between this technique and the standard PCR using the outer primers of the LAMP reaction. In both cases the limit o...

  18. Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes.

    Science.gov (United States)

    Treder, Krzysztof; Chołuj, Joanna; Zacharzewska, Bogumiła; Babujee, Lavanya; Mielczarek, Mateusz; Burzyński, Adam; Rakotondrafara, Aurélie M

    2018-02-01

    Potato virus Y (PVY) infection has been a global challenge for potato production and the leading cause of downgrading and rejection of seed crops for certification. Accurate and timely diagnosis is a key for effective disease control. Here, we have optimized a reverse transcription loop-mediated amplification (RT-LAMP) assay to differentiate the PVY O and N serotypes. The RT-LAMP assay is based on isothermal autocyclic strand displacement during DNA synthesis. The high specificity of this method relies heavily on the primer sets designed for the amplification of the targeted regions. We designed specific primer sets targeting a region within the coat protein gene that contains nucleotide signatures typical for O and N coat protein types, and these primers differ in their annealing temperature. Combining this assay with total RNA extraction by magnetic capture, we have established a highly sensitive, simplified and shortened RT-LAMP procedure as an alternative to conventional nucleic acid assays for diagnosis. This optimized procedure for virus detection may be used as a preliminary test for identifying the viral serotype prior to investing time and effort in multiplex RT-PCR tests when a specific strain is needed.

  19. 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.

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

    Science.gov (United States)

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

    2017-10-01

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

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

    Directory of Open Access Journals (Sweden)

    S M Mazidur Rahman

    2017-10-01

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

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

    Science.gov (United States)

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

    2014-08-27

    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.

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

    Directory of Open Access Journals (Sweden)

    Elisa Serra-Casas

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

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

    Science.gov (United States)

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

    2017-08-01

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

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

    Science.gov (United States)

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

    2011-02-01

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

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

    Science.gov (United States)

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

    2016-01-01

    Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1–8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed. PMID:26967908

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

    Directory of Open Access Journals (Sweden)

    Naomi W Lucchi

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

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

    Directory of Open Access Journals (Sweden)

    Xiefeng Yao

    2016-08-01

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

  10. Visual loop-mediated isothermal amplification (LAMP) for the rapid diagnosis of Enterocytozoon hepatopenaei (EHP) infection.

    Science.gov (United States)

    T, Sathish Kumar; A, Navaneeth Krishnan; J, Joseph Sahaya Rajan; M, Makesh; K P, Jithendran; S V, Alavandi; K K, Vijayan

    2018-05-01

    The emerging microsporidian parasite Enterocytozoon hepatopenaei (EHP), the causative agent of hepatopancreatic microsporidiosis, has been widely reported in shrimp-farming countries. EHP infection can be detected by light microscopy observation of spores (1.7 × 1 μm) in stained hepatopancreas (HP) tissue smears, HP tissue sections, and fecal samples. EHP can also be detected by polymerase chain reaction (PCR) targeting the small subunit (SSU) ribosomal RNA (rRNA) gene or the spore wall protein gene (SWP). In this study, a rapid, sensitive, specific, and closed tube visual loop-mediated isothermal amplification (LAMP) protocol combined with FTA cards was developed for the diagnosis of EHP. LAMP primers were designed based on the SSU rRNA gene of EHP. The target sequence of EHP was amplified at constant temperature of 65 °C for 45 min and amplified LAMP products were visually detected in a closed tube system by using SYBR™ green I dye. Detection limit of this LAMP protocol was ten copies. Field and clinical applicability of this assay was evaluated using 162 field samples including 106 HP tissue samples and 56 fecal samples collected from shrimp farms. Out of 162 samples, EHP could be detected in 62 samples (47 HP samples and 15 fecal samples). When compared with SWP-PCR as the gold standard, this EHP LAMP assay had 95.31% sensitivity, 98.98% specificity, and a kappa value of 0.948. This simple, closed tube, clinically evaluated visual LAMP assay has great potential for diagnosing EHP at the farm level, particularly under low-resource circumstances.

  11. Rapid screening method for male DNA by using the loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Kitamura, Masashi; Kubo, Seiji; Tanaka, Jin; Adachi, Tatsushi

    2017-08-12

    Screening for male-derived biological material from collected samples plays an important role in criminal investigations, especially those involving sexual assaults. We have developed a loop-mediated isothermal amplification (LAMP) assay targeting multi-repeat sequences of the Y chromosome for detecting male DNA. Successful amplification occurred with 0.5 ng of male DNA under isothermal conditions of 61 to 67 °C, but no amplification occurred with up to 10 ng of female DNA. Under the optimized conditions, the LAMP reaction initiated amplification within 10 min and amplified for 20 min. The LAMP reaction was sensitive at levels as low as 1-pg male DNA, and a quantitative LAMP assay could be developed because of the strong correlation between the reaction time and the amount of template DNA in the range of 10 pg to 10 ng. Furthermore, to apply the LAMP assay to on-site screening for male-derived samples, we evaluated a protocol using a simple DNA extraction method and a colorimetric intercalating dye that allows detection of the LAMP reaction by evaluating the change in color of the solution. Using this protocol, samples of male-derived blood and saliva stains were processed in approximately 30 min from DNA extraction to detection. Because our protocol does not require much hands-on time or special equipment, this LAMP assay promises to become a rapid and simple screening method for male-derived samples in forensic investigations.

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

    Science.gov (United States)

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

    2017-09-01

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

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

    Science.gov (United States)

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

    2016-12-01

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

  14. Loop-mediated isothermal amplification (LAMP): early detection of Toxoplasma gondii infection in mice.

    Science.gov (United States)

    Kong, Qing-Ming; Lu, Shao-Hong; Tong, Qun-Bo; Lou, Di; Chen, Rui; Zheng, Bin; Kumagai, Takashi; Wen, Li-Yong; Ohta, Nobuo; Zhou, Xiao-Nong

    2012-01-03

    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. 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). 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.

  15. Hardware locks for a real-time Java chip multiprocessor

    DEFF Research Database (Denmark)

    Strøm, Torur Biskopstø; Puffitsch, Wolfgang; Schoeberl, Martin

    2016-01-01

    A software locking mechanism commonly protects shared resources for multithreaded applications. This mechanism can, especially in chip-multiprocessor systems, result in a large synchronization overhead. For real-time systems in particular, this overhead increases the worst-case execution time....... This improvement can allow a larger number of real-time tasks to be reliably scheduled on a multiprocessor real-time platform....

  16. Advanced real-time manipulation of video streams

    CERN Document Server

    Herling, Jan

    2014-01-01

    Diminished Reality is a new fascinating technology that removes real-world content from live video streams. This sensational live video manipulation actually removes real objects and generates a coherent video stream in real-time. Viewers cannot detect modified content. Existing approaches are restricted to moving objects and static or almost static cameras and do not allow real-time manipulation of video content. Jan Herling presents a new and innovative approach for real-time object removal with arbitrary camera movements.

  17. A Programmable Microkernel for Real-Time Systems

    Science.gov (United States)

    2003-06-01

    A Programmable Microkernel for Real - Time Systems Christoph M. Kirsch Thomas A. Henzinger Marco A.A. Sanvido Report No. UCB/CSD-3-1250 June 2003...TITLE AND SUBTITLE A Programmable Microkernel for Real - Time Systems 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...THIS PAGE unclassified Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 A Programmable Microkernel for Real - Time Systems ∗ Christoph M

  18. Design Specifications for Adaptive Real-Time Systems

    Science.gov (United States)

    1991-12-01

    TICfl \\ E CT E Design Specifications for JAN’\\ 1992 Adaptive Real - Time Systems fl Randall W. Lichota U, Alice H. Muntz - December 1991 \\ \\\\/ 0 / r...268-2056 Technical Report CMU/SEI-91-TR-20 ESD-91-TR-20 December 1991 Design Specifications for Adaptive Real - Time Systems Randall W. Lichota Hughes...Design Specifications for Adaptive Real - Time Systems Abstract: The design specification method described in this report treats a software

  19. Temporal Proof Methodologies for Real-Time Systems,

    Science.gov (United States)

    1990-09-01

    real time systems that communicate either through shared variables or by message passing and real time issues such as time-outs, process priorities (interrupts) and process scheduling. The authors exhibit two styles for the specification of real - time systems . While the first approach uses bounded versions of temporal operators the second approach allows explicit references to time through a special clock variable. Corresponding to two styles of specification the authors present and compare two fundamentally different proof

  20. Proceedings of the Real-Time Systems Engineering Workshop

    Science.gov (United States)

    2001-08-01

    real - time systems engineering. The workshop was held as part of the SEI Symposium in...Washington, DC, during September 2000. The objective of the workshop was to identify key issues and obtain feedback from attendees concerning real - time systems engineering...and interoperability. This report summarizes the workshop in terms of foundation, management, and technical topics, and it contains a discussion related to developing a community of interest for real - time systems

  1. Static Schedulers for Embedded Real-Time Systems

    Science.gov (United States)

    1989-12-01

    Because of the need for having efficient scheduling algorithms in large scale real time systems , software engineers put a lot of effort on developing...provide static schedulers for he Embedded Real Time Systems with single processor using Ada programming language. The independent nonpreemptable...support the Computer Aided Rapid Prototyping for Embedded Real Time Systems so that we determine whether the system, as designed, meets the required

  2. PERTS: A Prototyping Environment for Real-Time Systems

    Science.gov (United States)

    Liu, Jane W. S.; Lin, Kwei-Jay; Liu, C. L.

    1993-01-01

    PERTS is a prototyping environment for real-time systems. It is being built incrementally and will contain basic building blocks of operating systems for time-critical applications, tools, and performance models for the analysis, evaluation and measurement of real-time systems and a simulation/emulation environment. It is designed to support the use and evaluation of new design approaches, experimentations with alternative system building blocks, and the analysis and performance profiling of prototype real-time systems.

  3. Design Recovery Technology for Real-Time Systems.

    Science.gov (United States)

    1995-10-01

    RL-TR-95-208 Final Technical Report October 1995 DESIGN RECOVERY TECHNOLOGY FOR REAL TIME SYSTEMS The MITRE Corporation Lester J. Holtzblatt...92 - Jan 95 4. TTTLE AND SUBTITLE DESIGN RECOVERY TECHNOLOGY FOR REAL - TIME SYSTEMS 6. AUTHOR(S) Lester J. Holtzblatt, Richard Piazza, and Susan...behavior of real - time systems in general, our initial efforts have centered on recovering this information from one system in particular, the Modular

  4. Simultaneous detection of four garlic viruses by multiplex reverse transcription PCR and their distribution in Indian garlic accessions.

    Science.gov (United States)

    Majumder, S; Baranwal, V K

    2014-06-01

    Indian garlic is infected with Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and allexiviruses. Identity and distribution of garlic viruses in various garlic accessions from different geographical regions of India were investigated. OYDV and allexiviruses were observed in all the garlic accessions, while SLV and GarCLV were observed only in a few accessions. A multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection and identification of OYDV, SLV, GarCLV and Allexivirus infecting garlic accessions in India. This multiplex protocol standardized in this study will be useful in indexing of garlic viruses and production of virus free seed material. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Simultaneous detection of enteropathogenic viruses in buffalos faeces using multiplex reverse transcription-polymerase chain reaction (mRT-PCR

    Directory of Open Access Journals (Sweden)

    U. Pagnini

    2010-02-01

    Full Text Available A multiplex reverse transcription- polymerase chain reaction (mRT-PCR assay that detects Bovine Viral Diarrhoea Virus, Bovine Coronavirus, and Group A Rotaviruses in infected cell-culture fluids and clinical faecal samples is described. One hundred twenty faecal samples from buffalo calves with acute gastroenteritis were tested. The mRT-PCR was validated against simplex RT-PCR with published primers for Pestivirus, Coronavirus and Rotavirus. The multiplex RT-PCR was equally sensitive and specific in detecting viral infections compared with simplex RT-PCR. The mRT-PCR readily identified viruses by discriminating the size of their amplified gene products. This mRT-PCR may be a sensitive and rapid assay for surveillance of buffalo enteric viruses in field specimens. This novel multiplex RT-PCR is an attractive technique for the rapid, specific, and cost-effective laboratory diagnosis of acute gastroenteritis.

  6. Mixed-mode Operating System for Real-time Performance

    OpenAIRE

    M.M. Hasan; S. Sultana; C.K. Foo

    2017-01-01

    The purpose of the mixed-mode system research is to handle devices with the accuracy of real-time systems and at the same time, having all the benefits and facilities of a matured Graphic User Interface (GUI) operating system which is typically nonreal-time. This mixed-mode operating system comprising of a real-time portion and a non-real-time portion was studied and implemented to identify the feasibilities and performances in practical applications (in the context of scheduled the real-time...

  7. Timeliness and Predictability in Real-Time Database Systems

    National Research Council Canada - National Science Library

    Son, Sang H

    1998-01-01

    The confluence of computers, communications, and databases is quickly creating a globally distributed database where many applications require real time access to both temporally accurate and multimedia data...

  8. Verifying real-time systems against scenario-based requirements

    DEFF Research Database (Denmark)

    Larsen, Kim Guldstrand; Li, Shuhao; Nielsen, Brian

    2009-01-01

    We propose an approach to automatic verification of real-time systems against scenario-based requirements. A real-time system is modeled as a network of Timed Automata (TA), and a scenario-based requirement is specified as a Live Sequence Chart (LSC). We define a trace-based semantics for a kernel...... subset of the LSC language. By equivalently translating an LSC chart into an observer TA and then non-intrusively composing this observer with the original system model, the problem of verifying a real-time system against a scenario-based requirement reduces to a classical real-time model checking...

  9. Building Real-Time Collaborative Applications with a Federated Architecture

    Directory of Open Access Journals (Sweden)

    Pablo Ojanguren-Menendez

    2015-12-01

    Full Text Available Real-time collaboration is being offered by multiple libraries and APIs (Google Drive Real-time API, Microsoft Real-Time Communications API, TogetherJS, ShareJS, rapidly becoming a mainstream option for webservices developers. However, they are offered as centralised services running in a single server, regardless if they are free/open source or proprietary software. After re-engineering Apache Wave (former Google Wave, we can now provide the first decentralised and federated free/open source alternative. The new API allows to develop new real-time collaborative web applications in both JavaScript and Java environments.

  10. External Quality Assessment for the Detection of Measles Virus by Reverse Transcription-PCR Using Armored RNA.

    Directory of Open Access Journals (Sweden)

    Dong Zhang

    Full Text Available In recent years, nucleic acid tests for detection of measles virus RNA have been widely applied in laboratories belonging to the measles surveillance system of China. An external quality assessment program was established by the National Center for Clinical Laboratories to evaluate the performance of nucleic acid tests for measles virus. The external quality assessment panel, which consisted of 10 specimens, was prepared using armored RNAs, complex of noninfectious MS2 bacteriophage coat proteins encapsulated RNA of measles virus, as measles virus surrogate controls. Conserved sequences amplified from a circulating measles virus strain or from a vaccine strain were encapsulated into these armored RNAs. Forty-one participating laboratories from 15 provinces, municipalities, or autonomous regions that currently conduct molecular detection of measles virus enrolled in the external quality assessment program, including 40 measles surveillance system laboratories and one diagnostic reagent manufacturer. Forty laboratories used commercial reverse transcription-quantitative PCR kits, with only one laboratory applying a conventional PCR method developed in-house. The results indicated that most of the participants (38/41, 92.7% were able to accurately detect the panel with 100% sensitivity and 100% specificity. Although a wide range of commercially available kits for nucleic acid extraction and reverse transcription polymerase chain reaction were used by the participants, only two false-negative results and one false-positive result were generated; these were generated by three separate laboratories. Both false-negative results were obtained with tests performed on specimens with the lowest concentration (1.2 × 104 genomic equivalents/mL. In addition, all 18 participants from Beijing achieved 100% sensitivity and 100% specificity. Overall, we conclude that the majority of the laboratories evaluated have reliable diagnostic capacities for the detection

  11. Comparison of reverse transcriptase PCR, reverse transcriptase loop-mediated isothermal amplification, and culture-based assays for Salmonella detection from pork processing environments.

    Science.gov (United States)

    Techathuvanan, Chayapa; Draughon, Frances Ann; D'Souza, Doris Helen

    2011-02-01

    Novel rapid Salmonella detection assays without the need for sophisticated equipment or labor remain in high demand. Real-time reverse transcriptase PCR (RT-PCR) assays, though rapid and sensitive, require expensive thermocyclers, while a novel RT loop-mediated isothermal amplification (RT-LAMP) method requires only a simple water bath. Our objective was to compare the detection sensitivity of Salmonella Typhimurium from the pork processing environment by RT-LAMP, RT-PCR, and culture-based assays. Carcass and surface swabs and carcass rinses were obtained from a local processing plant. Autoclaved carcass rinses (500 ml) were spiked with Salmonella Typhimurium and filtered. Filters were placed in stomacher bags containing tetrathionate broth (TTB) and analyzed with or without 10-h enrichment at 37 °C. Natural swabs were stomached with buffered peptone water, and natural carcass rinses were filtered, preenriched, and further enriched in TTB. Serially-diluted enriched samples were enumerated by spread plating on xylose lysine Tergitol 4 agar. RNA was extracted from 5 ml of enriched TTB with TRIzol. RT-LAMP assay using previously described invA primers was conducted at 62 °C for 90 min in a water bath with visual detection and by gel electrophoresis. SYBR Green I-based-real-time RT-PCR was carried out with invA primers followed by melt temperature analysis. The results of RT-LAMP detection for spiked carcass rinses were comparable to those of RT-PCR and cultural plating, with detection limits of 1 log CFU/ml, although they were obtained significantly faster, within 24 h including preenrichment and enrichment. RT-LAMP showed 4 of 12 rinse samples positive, while RT-PCR showed 1 of 12 rinse samples positive. For swabs, 6 of 27 samples positive by RT-LAMP and 5 of 27 by RT-PCR were obtained. This 1-day RT-LAMP assay shows promise for routine Salmonella screening by the pork industry. Copyright ©, International Association for Food Protection

  12. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    International Nuclear Information System (INIS)

    Huang, S-H; Tsai, M-H; Lin, C-W; Yang, T-C; Chuang, P-H; Tsai, I-S; Lu, H-C; Wan Lei; Lin, Y-J; Lai, C-H

    2008-01-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples

  13. Evaluation of a multiplex real-time PCR assay for the detection of respiratory viruses in clinical specimens.

    Science.gov (United States)

    Rheem, Insoo; Park, Joowon; Kim, Tae-Hyun; Kim, Jong Wan

    2012-11-01

    In this study, we evaluated the analytical performance and clinical potential of a one-step multiplex real-time PCR assay for the simultaneous detection of 14 types of respiratory viruses using the AdvanSure RV real-time PCR Kit (LG Life Sciences, Korea). Three hundred and twenty clinical specimens were tested with the AdvanSure RV real-time PCR Kit and conventional multiplex reverse transcription (RT)-PCR assay. The assay results were analyzed and the one-step AdvanSure RV real-time PCR Kit was compared with the conventional multiplex RT-PCR assay with respect to the sensitivity and specificity of the detection of respiratory viruses. The limit of detection (LOD) was 1.31 plaque-forming units (PFU)/mL for human rhinoviruses (hRVs), 4.93 PFU/mL for human coronavirus HCoV-229E/NL63, 2.67 PFU/mL for human coronavirus HCoV-OC43, 18.20 PFU/mL for parainfluenza virus 1 (PIV)-1, 24.57 PFU/mL for PIV-2, 1.73 PFU/mL for PIV-3, 1.79 PFU/mL for influenza virus group (Flu) A, 59.51 PFU/mL for FluB, 5.46 PFU/mL for human respiratory syncytial virus (hRSV)-A, 17.23 PFU/mL for hRSV-B, 9.99 PFU/mL for human adenovirus (ADVs). The cross-reactivity test for this assay against 23 types of non-respiratory viruses showed negative results for all viruses tested. The agreement between the one-step AdvanSure multiplex real-time PCR assay and the conventional multiplex RT-PCR assay was 98%. The one-step AdvanSure RV multiplex real-time PCR assay is a simple assay with high potential for specific, rapid and sensitive laboratory diagnosis of respiratory viruses compared to conventional multiplex RT-PCR.

  14. GNSS global real-time augmentation positioning: Real-time precise satellite clock estimation, prototype system construction and performance analysis

    Science.gov (United States)

    Chen, Liang; Zhao, Qile; Hu, Zhigang; Jiang, Xinyuan; Geng, Changjiang; Ge, Maorong; Shi, Chuang

    2018-01-01

    Lots of ambiguities in un-differenced (UD) model lead to lower calculation efficiency, which isn't appropriate for the high-frequency real-time GNSS clock estimation, like 1 Hz. Mixed differenced model fusing UD pseudo-range and epoch-differenced (ED) phase observations has been introduced into real-time clock estimation. In this contribution, we extend the mixed differenced model for realizing multi-GNSS real-time clock high-frequency updating and a rigorous comparison and analysis on same conditions are performed to achieve the best real-time clock estimation performance taking the efficiency, accuracy, consistency and reliability into consideration. Based on the multi-GNSS real-time data streams provided by multi-GNSS Experiment (MGEX) and Wuhan University, GPS + BeiDou + Galileo global real-time augmentation positioning prototype system is designed and constructed, including real-time precise orbit determination, real-time precise clock estimation, real-time Precise Point Positioning (RT-PPP) and real-time Standard Point Positioning (RT-SPP). The statistical analysis of the 6 h-predicted real-time orbits shows that the root mean square (RMS) in radial direction is about 1-5 cm for GPS, Beidou MEO and Galileo satellites and about 10 cm for Beidou GEO and IGSO satellites. Using the mixed differenced estimation model, the prototype system can realize high-efficient real-time satellite absolute clock estimation with no constant clock-bias and can be used for high-frequency augmentation message updating (such as 1 Hz). The real-time augmentation message signal-in-space ranging error (SISRE), a comprehensive accuracy of orbit and clock and effecting the users' actual positioning performance, is introduced to evaluate and analyze the performance of GPS + BeiDou + Galileo global real-time augmentation positioning system. The statistical analysis of real-time augmentation message SISRE is about 4-7 cm for GPS, whlile 10 cm for Beidou IGSO/MEO, Galileo and about 30 cm

  15. Flexible Scheduling by Deadline Inheritance in Soft Real Time Kernels

    NARCIS (Netherlands)

    Jansen, P.G.; Wygerink, Emiel

    1996-01-01

    Current Hard Real Time (HRT) kernels have their timely behaviour guaranteed on the cost of a rather restrictive use of the available resources. This makes HRT scheduling techniques inadequate for use in Soft Real Time (SRT) environment where we can make a considerable profit by a better and more

  16. Real-Time Plasma Control Tools for Advanced Tokamak Operation

    International Nuclear Information System (INIS)

    Varandas, C. A. F.; Sousa, J.; Rodrigues, A. P.; Carvalho, B. B.; Fernandes, H.; Batista, A. J.; Cruz, N.; Combo, A.; Pereira, R. C.

    2006-01-01

    Real-time control will play an important role in the operation and scientific exploitation of the new generation fusion devices. This paper summarizes the real-time systems and diagnostics developed by the Portuguese Fusion Euratom Association based on digital signal processors and field programmable gate arrays

  17. Real-time software for the COMPASS tokamak plasma control

    International Nuclear Information System (INIS)

    Valcarcel, D.F.; Duarte, A.S.; Neto, A.; Carvalho, I.S.; Carvalho, B.B.; Fernandes, H.; Sousa, J.; Sartori, F.; Janky, F.; Cahyna, P.; Hron, M.; Panek, R.

    2010-01-01

    The COMPASS tokamak has started its operation recently in Prague and to meet the necessary operation parameters its real-time system, for data processing and control, must be designed for both flexibility and performance, allowing the easy integration of code from several developers and to guarantee the desired time cycle. For this purpose an Advanced Telecommunications Computing Architecture based real-time system has been deployed with a solution built on a multi-core x86 processor. It makes use of two software components: the BaseLib2 and the MARTe (Multithreaded Application Real-Time executor) real-time frameworks. The BaseLib2 framework is a generic real-time library with optimized objects for the implementation of real-time algorithms. This allowed to build a library of modules that process the acquired data and execute control algorithms. MARTe executes these modules in kernel space Real-Time Application Interface allowing to attain the required cycle time and a jitter of less than 1.5 μs. MARTe configuration and data storage are accomplished through a Java hardware client that connects to the FireSignal control and data acquisition software. This article details the implementation of the real-time system for the COMPASS tokamak, in particular the organization of the control code, the design and implementation of the communications with the actuators and how MARTe integrates with the FireSignal software.

  18. Mixed-mode Operating System for Real-time Performance

    Directory of Open Access Journals (Sweden)

    M.M. Hasan

    2017-11-01

    Full Text Available The purpose of the mixed-mode system research is to handle devices with the accuracy of real-time systems and at the same time, having all the benefits and facilities of a matured Graphic User Interface (GUI operating system which is typically nonreal-time. This mixed-mode operating system comprising of a real-time portion and a non-real-time portion was studied and implemented to identify the feasibilities and performances in practical applications (in the context of scheduled the real-time events. In this research an i8751 microcontroller-based hardware was used to measure the performance of the system in real-time-only as well as non-real-time-only configurations. The real-time portion is an 486DX-40 IBM PC system running under DOS-based realtime kernel and the non-real-time portion is a Pentium III based system running under Windows NT. It was found that mixed-mode systems performed as good as a typical realtime system and in fact, gave many additional benefits such as simplified/modular programming and load tolerance.

  19. An algorithm for learning real-time automata

    NARCIS (Netherlands)

    Verwer, S.E.; De Weerdt, M.M.; Witteveen, C.

    2007-01-01

    We describe an algorithm for learning simple timed automata, known as real-time automata. The transitions of real-time automata can have a temporal constraint on the time of occurrence of the current symbol relative to the previous symbol. The learning algorithm is similar to the redblue fringe

  20. Real-Time Adaptation of Influence Strategies in Online Selling

    NARCIS (Netherlands)

    Kaptein, M.C.; Parvinen, P.

    2014-01-01

    Real-time adjustments in online selling are becoming increasingly common. In this paper we describe a novel method of real-time adaptation, and introduce influence strategies as a useful level of analysis for personalization of online selling. The proposed method incorporates three perspectives on