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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...... virus of arctic origin virus can be detected using RT-LAMP and the method reported is more rapid than the real-time RT-PCR. Further arctic fox samples are under analysis in order to confirm these findings....

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  3. Rapid detection of peste des petits ruminants virus by a reverse transcription loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Li, Lin; Bao, Jingyue; Wu, Xiaodong; Wang, Zhiliang; Wang, Junwei; Gong, Mingxia; Liu, Chunju; Li, Jinming

    2010-12-01

    Peste des petits ruminants virus (PPRV) is the causative agent of peste des petits ruminants (PPR), an economically important viral disease of small ruminants. In this report, a one-step, single-tube, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PPRV. A set of six LAMP primers were designed based on the matrix gene sequence of PPRV to amplify the target RNA by incubation at 63°C for 60min with Bst DNA polymerase and reverse transcriptase. The amplified products could be observed by the naked eye. The specificity of the RT-LAMP assay was validated by amplifying eight strains of PPRV isolated in different geographical areas. No cross-reactivity with other related viruses, including rinderpest virus, canine distemper virus and measles virus, was detected. The sensitivity of the assay was similar to that of real-time reverse transcription polymerase chain reaction (RT-PCR) and 10-fold higher than that of conventional RT-PCR. Twenty clinical samples were evaluated by the RT-LAMP assay, and the results were consistent with those of real-time RT-PCR. As a simple, rapid and accurate detection method, this RT-LAMP assay has important potential applications in the clinical diagnosis of PPR and the surveillance of PPRV. Copyright © 2010 Elsevier B.V. All rights reserved.

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    Qi Xiaole

    2011-03-01

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

  6. Detection of Papaya leaf distortion mosaic virus by reverse-transcription loop-mediated isothermal amplification.

    Science.gov (United States)

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

    2014-01-01

    Papaya leaf distortion mosaic virus (PLDMV) can infect transgenic papaya resistant to a related pathogen, Papaya ringspot virus (PRSV), posing a substantial threat to papaya production in China. Current detection methods, however, are unable to be used for rapid detection in the field. Here, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PLDMV, using a set of four RT-LAMP primers designed based on the conserved sequence of PLDMV CP. The RT-LAMP method detected specifically PLDMV and was highly sensitive, with a detection limit of 1.32×10(-6) μg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR, while also requiring significantly less time and equipment. The effectiveness of RT-LAMP and one-step RT-PCR in detecting the virus were compared using 90 field samples of non-transgenic papaya and 90 field samples of commercialized PRSV-resistant transgenic papaya from Hainan Island. None of the non-transgenic papaya tested positive for PLDMV using either method. In contrast, 19 of the commercialized PRSV-resistant transgenic papaya samples tested positive by RT-LAMP assay, and 6 of those tested negative by RT-PCR. Therefore, the PLDMV-specific RT-LAMP is a simple, rapid, sensitive, and cost-effective tool in the field diagnosis and control of PLDMV.

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

    Science.gov (United States)

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

    2014-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Wang Youling

    2011-12-01

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

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

    Science.gov (United States)

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

    2015-09-01

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

  10. Application of Reverse Transcription-PCR and Real-Time PCR in Nanotoxicity Research

    Science.gov (United States)

    Mo, Yiqun; Wan, Rong; Zhang, Qunwei

    2016-01-01

    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. PMID:22975959

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  12. A reverse transcription loop-mediated isothermal amplification (LAMP) assay for the detection of feline Coronavirus

    National Research Council Canada - National Science Library

    Angelica Stranieri; Stefania Lauzi; Alessia Giordano; Saverio Paltrinieri

    2016-01-01

    ...). The addition of two loop primers allows the reaction time to be of one hour only (Nagamine et al., 2002). The aim of this study was to develop a reverse transcription LAMP assay for an easy and inexpensive detection of feline Coronavirus...

  13. Analysis of liver connexin expression using reverse transcription quantitative real-time polymerase chain reaction

    Science.gov (United States)

    Maes, Michaël; Willebrords, Joost; Crespo Yanguas, Sara; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Summary Although connexin production is mainly regulated at the protein level, altered connexin gene expression has been identified as the underlying mechanism of several pathologies. When studying the latter, appropriate methods to quantify connexin mRNA levels are required. The present chapter describes a well-established reverse transcription quantitative real-time polymerase chain reaction procedure optimized for analysis of hepatic connexins. The method includes RNA extraction and subsequent quantification, generation of complementary DNA, quantitative real-time polymerase chain reaction and data analysis. PMID:27207283

  14. Development and evaluation of a simple assay for Marburg virus detection using a reverse transcription-loop-mediated isothermal amplification method.

    Science.gov (United States)

    Kurosaki, Yohei; Grolla, Allen; Fukuma, Aiko; Feldmann, Heinz; Yasuda, Jiro

    2010-07-01

    Marburg virus (MARV) causes a severe hemorrhagic fever in humans with a high mortality rate. The rapid and accurate identification of the virus is required to appropriately provide infection control and outbreak management. Here, we developed and evaluated a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and simple detection of MARV. By combining two sets of primers specific for the Musoke and Ravn genetic lineages, a multiple RT-LAMP assay detected MARV strains of both lineages, and no cross-reactivity with other hemorrhagic fever viruses (Ebola virus and Lassa virus) was observed. The assay could detect 10(2) copies of the viral RNA per tube within 40 min by real-time monitoring of the turbidities of the reaction mixtures. The assay was further evaluated using viral RNA extracted from clinical specimens collected in the 2005 Marburg hemorrhagic fever outbreak in Angola and yielded positive results for samples containing MARV at greater than 10(4) 50% tissue culture infective doses/ml, exhibiting 78% (14 of 18 samples positive) consistency with the results of a reverse transcription-PCR assay carried out in the field laboratory. The results obtained by both agarose gel electrophoresis and naked-eye judgment indicated that the RT-LAMP assay developed in this study is an effective tool for the molecular detection of MARV. Furthermore, it seems suitable for use for field diagnostics or in laboratories in areas where MARV is endemic.

  15. Rapid detection of sacbrood virus (SBV by one-step reverse transcription loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Jin-Long Yang

    2012-02-01

    Full Text Available Abstract Background Sacbrood virus (SBV primarily infects honeybee broods, and in order to deal with the problem cost effective detection methods are required. Findings A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP assay was developed for the rapid identification of SBV. The data demonstrated that, in a simple water bath, SBV RNA could be detected as early as 20 min at 65°C, and a positive amplification reaction was visible to the naked eye due to a color change brought on by the addition of nucleic acid stain SYBR Green. Conclusions The current study presents a method for the rapid and simple detection of SBV by RT-LAMP with high sensitivity and analytic specificity.

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

    Science.gov (United States)

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

    2013-11-01

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

  17. Rapid detection of European orthobunyaviruses by reverse transcription loop-mediated isothermal amplification assays.

    Science.gov (United States)

    Camp, Jeremy V; Nowotny, Norbert

    2016-10-01

    The development of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assays are described herein for the detection of two orthobunyaviruses (Bunyaviridae), which represent the two main serogroups found in mosquitoes in Central Europe. The RT-LAMP assays were optimized for the detection of Ťahyňa virus (a California encephalitis group virus found in Aedes sp or Ochlerotatus sp mosquitoes) and Batai virus (also called Čalovo virus, a Bunyamwera group virus found in Anopheles maculipennis s.l. mosquitoes) nucleic acid using endemic European virus isolates. The sensitivity of the RT-LAMP assays was determined to be comparable to that of conventional tests, with a limit of detectionisothermal conditions using very simple equipment. Furthermore, it was possible to proceed with the assays without nucleic acid extraction, albeit at a 100-fold loss of sensitivity. The RT-LAMP assays are a sensitive, cost-efficient method for both arbovirus surveillance as well as diagnostic laboratories to detect the presence of these endemic orthobunyaviruses.

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2013-12-01

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

  1. Development and application of reverse transcription loop-mediated isothermal amplification for detecting live Shewanella putrefaciens in preserved fish sample.

    Science.gov (United States)

    Li, Chenghua; Ying, Qi; Su, Xiurong; Li, Taiwu

    2012-04-01

    Given that live Shewanella putrefaciens is one of the major causes of spoilage for aquatic products even in chill storage, the rapid and accurate detection process is the first priority. In the present study, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) detecting assay was developed by targeting internal transcribed spacer (ITS) sequence between 16S and 23S rRNA. At the same time, a new procaryotic mRNA isolation strategy was also established by introducing a polyA tail to RNA during cDNA synthesis step. Under the optimal reaction time (60 min) and temperature (64.1 °C), S. putrefaciens could be specially identified from a variety of other tested bacteria by RT-LAMP. The sensitivity analysis showed that RT-LAMP could be identified as lower as 5.4 copies per reaction, which is over 200-fold higher than that of standard PCR (1.08 × 10³ copies per reaction). The method could be effectively identified S. putrefaciens in artificially contaminated or spoilaged fish samples with dose-dependent manners. To our knowledge, this is the first report using RT-LAMP assay to detect live S. putrefaciens in fish. The study provided a rapid and accurate detection method for live bacteria in aquatic food and established a new procaryotic mRNA isolation strategy at the same time, which will be useful for food preservation. © 2012 Institute of Food Technologists®

  2. Critical analysis of rhinovirus RNA load quantification by real-time reverse transcription-PCR.

    Science.gov (United States)

    Schibler, Manuel; Yerly, Sabine; Vieille, Gaël; Docquier, Mylène; Turin, Lara; Kaiser, Laurent; Tapparel, Caroline

    2012-09-01

    Rhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations. Although results obtained by real-time reverse-transcription PCR (RT-PCR) assays are frequently converted to viral RNA loads, this presents several limitations regarding accurate virus RNA quantification, particularly given the need to reliably quantify all known rhinovirus genotypes with a single assay. Using an internal extraction control and serial dilutions of an in vitro-transcribed rhinovirus RNA reference standard, we validated a quantitative one-step real-time PCR assay. We then used chimeric rhinovirus genomes with 5'-untranslated regions (5'UTRs) originating from the three rhinovirus species and from one enterovirus to estimate the impact of the 5'UTR diversity. Respiratory specimens from infected patients were then also analyzed. The assay quantification ability ranged from 4.10 to 9.10 log RNA copies/ml, with an estimated error margin of ±10%. This variation was mainly linked to target variability and interassay variability. Taken together, our results indicate that our assay can reliably estimate rhinovirus RNA load, provided that the appropriate error margin is used. In contrast, due to the lack of a universal rhinovirus RNA standard and the variability related to sample collection procedures, accurate absolute rhinovirus RNA quantification in respiratory specimens is currently hardly feasible.

  3. Rapid detection of all known ebolavirus species by reverse transcription-loop-mediated isothermal amplification (RT-LAMP).

    Science.gov (United States)

    Oloniniyi, Olamide K; Kurosaki, Yohei; Miyamoto, Hiroko; Takada, Ayato; Yasuda, Jiro

    2017-03-26

    Ebola virus disease (EVD), a highly virulent infectious disease caused by ebolaviruses, has a fatality rate of 25-90%. Without a licensed chemotherapeutic agent or vaccine for the treatment and prevention of EVD, control of outbreaks requires accurate and rapid diagnosis of cases. In this study, five sets of six oligonucleotide primers targeting the nucleoprotein gene were designed for specific identification of each of the five ebolavirus species using reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay. The detection limits of the ebolavirus species-specific primer sets were evaluated using in vitro transcribed RNAs. The detection limit of species-specific RT-LAMP assays for Zaire ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus, and Bundibugyo ebolavirus was 256 copies/reaction, while the detection limit for Reston ebolavirus was 64 copies/reaction, and the detection time for each of the RT-LAMP assays was 13.3±3.0, 19.8±4.6, 14.3±0.6, 16.1±4.7, and 19.8±2.4min (mean±SD), respectively. The sensitivity of the species-specific RT-LAMP assays were similar to that of the established RT-PCR and quantitative RT-PCR assays for diagnosis of EVD and are suitable for field or point-of-care diagnosis. The RT-LAMP assays were specific for the detection of the respective species of ebolavirus with no cross reaction with other species of ebolavirus and other viral hemorrhagic fever viruses such as Marburg virus, Lassa fever virus, and Dengue virus. The species-specific RT-LAMP assays developed in this study are rapid, sensitive, and specific and could be useful in case of an EVD outbreak.

  4. One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV.

    Science.gov (United States)

    Lee, Se Hee; Baek, Yun Hee; Kim, Yang-Hoon; Choi, Young-Ki; Song, Min-Suk; Ahn, Ji-Young

    2016-01-01

    Due to the limitation of rapid development of specific antiviral drug or vaccine for novel emerging viruses, an accurate and rapid diagnosis is a key to manage the virus spread. We developed an efficient and rapid method with high specificity for the Middle East Respiratory Syndrome coronavirus (MERS-CoV), based on one-pot reverse transcription loop-mediated isothermal amplification (one-pot RT-LAMP). A set of six LAMP primers [F3, B3, FIP, BIP, LF (Loop-F), and LB (Loop-B)] were designed using the sequence of nucleocapsid (N) gene with optimized RT-LAMP enzyme conditions: 100 U M-MLV RTase and 4 U Bst polymerase, implying that the reaction was able to detect four infectious viral genome copies of MERS-CoV within a 60 min reaction time period. Significantly, EvaGreen dye has better signal read-out properties in one-pot RT-LAMP reaction and is more compatible with DNA polymerase than SYBR green I. Isothermally amplified specific N genes were further evaluated using field-deployable microchamber devices, leading to the specific identification of as few as 0.4 infectious viral genome copies, with no cross-reaction to the other acute respiratory disease viruses, including influenza type A (H1N1 and H3N2), type B, human coronavirus 229E, and human metapneumovirus. This sensitive, specific and feasible method provides a large-scale technical support in emergencies, and is also applied as a sample-to-detection module in Point of Care Testing devices.

  5. [Colorimetric detection of human influenza A H1N1 virus by reverse transcription loop mediated isothermal amplification].

    Science.gov (United States)

    Nie, Kai; Wang, Da-Yan; Qin, Meng; Gao, Rong-Bao; Wang, Miao; Zou, Shu-Mei; Han, Feng; Zhao, Xiang; Li, Xi-Yan; Shu, Yue-Long; Ma, Xue-Jun

    2010-03-01

    A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-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 agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Detection of Anaplasma platys in dogs using real-time loop-mediated isothermal amplification.

    Science.gov (United States)

    Li, Hua-tao; Sun, Ling-suang; Chen, Zhong-ming; Hu, Jing-si; Ye, Cun-dong; Jia, Kun; Wang, Heng; Yuan, Li-guo; Zhang, Gui-hong; Li, Shoujun

    2014-03-01

    Anaplasma platys is a parasite of canine platelets that causes infectious cyclic thrombocytopenia. In this study, a novel real-time loop-mediated isothermal amplification (RT-LAMP) method was developed to detect A. platys. RT-LAMP primer sets were designed using a citrate synthase gene sequence and the assay was performed at 63 °C for 30 min. No cross-reactivity was observed with other Anaplasma or Ehrlichia spp. and the method exhibited a similar level of sensitivity in detecting the organism in 58 canine blood samples to that of a nested PCR. This RT-LAMP is a rapid and potentially cost-effective method of diagnosing A. platys infection in dogs. Copyright © 2014. Published by Elsevier Ltd.

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

    Science.gov (United States)

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

    2014-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Ying Yu

    2015-07-01

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

  10. On-chip single-copy real-time reverse-transcription PCR in isolated picoliter droplets

    Energy Technology Data Exchange (ETDEWEB)

    Beer, N R; Wheeler, E; Lee-Houghton, L; Watkins, N; Nasarabadi, S; Hebert, N; Leung, P; Arnold, D; Bailey, C; Colston, B

    2007-12-19

    The first lab-on-chip system for picoliter droplet generation and RNA isolation, followed by reverse transcription, and PCR amplification with real-time fluorescence detection in the trapped droplets has been developed. The system utilized a shearing T-junction in a fused silica device to generate a stream of monodisperse picoliter-scale droplets that were isolated from the microfluidic channel walls and each other by the oil phase carrier. An off-chip valving system stopped the droplets on-chip, allowing thermal cycling for reverse transcription and subsequent PCR amplification without droplet motion. This combination of the established real-time reverse transcription-PCR assay with digital microfluidics is ideal for isolating single-copy RNA and virions from a complex environment, and will be useful in viral discovery and gene-profiling applications.

  11. Detection of Zika Virus in Desiccated Mosquitoes by Real-Time Reverse Transcription PCR and Plaque Assay

    Science.gov (United States)

    Savage, Harry M.

    2017-01-01

    We assayed Zika virus–infected mosquitoes stored at room temperature for <30 days for live virus by using plaque assay and virus RNA by using real-time reverse transcription PCR. Viable virus was detected in samples stored <10 days, and virus RNA was detected in samples held for 30 days. PMID:28075325

  12. A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections

    Science.gov (United States)

    Kemleu, Sylvie; Guelig, Dylan; Eboumbou Moukoko, Carole; Essangui, Estelle; Diesburg, Steven; Mouliom, Abas; Melingui, Bernard; Manga, Jeanne; Donkeu, Christiane; Epote, Annie; Texier, Gaëtan; LaBarre, Paul; Burton, Robert

    2016-01-01

    Highly sensitive and field deployable molecular diagnostic tools are critically needed for detecting submicroscopic, yet transmissible levels of malaria parasites prevalent in malaria endemic countries worldwide. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated in comparison with thick blood smear microscopy, an antigen-based rapid diagnostic test (RDT), and an in-house RT-PCR targeting the same RT-LAMP transcript. The optimized assay detected Plasmodium falciparum infections in as little as 0.25ng of total parasite RNA, and exhibited a detection limit of 0.08 parasites/ μL when tested directly on infected whole blood lysates, or ~0.0008 parasites/ μL when using RNA extracts. Assay positivity was observed as early as eight minutes from initiation of the RT-LAMP and in most cases the reaction was complete before twenty minutes. Clinical evaluation of the assay on 132 suspected malaria cases resulted in a positivity rate of 90% for RT-LAMP using extracted RNA, and 85% when using whole blood lysates. The positivity rates were 70% for P. falciparum-specific RDT, 83% for RT-PCR, and 74% for thick blood smear microscopy (Mean parasite density = 36,986 parasites/ μL). Concordance rates between the developed RT-LAMP and comparator tests were greater than 75%, the lowest being with light microscopy (78%, McNemar’s test: P = 0.0002), and the highest was with RT-PCR (87%, McNemar’s test: P = 0.0523). Compared to reference RT-PCR, assay sensitivity was 90% for RT-LAMP on whole blood, and 96% for RT-LAMP using corresponding RNA extracts. Electricity-free heaters were further developed and evaluated in comparison with a battery-operated isothermal amplification machine for use with the developed test in resource-limited settings. Taken together, the data highlight the benefits of targeting high abundant RNA transcripts in molecular diagnosis, as well as the potential usefulness of the developed RT-LAMP-assay in

  13. Utility of IgM ELISA, TaqMan real-time PCR, reverse transcription PCR, and RT-LAMP assay for the diagnosis of Chikungunya fever.

    Science.gov (United States)

    Reddy, Vijayalakshmi; Ravi, Vasanthapuram; Desai, Anita; Parida, Manmohan; Powers, Ann M; Johnson, Barbara W

    2012-11-01

    Chikungunya fever a re-emerging infection with expanding geographical boundaries, can mimic symptoms of other infections like dengue, malaria which makes the definitive diagnosis of the infection important. The present study compares the utility of four laboratory diagnostic methods viz. IgM capture ELISA, an in house reverse transcription PCR for the diagnosis of Chikungunya fever, TaqMan real-time PCR, and a one step reverse transcription-loop mediated isothermal amplification assay (RT-LAMP). Out of the 70 serum samples tested, 29 (41%) were positive for Chikungunya IgM antibody by ELISA and 50 (71%) samples were positive by one of the three molecular assays. CHIKV specific nucleic acid was detected in 33/70 (47%) by reverse transcription PCR, 46/70 (66%) by TaqMan real-time PCR, and 43/70 (62%) by RT-LAMP assay. A majority of the samples (62/70; 89%) were positive by at least one of the four assays used in the study. The molecular assays were more sensitive for diagnosis in the early stages of illness (2-5 days post onset) when antibodies were not detectable. In the later stages of illness, the IgM ELISA is a more sensitive diagnostic test. In conclusion we recommend that the IgM ELISA be used as an initial screening test followed one of the molecular assays in samples that are collected in the early phase of illness and negative for CHIKV IgM antibodies. Such as approach would enable rapid confirmation of the diagnosis and implementation of public health measures especially during outbreaks. Copyright © 2012 Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

    2014-09-01

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

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

    OpenAIRE

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

    2015-01-01

    Background & objectives: 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. Methods: Specific primers and probes for rRT-PCR were designed based on VP1 sequences of WPV1 and WPV3 isolat...

  16. Development of a reverse transcription loop-mediated isothermal amplification assay for the rapid diagnosis of avian influenza A (H7N9) virus infection.

    Science.gov (United States)

    Nakauchi, Mina; Takayama, Ikuyo; Takahashi, Hitoshi; Tashiro, Masato; Kageyama, Tsutomu

    2014-08-01

    A genetic diagnosis system for detecting avian influenza A (H7N9) virus infection using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) technology was developed. The RT-LAMP assay showed no cross-reactivity with seasonal influenza A (H3N2 and H1N1pdm09) or influenza B viruses circulating in humans or with avian influenza A (H5N1) viruses. The sensitivity of the RT-LAMP assay was 42.47 copies/reaction. Considering the high specificity and sensitivity of the assay for detecting the avian influenza A (H7N9) virus and that the reaction was completed within 30 min, the RT-LAMP assay developed in this study is a promising rapid diagnostic tool for avian influenza A (H7N9) virus infection.

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

    Science.gov (United States)

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

    2016-10-01

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

  18. Detection and differentiation of Japanese encephalitis virus genotype I and genotype III by reverse transcription loop-mediated isothermal amplification combined with restriction fragment length polymorphism.

    Science.gov (United States)

    Zhang, Liang; Cao, Sanjie; Wu, Rui; Zhu, Shuquan; Liu, Hanyang; Yuan, Lei; Shi, Shuangyan; Zhang, Dan; Huang, Xiaobo; Wen, Xintian; Wen, Yiping; Yan, Qigui; Huang, Yong; Ma, Xiaoping

    2015-04-01

    Japanese encephalitis (JE), which is a mosquito-borne arboviral infection, is the leading cause of viral encephalitis in Asian countries. The causative agent of JE is Japanese encephalitis virus (JEV), in which the predominant genotype has changed from genotype III (G III) to genotype I (G I). However, a method for the rapid differentiation between JEV G I and G III remains unavailable. This study aimed to establish a rapid JEV genotyping method using reverse transcription loop-mediated isothermal amplification (RT-LAMP). An Spe I site, which was located in the target sequence (C gene) of JEV G III strains but not in JEV G I strains, was selected as the RT-LAMP target. After testing 64 specimens, results showed that RT-LAMP can detect and differentiate JEV G I and G III specifically. Thus, a novel RT-LAMP system for the rapid detection and differentiation of JEV G I and G III was developed successfully.

  19. 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.

  20. To Normalize the Using of Quantitative Real-time Reverse Transcription PCR%Real time RT-qPCR 检测规范化

    Institute of Scientific and Technical Information of China (English)

    马俊彦; 林俊

    2010-01-01

    Real time RT-qPCR(real-time quantitatire reverse transcription-PCR)是一种快速、简便、准确、灵敏、成本低廉的基因检测技术,被认为是目前检测基因在转录水平表达的金标准.研究发现Real time RT-qPCR的检测结果会受到实验设计、引物、模板的质量、内参的选择及数据分析的方法等多个因素的影响,规范实验设计及操作是得到可靠结论的前提和必要条件.对近年来国内外涉及Real time RT-qPCR整个实验流程及数据发表的一些规范及标准进行综述,以便规范实验设计及操作,加强实验质量控制,促进研究结果的推广,和更好地发挥其应用价值.

  1. Detection of Avian bornavirus in multiple tissues of infected psittacine birds using real-time reverse transcription polymerase chain reaction.

    Science.gov (United States)

    Delnatte, Pauline; Mak, Matthew; Ojkic, Davor; Raghav, Raj; DeLay, Josepha; Smith, Dale A

    2014-03-01

    Avian bornavirus (ABV), the cause of proventricular dilation disease in psittacine birds, has been detected in multiple tissues of infected birds using immunohistochemical staining (IHC) and reverse transcription polymerase chain reaction (RT-PCR). In the current study, real-time RT-PCR, using primers targeting the ABV matrix gene, was used to detect ABV in 146 tissues from 7 ABV-infected psittacine birds. Eighty-six percent of the samples tested positive, with crossing point values ranging from 13.82 to 37.82 and a mean of 22.3. These results were compared to the findings of a previous study using gel-based RT-PCR and IHC on the same samples. The agreement between the 2 RT-PCR techniques was 91%; when tests disagreed it was because samples were negative using gel-based RT-PCR but positive on real-time RT-PCR. Agreement with IHC was 77%; 16 out of 74 samples were negative using IHC but positive on real-time RT-PCR. The results suggest that real-time RT-PCR is a more sensitive technique than gel-based RT-PCR and IHC to detect ABV in tissues. The tissues that were ranked most frequently as having a high amount of viral RNA were proventriculus, kidney, colon, cerebrum, and cerebellum. Skeletal muscle, on the other hand, was found to have a consistently low amount of viral RNA.

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

    Directory of Open Access Journals (Sweden)

    Sanchita Bhadra

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

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

    Science.gov (United States)

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

    2015-02-01

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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Yiyue Ge

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

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

    Science.gov (United States)

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

    2015-01-01

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

  7. Development of Reverse Transcription Loop-Mediated Isothermal Amplification for Simple and Rapid Detection of Promyelocytic Leukemia–Retinoic Acid Receptor α mRNA

    Science.gov (United States)

    Hashimoto, Yuki; Hatayama, Yuki; Kojima, Nao; Morishita, Shota; Matsumoto, Satoko; Hosoda, Yuzuru; Hara, Ayako; Motokura, Toru

    2016-01-01

    Background Acute promyelocytic leukemia (APL) is a disease characterized by expression of Promyelocytic Leukemia–Retinoic Acid Receptor α (PML-RARα) chimeric mRNA. Although APL is curable, early death due to hemorrhage is a major problem. Here, we report the development of a simple and rapid diagnostic method for APL based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). Methods An RT-LAMP primer set was designed to detect three types of PML-RARα mRNA in a single reaction. Serial dilutions of plasmid DNA containing bcr1, bcr2, or bcr3 PML-RARα sequences and RNA extracted from bone marrow aspirates of 6 patients with APL were used to compare the results of RT-LAMP and nested PCR assays. Results Plasmid DNA was amplified by RT-LAMP, for which the reaction time was > 4 h shorter and the lower detection limit was higher than for nested RT-PCR. Six of 7 samples tested positive by both methods. Conclusion We developed an RT-LAMP assay for simple and rapid PML-RARα mRNA detection that may be clinically useful for point-of-care testing and APL diagnosis. PMID:28070163

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Zengguo eCao

    2016-04-01

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

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

    Science.gov (United States)

    Kamala, S; Makeshkumar, T

    2015-09-15

    Dasheen mosaic virus (DsMV), the pathogen causing mosaic disease of elephant foot yam (Amorphophallus paeoniifoilius) is disseminated mainly through vegetative propagation of the tubers. For the rapid and sensitive detection of the virus, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay based on the coat protein gene has been developed. A final concentration of 5.4 mM magnesium sulphate and 0.7 M betaine in the reaction mixture was found to be optimum for getting characteristic ladder like bands of the amplified product after gel electrophoresis. The reaction was set at 65°C for 50 min followed by reaction termination at 86°C for 5 min in a water bath. The sensitivity of the assay was found to be 100 times higher than that of RT-PCR. The virus was indexed successfully from tubers of elephant foot yam. In tube detection of the DsMV was carried out using fluorescence detection reagents. The assay was validated with field samples from various regions of Kerala state, India. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. [Visual detection of H1 subtype and identification of N1, N2 subtype of avian influenza virus by reverse transcription loop-mediated isothermal amplification assay].

    Science.gov (United States)

    Peng, Yi; Xie, Zhi-Xun; Guo, Jie; Zhou, Chen-Yu; Liu, Jia-Bo; Pang, Yao-Shan; Deng, Xian-Wen; Xie, Zhi-Qin; Xie, Li-Ji; Fan, Qing; Luo, Si-Si

    2013-03-01

    In order to visually detect H1, N1 and N2 subtype of avian influenza virus (AIV), three reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed. According to the sequences of AIV gene available in GenBank, three degenerate primer sets specific to HA gene of H1 subtype AIV, NA gene of N1 and N2 subtype AIV were designed, and the reaction conditions were optimized. The results showed that all the assays had no cross-reaction with other subtype AIV and other avian respiratory pathogens, and the detection limit was higher than that of conventional RT-PCR. These assays were performed in water bath within 50 minutes. Without opening tube, the amplification result could be directly determined by inspecting the color change of reaction system as long as these assays were fin-ished. Fourteen specimens of H1N1 subtype and eight specimens of H1N2 subtype of AIV were identified from the 120 clinical samples by RT-LAMP assays developed, which was consistent with that of virus isolation. These results suggested that the three newly developed RT-LAMEP assays were simple, specific and sensitive and had potential for visual detection of H1, N1 and N2 subtype of AIV in field.

  13. Rapid and simple detection of Japanese encephalitis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick.

    Science.gov (United States)

    Deng, Jieru; Pei, Jingjing; Gou, Hongchao; Ye, Zuodong; Liu, Cuicui; Chen, Jinding

    2015-03-01

    Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in geographical areas, such as Asia and Western Pacific, where it is a threat to human and animal health. To control this disease, it is necessary to develop a rapid, simple, accurate method for diagnosis. In this study, a method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) coupled with a lateral flow dipstick (LFD) has been developed to detect JEV (JEV RT-LAMP-LFD). The entire assay can be completed within 70 min, and in this study, no false positive results were observed when other pathogens were tested, indicating that the assay is a highly specific method for the detection of JEV. Additionally, the sensitivity of the RT-LAMP-LFD assay for SA14-14-2 strain was 50 pg of RNA, which was similar to that of RT-PCR and RT-LAMP combined with gel electrophoresis, and was 10-fold more sensitive than RT-LAMP combined with calcein. The limit of detection for this assay was 5 pg of RNA. In addition, no false positive results were obtained with 14 serum samples. Our results indicate that this RT-LAMP-LFD assay will be of great value for JEV infection testing due to its rapid and highly specific and sensitive properties. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Yin Jianhua

    2011-07-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Stephen Johnston; Zachary Gallaher; Krzysztof Czaja

    2012-01-01

    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-ΔΔCt 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.

  16. Real-Time Quantitative PCR (QPCR) and Reverse Transcription-QPCR for Detection and Enumeration of Total Yeasts in Wine▿

    Science.gov (United States)

    Hierro, Núria; Esteve-Zarzoso, Braulio; González, Ángel; Mas, Albert; Guillamón, Jose M.

    2006-01-01

    Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage. PMID:17088381

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

    Science.gov (United States)

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

    2013-11-27

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

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

    Directory of Open Access Journals (Sweden)

    Wei Qi-Wei

    2012-09-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Design and Assessment of a Real Time Reverse Transcription-PCR Method to Genotype Single-Stranded RNA Male-Specific Coliphages (Family Leviviridae).

    Science.gov (United States)

    A real-time, reverse transcription-PCR (RT-qPCR) assay was developed to differentiate the four genogroups of male-specific ssRNA coliphages (FRNA) (family Leviviridae). As FRNA display a trend of source-specificity (human sewage or animal waste) at the genogroup level, this assa...

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

    NARCIS (Netherlands)

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

    2015-01-01

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

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

    NARCIS (Netherlands)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Kiddle Guy

    2012-04-01

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

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

    Science.gov (United States)

    Kiddle, Guy; Hardinge, Patrick; Buttigieg, Neil; Gandelman, Olga; Pereira, Clint; McElgunn, Cathal J; Rizzoli, Manuela; Jackson, Rebecca; Appleton, Nigel; Moore, Cathy; Tisi, Laurence C; Murray, James A H

    2012-04-30

    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. 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. LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must be controlled to ensure

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

    Directory of Open Access Journals (Sweden)

    Ryan A Waters

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

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of African Horse Sickness Virus.

    Science.gov (United States)

    Fowler, V L; Howson, E L A; Flannery, J; Romito, M; Lubisi, A; Agüero, M; Mertens, P; Batten, C A; Warren, H R; Castillo-Olivares, J

    2017-10-01

    African horse sickness (AHS) is a disease of equids caused by African Horse Sickness Virus (AHSV) and is transmitted by Culicoides midges. AHS is endemic in sub-Saharan Africa, but during the past century, outbreaks of significant economic importance and elevated mortality have been recorded in Northern African countries, the Iberian and Arabian Peninsula, the Middle East and the Indian subcontinent. Effective control combines the application of early warning systems, accurate laboratory diagnosis and reporting, animal movement restrictions, suitable vaccination and surveillance programs, and the coordination of all these measures by efficient veterinary services. Conventional reverse-transcriptase (RT) PCR (RT-PCR) and real-time RT-PCR (rRT-PCR) assays have improved the sensitivity and rapidity of diagnosing AHS, resulting in the adoption of these methods as recommended tests by the World Organisation for Animal Health (OIE). However, currently these assays are only performed within laboratory settings; therefore, the development of field diagnostics for AHS would improve the fast implementation of control policies. Loop-mediated isothermal amplification (LAMP) is an isothermal, autocycling, strand-displacement nucleic acid amplification technique which can be performed in the field. LAMP assays are attractive molecular assays because they are simple to use, rapid, portable and have sensitivity and specificity within the range of rRT-PCR. This study describes the development of a novel RT-LAMP assay for the detection of AHSV. The AHSV RT-LAMP assay has an analytical sensitivity of 96.1% when considering an rRT-PCR cut-off value of CT  > 36, or 91.3% when no rRT-PCR cut-off is applied. Diagnostic sensitivity and specificity were 100%. This assay provides for a rapid and low cost AHS diagnostic for use in the field. © 2016 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.

  8. 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

    The aims of the present investigation were to develop and test a sensitive and reproducible method for the study of gene expression in the porcine lung pathogen Actinobacillus pleuropneumoniae by real-time quantitative reverse transcription (RT)-PCR and to evaluate a number of suitable internal...... 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...

  9. 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.

  10. Improvement and optimization of a multiplex real-time reverse transcription polymerase chain reaction assay for the detection and typing of Vesicular stomatitis virus.

    Science.gov (United States)

    Hole, Kate; Velazquez-Salinas, Lauro; Velazques-Salinas, Lauro; Clavijo, Alfonso

    2010-05-01

    An improvement to a previously reported real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay for the detection of Vesicular stomatitis virus (VSV) is described. Results indicate that the new assay is capable of detecting a panel of genetically representative strains of VSV present in North, Central, and South America. The assay is specific for VSV and allows for simultaneous differentiation between Vesicular stomatitis Indiana virus and Vesicular stomatitis New Jersey virus. This real-time RT-PCR is able to detect current circulating strains of VSV and can be used for rapid diagnosis of VSV and differentiation of VSV from other vesicular diseases, such as foot-and-mouth disease.

  11. 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...

  12. 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.

  13. 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.

  14. Establishment and Application of a TaqMan Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Rubella Virus RNA

    Institute of Scientific and Technical Information of China (English)

    Li-Hong ZHAO; Yu-Yan MA; Hong WANG; Shu-Ping ZHAO; Wei-Ming ZHAO; Hua LI; Lei-Yi WANG

    2006-01-01

    The aim of this study was to establish and apply a real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) for rubella virus (RV) RNA. First, the primer and TaqMan probe concentrations, as well as reaction temperatures were optimized to establish an efficient real-time quantitative RT-PCR assay for RV RNA. Next, an RV-specific PCR amplicon was made as an external standard to estimate the linearity, amplification efficiency, analytical sensitivity and reproducibility of the real time quantitative assay. Finally, the assay was applied to quantify RVRNA in clinical samples for rubella diagnosis.The RV-specific PCR amplicon was prepared for evaluation of the assay at 503 bp, and its original concentration was 2.75×109 copies/μl. The real time quantitative assay was shown to have good linearity (R2=0.9920), high amplification efficiency (E=1.91), high sensitivity (275 copies/ml), and high reproducibility (variation coefficient range, from 1.25% to 3.58%). Compared with the gold standard, the specificity and sensitivity of the assay in clinical samples was 96.4% and 86.4%, respectively. Therefore, the established quantitative RT-PCR method is a simple, rapid, less-labored, quantitative, highly specific and sensitive assay for RV RNA.

  15. Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

    Science.gov (United States)

    Huo, P; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P

    2015-12-01

    Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya.

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

    Science.gov (United States)

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

    2015-01-01

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

  17. 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.

  18. 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.

  19. Evaluation of loop-mediated isothermal amplification method (LAMP) for pathogenic Leptospira spp. detection with leptospires isolation and real-time PCR.

    Science.gov (United States)

    Suwancharoen, Duangjai; Sittiwicheanwong, Busara; Wiratsudakul, Anuwat

    2016-09-01

    Leptospirosis has been one of the worldwide zoonotic diseases caused by pathogenic Leptospira spp. Many molecular techniques have consecutively been developed to detect such pathogen including loop-mediated isothermal amplification method (LAMP). The objectives of this study were to evaluate the diagnostic accuracy of LAMP assay and real-time PCR using bacterial culture as the gold standard and to assess the agreement among these three tests using Cohen's kappa statistics. In total, 533 urine samples were collected from 266 beef and 267 dairy cattle reared in central region of Thailand. Sensitivity and specificity of LAMP were 96.8% (95% CI 81.5-99.8) and 97.0% (95% CI 94.9-98.2), respectively. The accuracy of LAMP (97.0%) was significantly higher than that of real-time PCR (91.9%) at 95% CI. With Cohen's kappa statistics, culture method and LAMP were substantially agreed with each other (77.4%), whereas real-time PCR only moderately agreed with culture (47.7%) and LAMP (45.3%), respectively. Consequently, LAMP was more effective than real-time PCR in detecting Leptospira spp. in the urine of cattle. Besides, LAMP had less cost and was simpler than real-time PCR. Thus, LAMP was an excellent alternative for routine surveillance of leptospirosis in cattle.

  20. 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.

  1. Quantitative Determination of Cucumber Mosaic Virus Genome RNAs in Virions by Real-Time Reverse Transcription-Polymerase Chain Reaction

    Institute of Scientific and Technical Information of China (English)

    Jun-Li FENG; Shao-Ning CHEN; Xiang-Shan TANG; Xian-Feng DING; Zhi-You DU; Ji-Shuang CHEN

    2006-01-01

    A real-time RT-PCR procedure using the green fluorescent dye SYBR Green I was developed for determining the absolute and relative copies of cucumber mosaic virus (CMV) genomic RNAs contained in purified virions. Primers specific to each CMV ORF were designed and selected. Sequences were then amplified with length varying from 61 to 153 bp. Using dilution series of CMV genome RNAs prepared by in vitro transcription as the standard samples, a good linear correlation was observed between their threshold cycle (Ct)values and the logarithms of the initial template amounts. The copies of genomic RNA 1, RNA 2,RNA 3 and the subgenomic RNA 4 in CMV virions were quantified by this method, and the ratios were about Our work is the first report concerning the relative amounts of different RNA fragments in CMV virions as a virus with tripartite genome.

  2. Detection of Zaire Ebola virus by real-time reverse transcription-polymerase chain reaction, Sierra Leone, 2014.

    Science.gov (United States)

    Liu, Licheng; Sun, Yang; Kargbo, Brima; Zhang, Chuntao; Feng, Huahua; Lu, Huijun; Liu, Wenseng; Wang, Chengyu; Hu, Yi; Deng, Yongqiang; Jiang, Jiafu; Kang, Xiaoping; Yang, Honglei; Jiang, Yongqiang; Yang, Yinhui; Kargbo, David; Qian, Jun; Chen, Weijun

    2015-09-15

    During the 2014 Ebola virus disease (EVD) outbreak, a real-time quantitative polymerase chain reaction was established to detect and identify the Zaire Ebola virus. We describe the use of this assay to screen 315 clinical samples from EVD suspected person in Sierra Leone. The detection rate in blood samples was 77.81% (207/266), and there were relatively higher detection rate (79.32% and 81.42%, respectively) during the first two weeks after onset of symptoms. In the two weeks that followed, the detection rate declined to 66.67% and 25.00%, respectively. There was the highest virus load at the first week and then decreased. The detection rate in swab samples was 89.79% (44/49). This may be benefit from the included patients. 46 of 49 swab samples were collected from died patients. Taken together, the results presented here indicate that the assay specifically and sensitively detects Zaire Ebola virus.

  3. 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.

  4. 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

    by a combined assay that detected all samples as positive. In addition, the introduction of labeled positive controls (LPC) increased the diagnostic safety of the single as well as the combined assay. Based on the newly developed, alternative assay for the detection of rabies virus and the application of LPCs......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...

  5. Simultaneous detection of hemagglutinin and neuraminidase genes of novel influenza A (H7N9) by duplex real-time reverse transcription polymerase chain reaction.

    Science.gov (United States)

    Li, Yan; Wu, Tao; Qi, Xian; Ge, Yiyue; Guo, Xiling; Wu, Bin; Yu, Huiyan; Zhu, Yefei; Shi, Zhiyang; Wang, Hua; Cui, Lunbiao; Zhou, Minghao

    2013-12-01

    A novel reassortant influenza A (H7N9) virus emerged recently in China. In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for the simultaneous detection of hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 influenza viruses. The sensitivity of the assay was determined to be 10 RNA copies per reaction for both HA and NA genes. No cross-reactivity was observed with other influenza virus subtypes or respiratory tract viruses. One hundred and forty-six clinical and environmental specimens were tested and compared with reference methods and were found to be consistent. The assay is suitable for large-scale screening due to short turnaround times and high specificity, sensitivity, and reproducibility.

  6. Probe-free real-time reverse transcription polymerase chain reaction assays for the detection and typing of porcine reproductive and respiratory syndrome virus in Canada.

    Science.gov (United States)

    Eschbaumer, Michael; Li, Wansi May; Wernike, Kerstin; Marshall, Frank; Czub, Markus

    2015-07-01

    Porcine reproductive and respiratory syndrome (PRRS) has tremendous impact on the pork industry in North America. The molecular diagnosis of infection with PRRS virus (PRRSV) is hampered by its considerable strain diversity. In this study, 43 previously published or newly developed primers for probe-free real-time reverse transcription polymerase chain reaction (RT-PCR) were evaluated on their sensitivity, specificity, reproducibility, and repeatability, using a diverse panel of 36 PRRSV strains as well as other arteriviruses and unrelated porcine viruses. Three primer pairs had excellent diagnostic and analytical sensitivity on par with a probe-based reference assay, absolute specificity to virus genotype and species, as well as over 95% reproducibility and repeatability across a wide dynamic range.

  7. 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.

  8. A capsid gene-based real-time reverse transcription polymerase chain reaction assay for the detection of marine vesiviruses in the Caliciviridae

    Science.gov (United States)

    McClenahan, Shasta D.; Bok, Karin; Neill, John D.; Smith, Alvin W.; Rhodes, Crystal R.; Sosnovtsev, Stanislav V.; Green, Kim Y.; Romero, Carlos H.

    2009-01-01

    A real-time reverse transcription polymerase chain reaction (rtRT-PCR) assay was developed for the identification of marine vesiviruses. The primers were designed to target a 176-nucleotide fragment within a highly conserved region of the San Miguel sea lion viruses (SMSVs) capsid gene. The assay detected viral RNA from nine marine vesivirus serotypes described previously, including two serotypes (SMSV-8 and SMSV-12) not identified with presently available molecular assays, a highly-related bovine vesivirus strain (Bos-1), a mink vesivirus strain (MCV), and two novel genotypes isolated recently from Steller sea lions (SSL V810 and V1415). The real-time assay did not amplify sequences from the corresponding genomic regions of feline calicivirus (also in the genus Vesivirus) and representative members of the genus Norovirus. The rtRT-PCR assay described below may prove useful as a diagnostic tool for the detection of currently circulating, emerging and previously described marine vesiviruses in clinical samples, especially when large numbers are screened in surveillance studies of these restricted viruses. PMID:19410604

  9. Up-regulation of Cartilage Oligomeric Matrix Protein Gene Expression by Insulin-like Growth Factor-I Revealed by Real Time Reverse Transcription-Polymerase Chain Reaction

    Institute of Scientific and Technical Information of China (English)

    Hua TIAN; Ioannis STOGIANNIDIS

    2006-01-01

    Cartilage oligomeric matrix protein (COMP) strengthens cartilage by binding to type Ⅱ and typeⅨ collagen-forming bridges between collagen fibrils. It was hypothesized that perhaps one or more anabolic growth factors such as insulin-like growth factor-I (IGF-I), fibroblast growth factor-1 (FGF-1) or platelet derived growth factor-BB (PDGF-BB) increase COMP gene expression. Their effects on primary human chondrocytes and the chondrogenic cell line ATDC5 were studied using real time reverse transcript-polymerase chain reaction (RT-PCR) for quantification. IGF-I, but not the FGF-1 or PDGF-BB, up-regulated COMP gene expression by approximate 5-fold in human adult chondrocytes in a dose- and time-dependent manner. IGF-I exerted similar effects on ATDC5 cells. Results from these real time RT-PCR experiments were confirmed by transfecting into ATDC5 cells a full-length mouse COMP promoter cloned upstream of a luciferase reporter gene. On stimulation with IGF-I, the luciferase reporter activity increased by about eight times. In conclusion, IGF-I seems to be an important positive regulator of COMP, which may play an important role in an attempted repair of either traumatized or degenerated cartilage.

  10. Lanthanide chelate complementation and hydrolysis enhanced luminescent chelate in real-time reverse transcription polymerase chain reaction assays for KLK3 transcripts.

    Science.gov (United States)

    Alinezhad, Saeid; Väänänen, Riina-Minna; Lehmusvuori, Ari; Karhunen, Ulla; Soukka, Tero; Kähkönen, Esa; Taimen, Pekka; Alanen, Kalle; Pettersson, Kim

    2014-01-01

    The requirement for high-performance reporter probes in real-time detection of polymerase chain reaction (PCR) has led to the use of time-resolved fluorometry of lanthanide chelates. The aim of this study was to investigate the applicability of the principle of lanthanide chelate complementation (LCC) in comparison with a method based on hydrolysis enhancement and quenching of intact probes. A real-time reverse transcription (RT) PCR assay for kallikrein-related peptidase 3 (KLK3, model analyte) was developed by using the LCC detection method. Both detection methods were tested with a standard series of purified PCR products, 20 prostatic tissues, 20 healthy and prostate cancer patient blood samples, and female blood samples spiked with LNCaP cells. The same limit of detection was obtained with both methods, and two cycles earlier detection with the LCC method was observed. KLK3 messenger RNA (mRNA) was detected in all tissue samples and in 1 of 20 blood samples identically with both methods. The background was 30 times lower, and the signal-to-background (S/B) ratio was 3 times higher, when compared with the reference method. Use of the new reporter method provided similar sensitivity and specificity as the reference method. The lower background, the improved S/B ratio, and the possibility of melting curve analysis and single nucleotide polymorphism (SNP) detection could be advantages for this new reporter probe.

  11. Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

    Science.gov (United States)

    Choudhary, Nandlal; Wei, G; Govindarajulu, A; Roy, Avijit; Li, Wenbin; Picton, Deric D; Nakhla, M K; Levy, L; Brlansky, R H

    2015-11-01

    Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence.

  12. 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.

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

    Science.gov (United States)

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

    2011-10-01

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

  14. Synergy between cucumber mosaic virus and zucchini yellow mosaic virus on Cucurbitaceae hosts tested by real-time reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Zeng, Rong; Liao, Qiansheng; Feng, Junli; Li, Dingjun; Chen, Jishuang

    2007-06-01

    Cucumber mosaic virus (CMV) and zucchini yellow mosaic virus (ZYMV) are two principal viruses infecting cucurbitaceous crops, and their synergy has been repeatedly observed. In our present work, a real-time reverse transcription-polymerase chain reaction procedure was established to study the accumulation kinetics of these two viruses in single and combined infections at the molecular level. The accumulations of open reading frames (ORFs) for 1a, 2a, 3a and coat protein (CP) of CMV and CP of ZYMV were tested. In the single infection, CMV-Fny ORFs accumulated to their maxima in cucumber or bottle gourd at 14 d post-inoculation (dpi), and gradually declined thereafter. ZYMV-SD CP ORF reached maximal accumulation at 14 and 28 dpi on cucumber and bottle gourd, respectively. However, when co-infected with CMV-Fny and ZYMV-SD, the maximal accumulation levels of all viral ORFs were delayed. CMV-Fny ORFs reached their maxima at 21 dpi on both hosts, and ZYMV-SDCP ORF reached maximal accumulation at 21 and 28 dpi on cucumber and bottle gourd, respectively. Generally, the accumulation levels of CMV-Fny ORFs in the co-infection were higher than those in the single infection, whereas the accumulation of ZYMV-SD CP ORF showed a reverse result.

  15. A duplex real-time reverse transcription polymerase chain reaction for the detection and quantitation of avian leukosis virus subgroups A and B.

    Science.gov (United States)

    Zhou, Gang; Cai, Wenbo; Liu, Xiaolei; Niu, Chengming; Gao, Caixia; Si, Changde; Zhang, Wei; Qu, Liandong; Han, Lingxia

    2011-05-01

    Avian leukosis is a disease that is spreading widely in the world causing large economic losses to the poultry industry. In this study, a duplex quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay was developed to detect and quantify avian leukosis virus subgroups A and B (ALVA/B). The assay was optimised to measure viral gp85 and chicken housekeeping (β-actin) genes. The result showed that the assay was specific for reference strains of ALVA/B subtype and no cross-reaction was detected with ALV subtypes E and J or with four other non-ALV viruses. The assay detected as few as 56 gp85 cDNA copies and was 100-fold more sensitive than a conventional RT-PCR. Seventy clinical blood samples were evaluated by both the qRT-PCR and the conventional RT-PCR assay, and the results show that 65 samples were positive by the qRT-PCR compared with 43 by the conventional RT-PCR. When this assay was used to quantify the viral load in ALV-inoculated embryos from three congenic chicken lines, the embryos from the B21 line showed the highest viral load, whereas the lowest load was found in the B5 line. This assay provides a powerful tool for quantitative detection of the ALVA/B and for the study of host genetic resistance to avian leukosis.

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

    Science.gov (United States)

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

    2014-01-01

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

  17. Analytical validation of a real-time reverse transcription polymerase chain reaction test for Pan-American lineage H7 subtype Avian influenza viruses

    Science.gov (United States)

    Spackman, Erica; Ip, H.S.; Suarez, D.L.; Slemons, R.D.; Stallknecht, D.E.

    2008-01-01

    A real-time reverse transcription polymerase chain reaction test for the identification of the H7 subtype in North American Avian influenza viruses (AIVs) was first reported in 2002; however, recent AIV surveillance efforts in wild birds and H7 outbreaks in poultry demonstrated that the 2002 test did not detect all H7 AIVs present in North and South America. Therefore, a new test, the 2008 Pan-American H7 test, was developed by using recently available H7 nucleotide sequences. The analytical specificity of the new assay was characterized with an RNA panel composed of 19 H7 viruses from around the world and RNA from all hemagglutinin subtypes except H16. Specificity for North and South American lineage H7 viruses was observed. Assay limits of detection were determined to be between 103 and 104 gene copies per reaction with in vitro transcribed RNA, and 100.0 and 10 0.8 50% egg infectious doses per reaction. The 2008 Pan-American H7 test also was shown to perform similarly to the 2002 test with specimens from chickens experimentally exposed to A/Chicken/BritishColumbia/314514-2/04 H7N3 highly pathogenic AIV. Furthermore, the 2008 test was able to detect 100% (n = 27) of the H7 AIV isolates recovered from North American wild birds in a 2006-2007 sample set (none of which were detected by the 2002 H7 test).

  18. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time reverse-transcription polymerase chain reaction and virus isolation.

    Science.gov (United States)

    Goodell, Christa K; Zhang, Jianqiang; Strait, Erin; Harmon, Karen; Patnayak, Devi; Otterson, Tracy; Culhane, Marie; Christopher-Hennings, Jane; Clement, Travis; Leslie-Steen, Pamela; Hesse, Richard; Anderson, Joe; Skarbek, Kevin; Vincent, Amy; Kitikoon, Pravina; Swenson, Sabrina; Jenkins-Moore, Melinda; McGill, Jodi; Rauh, Rolf; Nelson, William; O'Connell, Catherine; Shah, Rohan; Wang, Chong; Main, Rodger; Zimmerman, Jeffrey J

    2016-01-01

    The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 assays based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) and 7 assays based on virus isolation (VI). The OF specimens were inoculated with H1N1 or H3N2 IAV and serially diluted 10-fold (10(-1) to 10(-8)). Eight participating laboratories received 180 randomized OF samples (10 replicates × 8 dilutions × 2 IAV subtypes plus 20 IAV-negative samples) and performed the rRT-PCR and VI procedure(s) of their choice. Analysis of the results with a mixed-effect logistic-regression model identified dilution and assay as variables significant (P < 0.0001) for IAV detection in OF by rRT-PCR or VI. Virus subtype was not significant for IAV detection by either rRT-PCR (P = 0.457) or VI (P = 0.101). For rRT-PCR the cycle threshold (Ct) values increased consistently with dilution but varied widely. Therefore, it was not possible to predict VI success on the basis of Ct values. The success of VI was inversely related to the dilution of the sample; the assay was generally unsuccessful at lower virus concentrations. Successful swine health monitoring and disease surveillance require assays with consistent performance, but significant differences in reproducibility were observed among the assays evaluated.

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

    Science.gov (United States)

    Wernike, Kerstin; Bonilauri, Paolo; Dauber, Malte; Errington, Jane; LeBlanc, Neil; Revilla-Fernández, Sandra; Hjulsager, Charlotte; Isaksson, Mats; Stadejek, Tomasz; Beer, Martin; Hoffmann, Bernd

    2012-09-01

    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 or commercial kits used in the ring trial could identify all different PRRSV strains with an optimal analytical and diagnostic sensitivity. The genetic variability of the PRRSV strains, which is supposed to hinder the diagnostic of the RT-PCR because of mutations at the primer binding sites, was also confirmed by sequencing and subsequent phylogenetic analysis. In summary, a major problem in PRRSV diagnostics by RT-qPCR is false-negative results. To achieve maximum safety in the molecular diagnosis of PRRSV, the combined usage of different assays or kits is highly recommended.

  20. Development of conventional and real-time reverse transcription polymerase chain reaction assays to detect Tembusu virus in Culex tarsalis mosquitoes.

    Science.gov (United States)

    Petz, Lawrence N; Turell, Michael J; Padilla, Susana; Long, Lewis S; Reinbold-Wasson, Drew D; Smith, Darci R; O'Guinn, Monica L; Melanson, Vanessa R; Lee, John S

    2014-10-01

    Tembusu virus (TMUV) is an important emerging arthropod-borne virus that may cause encephalitis in humans and has been isolated in regions of southeast Asia, including Malaysia, Thailand, and China. Currently, detection and identification of TMUV are limited to research laboratories, because quantitative rapid diagnostic assays for the virus do not exist. We describe the development of sensitive and specific conventional and real-time quantitative reverse transcription polymerase chain reaction assays for detecting TMUV RNA in infected cell culture supernatant and Culex tarsalis mosquitoes. We used this assay to document the replication of TMUV in Cx. tarsalis, where titers increased 1,000-fold 5 days after inoculation. These assays resulted in the detection of virus-specific RNA in the presence of copurified mosquito nucleic acids. The use of these rapid diagnostic assays may have future applications for field pathogen surveillance and may assist in early detection, diagnosis, and control of the associated arthropod-borne pathogens. © The American Society of Tropical Medicine and Hygiene.

  1. Comparative analysis of quantitative reverse transcription real-time PCR and commercial enzyme imunoassays for detection of enterotoxigenic Bacillus thuringiensis isolates.

    Science.gov (United States)

    Kaminska, Paulina S; Yernazarova, Aliya; Murawska, Emilia; Swiecicki, Jakub; Fiedoruk, Krzysztof; Bideshi, Dennis K; Swiecicka, Izabela

    2014-08-01

    Entomopathogenic Bacillus thuringiensis is closely related to Bacillus cereus, a human pathogen known to cause emesis and diarrhea. Standard detection methods do not distinguish these bacilli. Hemolysin BL (hbl) and non-hemolytic enterotoxin (nhe) genes that encode, respectively, HBL and NHE enterotoxins, are known to be harbored in both bacterial species, suggesting that differentiation of these bacilli is clinically and epidemiologically relevant. In this study the reliability of quantitative reverse transcription real-time PCR (qRT-PCR) and enzyme immunoassays (EIAs) in detecting hbl and nhe transcripts and corresponding toxins in environmental B. thuringiensis isolates was assessed. At least one enterotoxin gene was present in each isolate, and nhe or hbl genes were found in 85% and 55% of the strains, respectively. Based on statistical analyses, both BCET-RPLA and Duopath detected HBL at similar levels, and TECRA and Duopath can be used interchangeably for the detection of NHE, although TECRA has significantly lower sensitivity than Duopath. Thus, as potential enterotoxic B. thuringiensis strains occur in the natural environment, and EIA results may not correspond with the presence of enterotoxin genes and their expression, we suggest that reliable interpretation will be significantly enhanced by including qRT-PCR to support inferences based on EIAs.

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Science.gov (United States)

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

    2014-09-01

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

  4. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time, reverse transcription polymerase chain reaction (rRT-PCR) and virus isolation

    Science.gov (United States)

    The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 real-time, reverse transcription polymerase chain reaction (rRT-PCR) and 7 virus isolation (VI) assays. To conduct the study, OF was inoculated with H1N1 or H3N2 IAV and serially 10-fold d...

  5. Comparison of Real-Time PCR, Reverse Transcriptase Real-Time PCR, Loop-Mediated Isothermal Amplification, and the FDA Conventional Microbiological Method for the Detection of Salmonella spp. in Produce ▿ †

    Science.gov (United States)

    Zhang, Guodong; Brown, Eric W.; González-Escalona, Narjol

    2011-01-01

    Contamination of foods, especially produce, with Salmonella spp. is a major concern for public health. Several methods are available for the detection of Salmonella in produce, but their relative efficiency for detecting Salmonella in commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection of Salmonella in six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated with Salmonella serovars at two different levels (105 and Bacteriological Analytical Manual) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU of Salmonella cells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only live Salmonella cells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection of Salmonella spp. in six high-risk produce commodities. PMID:21803916

  6. Comparison of real-time PCR, reverse transcriptase real-time PCR, loop-mediated isothermal amplification, and the FDA conventional microbiological method for the detection of Salmonella spp. in produce.

    Science.gov (United States)

    Zhang, Guodong; Brown, Eric W; González-Escalona, Narjol

    2011-09-01

    Contamination of foods, especially produce, with Salmonella spp. is a major concern for public health. Several methods are available for the detection of Salmonella in produce, but their relative efficiency for detecting Salmonella in commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection of Salmonella in six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated with Salmonella serovars at two different levels (10(5) and Bacteriological Analytical Manual) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU of Salmonella cells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only live Salmonella cells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection of Salmonella spp. in six high-risk produce commodities.

  7. Real-time quantitative reverse transcription-polymerase chain reaction to detect propionibacterial ribosomal RNA in the lymph nodes of Chinese patients with sarcoidosis.

    Science.gov (United States)

    Zhou, Y; Wei, Y-R; Zhang, Y; Du, S-S; Baughman, R P; Li, H-P

    2015-09-01

    The aim of this study was to investigate the diagnostic value of using the copy number of propionibacterial rRNA as a biomarker for sarcoidosis. Ribosomal RNA of Propionibacterium acnes and P. granulosum was measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) using formalin-fixed and paraffin-embedded tissue of lymph node biopsy from 65 Chinese patients with sarcoidosis, 45 with tuberculosis and 50 controls with other diseases (23 with non-specific lymphadenitis and 27 with mediastinal lymph node metastasis from lung cancer). The receiver operating characteristic (ROC) curve was analysed to determine an optimal cut-off value for diagnosis, and the diagnostic accuracy of the cut-off value was evaluated in additional tissue samples [24 patients with sarcoidosis and 22 with tuberculosis (TB)]. P. acnes or P. granulosum rRNA was detected in 48 of the 65 sarcoidosis samples but only in four of the 45 TB samples and three of the 50 control samples. Analysis of the ROC curve revealed that an optimal cut-off value of the copy number of propionibacterial rRNA for diagnosis of sarcoidosis was 50·5 copies/ml with a sensitivity and specificity of 73·8 and 92·6%, respectively. Based on the cut-off value, 19 of the 24 additional sarcoidosis samples exhibited positive P. acnes or P. granulosum, whereas only one of the 22 additional TB samples was positive, resulting in a sensitivity and specificity of 79·2 and 95·5%, respectively. These findings suggest that propionibacteria might be associated with sarcoidosis granulomatous inflammation. Detection of propionibacterial rRNA by RT-PCR might possibly distinguish sarcoidosis from TB.

  8. 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.

  9. A diagnostic one-step real-time reverse transcription polymerase chain reaction method for accurate detection of influenza virus type A

    Science.gov (United States)

    Behzadi, Mohammad Amin; Alborzi, Abdolvahab

    2016-01-01

    Introduction Influenza A is known as a public health concern worldwide. In this study, a novel one-step real-time reverse transcription polymerase chain reaction (rtRT-PCR) assay was designed and optimized for the detection of influenza A viruses. Material and methods The primers and probe were designed based on the analysis of 90 matrix nucleotide sequence data of influenza type A subtypes from the GenBank database of the National Center for Biotechnology Information (NCBI). The influenza virus A/Tehran/5652/2010 (H1N1 pdm09) was used as a reference. The rtRT-PCR assay was optimized, compared with that of the World Health Organization (WHO), and its analytical sensitivity, specificity and reproducibility were evaluated. In total, 64 nasopharyngeal swabs from patients with influenza-like illness (ILI) and 41 samples without ILI symptoms were tested for the virus, using conventional cell culture, direct immunofluorescence antibody (DFA) methods, and one-step rtRT-PCR with the designed primer set and probe and the WHO’s. Results The optimized assay results were similar to the WHO’s. The optimized assay results were similar to WHO’s, with non-significant differences for 10–103 copies of viral RNA/reaction (p > 0.05). It detected 10 copies of viral RNA/reaction with high reproducibility and no cross reactivity with other respiratory viruses. A specific cytopathic effect was observed in 6/64 (9.37%) of the ILI group using conventional culture and DFA staining methods; however, it was not seen in non-ILI. Also, the results of our assay and the WHO’s were similar to those of viral isolation and DFA staining. Conclusions Given the high specificity, sensitivity and reproducibility of this novel assay, it can serve as a reliable diagnostic tool for the detection of influenza A viruses in clinical specimens and lab experiments. PMID:27904520

  10. Comparison of nasopharyngeal and oropharyngeal swabs for the diagnosis of eight respiratory viruses by real-time reverse transcription-PCR assays.

    Directory of Open Access Journals (Sweden)

    Curi Kim

    Full Text Available BACKGROUND: Many acute respiratory illness surveillance systems collect and test nasopharyngeal (NP and/or oropharyngeal (OP swab specimens, yet there are few studies assessing the relative measures of performance for NP versus OP specimens. METHODS: We collected paired NP and OP swabs separately from pediatric and adult patients with influenza-like illness or severe acute respiratory illness at two respiratory surveillance sites in Kenya. The specimens were tested for eight respiratory viruses by real-time reverse transcription-polymerase chain reaction (qRT-PCR. Positivity for a specific virus was defined as detection of viral nucleic acid in either swab. RESULTS: Of 2,331 paired NP/OP specimens, 1,402 (60.1% were positive for at least one virus, and 393 (16.9% were positive for more than one virus. Overall, OP swabs were significantly more sensitive than NP swabs for adenovirus (72.4% vs. 57.6%, p<0.01 and 2009 pandemic influenza A (H1N1 virus (91.2% vs. 70.4%, p<0.01. NP specimens were more sensitive for influenza B virus (83.3% vs. 61.5%, p = 0.02, parainfluenza virus 2 (85.7%, vs. 39.3%, p<0.01, and parainfluenza virus 3 (83.9% vs. 67.4%, p<0.01. The two methods did not differ significantly for human metapneumovirus, influenza A (H3N2 virus, parainfluenza virus 1, or respiratory syncytial virus. CONCLUSIONS: The sensitivities were variable among the eight viruses tested; neither specimen was consistently more effective than the other. For respiratory disease surveillance programs using qRT-PCR that aim to maximize sensitivity for a large number of viruses, collecting combined NP and OP specimens would be the most effective approach.

  11. 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.

  12. Quantitative real-time reverse transcription-PCR analysis reveals stable and prolonged neurotoxin cluster gene activity in a Clostridium botulinum type E strain at refrigeration temperature.

    Science.gov (United States)

    Chen, Ying; Korkeala, Hannu; Lindén, Jere; Lindström, Miia

    2008-10-01

    The relative expression levels of six botulinum neurotoxin cluster genes in a group II Clostridium botulinum type E strain grown at 10 or 30 degrees C were investigated using quantitative real-time reverse transcription-PCR. An enzyme-linked immunosorbent assay was used to confirm neurotoxin expression. Distinct mRNA and toxin production patterns were observed at the two temperatures. The average relative mRNA levels at 10 degrees C were higher than (ntnh and p47), similar to (botE), or lower than (orfx1, orfx2, orfx3) those at 30 degrees C. The maximum botE expression levels and average neurotoxin levels at 10 degrees C were 45 to 65% of those at 30 degrees C. The relative mRNA levels at 10 degrees C declined generally slowly within 8 days, as opposed to the rapid decline observed at 30 degrees C within 24 h. Distinct expression patterns of the six genes at the two temperatures suggest that the type E neurotoxin cluster genes are transcribed as two tricistronic operons at 30 degrees C, whereas at 10 degrees C monocistronic (botE or orfx1 alone) and bicistronic (ntnh-p47 and orfx2-orfx3) transcription may dominate. Thus, type E botulinum neurotoxin production may be involved with various temperature-dependent regulatory events. In light of group II C. botulinum type E being a dangerous food-borne pathogen, these findings may be important in terms of the safety of refrigerated packaged foods of extended durability.

  13. Detection of respiratory viruses and bacteria in children using a twenty-two target reverse-transcription real-time PCR (RT-qPCR) panel.

    Science.gov (United States)

    Ellis, Chelsey; Misir, Amita; Hui, Charles; Jabbour, Mona; Barrowman, Nicholas; Langill, Jonathan; Bowes, Jennifer; Slinger, Robert

    2016-05-01

    Rapid detection of the wide range of viruses and bacteria that cause respiratory infection in children is important for patient care and antibiotic stewardship. We therefore designed and evaluated a ready-to-use 22 target respiratory infection reverse-transcription real-time polymerase chain reaction (RT-qPCR) panel to determine if this would improve detection of these agents at our pediatric hospital. RT-qPCR assays for twenty-two target organisms were dried-down in individual wells of 96 well plates and saved at room temperature. Targets included 18 respiratory viruses and 4 bacteria. After automated nucleic acid extraction of nasopharyngeal aspirate (NPA) samples, rapid qPCR was performed. RT-qPCR results were compared with those obtained by the testing methods used at our hospital laboratories. One hundred fifty-nine pediatric NPA samples were tested with the RT-qPCR panel. One or more respiratory pathogens were detected in 132/159 (83%) samples. This was significantly higher than the detection rate of standard methods (94/159, 59%) (Pviruses, bocavirus, and coronaviruses. The panel internal control assay performance remained stable at room temperature storage over a two-month testing period. The RT-qPCR panel was able to identify pathogens in a high proportion of respiratory samples. The panel detected more positive specimens than the methods in use at our hospital. The pre-made panel format was easy to use and rapid, with results available in approximately 90 minutes. We now plan to determine if use of this panel improves patient care and antibiotic stewardship.

  14. Detection of avian influenza A/H7N9/2013 virus by real-time reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Kang, Xiaoping; Wu, Weili; Zhang, Chuntao; Liu, Licheng; Feng, Huahua; Xu, Lizhi; Zheng, Xin; Yang, Honglei; Jiang, Yongqiang; Xu, Bianli; Xu, Jin; Yang, Yinhui; Chen, Weijun

    2014-09-01

    The first case of avian influenza A/H7N9 infection was reported in Shanghai in mid-February, 2013; by May 1, 2013, it had infected 127 people and caused 26 deaths in 10 provinces in China. Therefore, it is important to obtain reliable epidemiological data on the spread of this new infectious agent, a need that may be best met by the development of novel molecular methods. Here, a new method was described for the detection of avian influenza A/H7N9 using real-time reverse transcription-polymerase chain reaction (rRT-PCR). Using serial dilutions of avian influenza A H7N9 cultures, the detection limit of the assay was determined to be approximately 3.2×10(-4) HAUs (hemagglutination units) for the H7 gene and 6.4×10(-4) HAUs for N9 gene. In tests of serial dilutions of in vitro-transcribed avian influenza A H7 and N9 gene RNA, positive results were obtained for target RNA containing at least three copies of the H7 gene and six copies of the N9 gene. Thirteen throat swabs from H7N9 patients were tested; all tested positive in the assay. Specificity was evaluated by testing 18 other subtypes of influenza viruses; all tested negative. A total of 180 throat swabs from patients infected with influenza virus, including 60 from patients infected with seasonal influenza A/H1N1 virus, 60 from patients infected with pandemic influenza A/H1N1/2009 virus, 30 from patients infected with seasonal influenza A/H3N2 virus and 30 from patients infected with influenza B virus, were also tested; all tested negative.

  15. Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay

    Institute of Scientific and Technical Information of China (English)

    Zhu Yang; Guoliang Mao; Yujun Liu; Yuan-Chuan Chen; Chengjing Liu; Jun Luo; Xihan Li

    2013-01-01

    A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N 1/2009 influenza A virus.In this study,we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus.The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers.The qRT-PCR assays with the newly designed primers are highly specific,and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human,swine,and raccoon dog origin.Furthermore,the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction,respectively.When tested with 83 clinical samples,32 were detected to be positive using the qRT-PCR assays with our designed primers,while only 25 were positive by the assays with the WHO-recommended primers.These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection.

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

    Science.gov (United States)

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

    2017-04-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2014-05-02

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

  19. Rapid and quantitative detection of Fusarium oxysporum f. sp. cubense race 4 in soil by real-time fluorescence loop-mediated isothermal amplification.

    Science.gov (United States)

    Peng, Jun; Zhang, He; Chen, Fengping; Zhang, Xin; Xie, Yixian; Hou, Xianwen; Li, Guangyi; Pu, Jinji

    2014-12-01

    In this study, a real-time fluorescence loop-mediated isothermal amplification (RealAmp) was developed and evaluated for the rapid and quantitative detection of Fusarium oxysporum f. sp. cubense race 4 (R4) in soil. The LAMP primer set was designed based on previously verified RAPD marker sequences, and the RealAmp assay could specifically detect and distinguish R4 isolates from other related species. The detection sensitivity of the RealAmp assay was approx. 3·82 × 10(3) copies of plasmid DNA or 10(3) of spores per gram in artificially infested soil, indicating that the method is highly tolerant to inhibitor substances in soil compared to real-time PCR. Combining previously published TR4-specific detection methods with the newly established R4-specific RealAmp assay, an indirect approach to detect and differentiate ST4 isolates was achieved by comparing the detection results of R4 and TR4 simultaneously. The existence of ST4 isolates in China was subsequently confirmed through the developed approach. The developed RealAmp assay has been confirmed to be a simple, rapid and effective method to detect R4 in soil, which facilitates to further identify and distinguish ST4 isolates through the comparative analysis of detection results between TR4 and R4 simultaneously. The technique is an alternative quantitative detection method, which will be used for a routine detection service for the soil-borne pathogen in China. © 2014 The Society for Applied Microbiology.

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

    Directory of Open Access Journals (Sweden)

    Yi eWang

    2015-12-01

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

  1. New in situ capture quantitative (real-time) reverse transcription-PCR method as an alternative approach for determining inactivation of Tulane virus.

    Science.gov (United States)

    Wang, Dapeng; Xu, Shuxia; Yang, David; Young, Glenn M; Tian, Peng

    2014-04-01

    Human noroviruses (HuNoVs) are the major cause of epidemic nonbacterial gastroenteritis. Although quantitative (real-time) reverse transcription-PCR (qRT-PCR) is widely used for detecting HuNoVs, it only detects the presence of viral RNA and does not indicate viral infectivity. Human blood group antigens (HBGAs) have been identified as receptors/co-receptors for both HuNoVs and Tulane virus (TV) and are crucial for viral infection. We propose that viral infectivity can be evaluated with a molecular assay based on receptor-captured viruses. In this study, we employed TV as an HuNoV surrogate to validate the HBGA-based capture qRT-PCR method against the 50% tissue culture infectious dose (TCID50) method. We employed type B HBGA on an immuno-well module to concentrate TV, followed by amplification of the captured viral genome by in situ qRT-PCR. We first demonstrated that this in situ capture qRT-PCR (ISC-qRT-PCR) method could effectively concentrate and detect TV. We then treated TV under either partial or full inactivation conditions and measured the remaining infectivity by ISC-qRT-PCR and a tissue culture-based amplification method (TCID50). We found that the ISC-qRT-PCR method could be used to evaluate virus inactivation deriving from damage to the capsid and study interactions between the capsid and viral receptor. Heat, chlorine, and ethanol treatment primarily affect the capsid structure, which in turns affects the ability of the capsid to bind to viral receptors. Inactivation of the virus by these methods could be reflected by the ISC-qRT-PCR method and confirmed by TCID50 assay. However, the loss of the infectivity caused by damage to the viral genome (such as that from UV irradiation) could not be effectively reflected by this method. Despite this limitation, the ISC-qRT-PCR provides an alternative approach to determine inactivation of Tulane virus. A particular advantage of the ISC-qRT-PCR method is that it is also a faster and easier method to effectively

  2. 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.

  3. 直接RT-LAMP检测EV71方法的建立和评价%Development and assessment of direct reverse transcription loop-mediated isothermal amplification for EV71 detection

    Institute of Scientific and Technical Information of China (English)

    阮美生; 郭龙华; 周天龙; 黎荣

    2015-01-01

    Objective To develop a simple and rapid direct reverse transcription loop-mediated isothermal am-plification (direct RT-LAMP) experimental method for detecting enterovirus 71 (EV71) infection, and the sensitivity and specificity of this method using clinical nasopharyngeal swab specimens for the detection of EV71 were evaluated. Methods Direct RT-LAMP without RNA extraction and with heat-treatment was used to detect EV71 infection in 290 nasopharyngeal swab specimens. The sensitivity and specificity were evaluated. Results The accordance rate of direct RT-LAMP and RT-LAMP was 92.4%(268/290), and that of direct RT-LAMP and qRT-PCR was 88.9%. No false posi-tive was found in direct RT-LAMP or RT-LAMP. The clinical performance demonstrated the sensitivity and specificity of direct RT-LAMP was 90.3%and 100%compared to RT-LAMP, and 86.8%and 100%compared to qRT-PCR, re-spectively. Conclusion Direct RT-LAMP method can potentially be developed for simple and rapid screening of EV71 and other pathogens.%目的:建立并评价一种简单快速的逆转录环-介导的等温扩增(直接RT-LAMP)检测人肠道病毒71(EV71)的实验方法。方法无需RNA提取,用热处理标本后用直接RT-LAMP方法检测EV71,并用临床收集的290份咽试标本评价其灵敏度和特异性。结果直接RT-LAMP和RT-LAMP实验完全符合率为92.4%(268/290),直接RT-LAMP和qRT-PCR完全符合率为88.9%。在RT-LAMP或直接RT-LAMP实验中没有发现假阳性。与RT-LAMP比较,直接RT-LAMP的灵敏度和特异性分别为90.3%和100%;与qRT-PCR比较,其敏感性和特异性分别为86.8%和100%。结论直接RT-LAMP方法能被开发成简单快速检测EV71和其他病原体的方法。

  4. Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP system for a highly sensitive detection of enterovirus in the stool samples of acute flaccid paralysis cases

    Directory of Open Access Journals (Sweden)

    Wakita Takaji

    2009-12-01

    Full Text Available Abstract Background In the global eradication program for poliomyelitis, the laboratory diagnosis plays a critical role by isolating poliovirus (PV from the stool samples of acute flaccid paralysis (AFP cases. In this study, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP system for a rapid and highly sensitive detection of enterovirus including PV to identify stool samples positive for enterovirus including PV. Methods A primer set was designed for RT-LAMP to detect enterovirus preferably those with PV-like 5'NTRs of the viral genome. The sensitivity of RT-LAMP system was evaluated with prototype strains of enterovirus. Detection of enterovirus from stool extracts was examined by using RT-LAMP system. Results We detected at least 400 copies of the viral genomes of PV(Sabin strains within 90 min by RT-LAMP with the primer set. This RT-LAMP system showed a preference for Human enterovirus species C (HEV-C strains including PV, but exhibited less sensitivity to the prototype strains of HEV-A and HEV-B (detection limits of 7,400 to 28,000 copies. Stool extracts, from which PV, HEV-C, or HEV-A was isolated in the cell culture system, were mostly positive by RT-LAMP method (positive rates of 15/16 (= 94%, 13/14 (= 93%, and 4/4 (= 100%, respectively. The positive rate of this RT-LAMP system for stool extracts from which HEV-B was isolated was lower than that of HEV-C (positive rate of 11/21 (= 52%. In the stool samples, which were negative for enterovirus isolation by the cell culture system, we found that two samples were positive for RT-LAMP (positive rates of 2/38 (= 5.3%. In these samples, enterovirus 96 was identified by sequence analysis utilizing a seminested PCR system. Conclusions RT-LAMP system developed in this study showed a high sensitivity comparable to that of the cell culture system for the detection of PV, HEV-A, and HEV-C, but less sensitivity to HEV-B. This RT-LAMP system would be useful for the

  5. 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

    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......-PCR protocols and a standardized protocol commonly used by the Friedrich-Loeffler-Institute (FLI) on a panel of well-characterized samples. In general, all participants produced results within the acceptable range. The FLI assay, several in-house assays, and the commercial kits had high analytical sensitivity...... 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...

  6. Use of automated real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor experimental swine vesicular disease virus infection in pigs

    DEFF Research Database (Denmark)

    Reid, S.M.; Paton, D.J.; Wilsden, G.;

    2004-01-01

    of the template extraction method was required to counteract the effects of RT-PCR inhibitors in faeces. It was concluded that the automated real-time RT-PCR is a useful diagnostic method for SVD in clinically or subclinically affected pigs and contributed to the study of the pathogenesis of SVD in the pigs....

  7. Assessment of the inhibitory effect of ribavirin on the rainbow trout rhabdovirus VHSV by real-time reverse-transcription PCR.

    Science.gov (United States)

    Marroquí, Laura; Estepa, Amparo; Perez, Luis

    2007-05-16

    Viral hemorrhagic septicemia virus (VHSV) is one of the most ubiquitous viruses in salmonid aquaculture in Europe. This infectious disease results in significant losses in the farming industry and therefore effective therapeutic agents are needed to control outbreaks caused by this pathogen. Thus, accurate methods to test new antiviral compounds need to be developed. Our goal was to establish a model system for testing novel antivirals with potential applications to aquaculture. In a previous study, a TaqMan real-time RT-PCR assay was designed to detect and quantitate VHSV in rainbow trout tissues [Chico, V., Gomez, N., Estepa, A., Perez, L., 2006. Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR. J. Virol. Methods 132, 154-159]. In this report, we applied the real-time RT-PCR assay to the evaluation of the inhibitory effect of ribavirin, a well-known broad spectrum antiviral drug, in a cell culture system. When added from the beginning of the infection, ribavirin caused a dose-dependent reduction of VHSV RNA accumulation. Real-time RT-PCR measurements showed 99.8% inhibition at 25 microg/ml ribavirin, with an IC50 of 0.43 microg/ml. Ribavirin maintained its inhibitory activity against VHSV when added at 6 h post-infection. Quantitation of N protein messenger RNA and plus-stranded RNA showed a substantial decrease of viral transcription in ribavirin-treated cells. Partial reversion of the effect of ribavirin by addition of GTP was observed, confirming that ribavirin targets the synthesis of guanidine nucleotides in the cells. This is the first report of a real-time PCR-based assay for addressing the efficacy and mechanism of action of an antiviral agent for rainbow trout.

  8. 克里米亚-刚果出血热病毒可视化逆转录环介导等温扩增快速检测法的建立%A reverse transcription loop-mediated isothermal amplification for rapid visual detection of Crimean-Congo hemorrhagic fever virus

    Institute of Scientific and Technical Information of China (English)

    韩一芳; 钟璟皓; 张晨; 张琪; 吕恒; 胡丹; 张锦海; 王长军

    2016-01-01

    目的 建立克里米亚刚果出血热病毒(Crimean-Congo hemorrhagic fever virus,CCHFV)的可视化逆转录环介导等温扩增(reverse transcription loop-mediated isothermal amplification,RTLAMP)快速检测法.方法 体外合成CCHFV的S基因,构建重组质粒,体外转录为模板RNA,在线设计3组LAMP引物,使用实时浊度仪筛选出最佳引物,以羟基萘酚蓝(Hydroxynaphthol blue,HNB)作为指示剂,建立可视化RT-LAMP反应体系.结果 实时浊度检测结果显示合成的3组LAMP引物中第1组引物的扩增效率最高,峰值出现于20 min.添加环引物后,扩增效率进一步提高,13 min即可达到峰值.利用最佳引物建立的CCHFV可视化RT-LAMP检测法最低检测限浓度为10拷贝/μl,检出时间为30 min,较巢式RT-PCR法和实时定量RT-PCR法分别高1-3个数量级.该方法稳定性好,不会与症状相近的病原体如肾综合征出血热汉坦病毒(汉滩型和汉城型)、马尔堡病毒、新型布尼亚病毒、埃博拉病毒(GP蛋白和VP蛋白)产生交叉反应.结论 建立的CCHFV可视化RT-LAMP检测法,灵敏度好、特异度高、快速廉价、操作简单,无需开盖即可直接观察结果,适用于条件有限的基层单位和偏远地区.但仍需临床样本进行进一步验证.%Objective To develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid and visual detection of Crimean-Congo hemorrhagic fever virus.Methods The recombinant plasmid containing the S gene of Crimean-Congo hemorrhagic fever virus was constructed using in vitro gene synthesis.The most effective primers of the three sets of RT-LAMP primers,designed by Primer Explorer software 4.0,was selected using real-time turbidimetry.Hydroxynaphthol blue (HNB) as the indicator was used to judge the result.Results The result of real-time turbidimetry showed that the first set of primers had the highest amplification efficiency with a peak time of 20 min.The amplification efficiency was

  9. Use of automated real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor experimental swine vesicular disease virus infection in pigs

    DEFF Research Database (Denmark)

    Reid, S.M.; Paton, D.J.; Wilsden, G.

    2004-01-01

    Automated real-time RT-PCR was evaluated as a diagnostic tool for swine vesicular disease virus (SVDV) infection on a range of samples (vesicular epithelium, serum, nasal swabs, faeces) from four inoculated and three in-contact pigs over a period of 28 days. Traditional diagnostic procedures (virus....... The RT-PCR and virus isolation were generally comparable in detecting SVDV in the serum and nasal swabs from inoculated and in-contact pigs up to day 6 after infection; it was possible, however, to isolate virus for a longer period from the faeces of a few pigs. This suggested that further optimization...... of the template extraction method was required to counteract the effects of RT-PCR inhibitors in faeces. It was concluded that the automated real-time RT-PCR is a useful diagnostic method for SVD in clinically or subclinically affected pigs and contributed to the study of the pathogenesis of SVD in the pigs....

  10. Development of Conventional and Real-Time Reverse Transcription Polymerase Chain Reaction Assays to Detect Tembusu Virus in Culex tarsalis Mosquitoes

    Science.gov (United States)

    2014-08-11

    appropri- ate preventive measures can be taken to prevent morbidity and mortality associated with infection with these pathogens. Development of...field-based real-time quantitative RT-PCR (qRT-PCR) assays for rapid detection of virus-infected mos- quitoes would be a great assistance to preventive ...specimens were propagated in primary duck embryo, C6/36 ( Aedes albopictus), or Vero (African green monkey kidney) cells to produce stock viruses for

  11. Application of real-time reverse transcription polymerase chain reaction to the detection the matrix, H5 and H7 genes of avian influenza viruses in field samples from South Korea.

    Science.gov (United States)

    Kim, Hye-Ryoung; Oem, Jae-Ku; Bae, You-Chan; Kang, Min-Su; Lee, Hee-Soo; Kwon, Yong-Kuk

    2013-03-14

    The rapid and accurate identification of the H5 and H7 subtypes of avian influenza (AI) virus is an important step for the control and eradication of highly pathogenic AI outbreaks and for the surveillance of AI viruses that have the potential to undergo changes in pathogenicity in poultry and wild birds. Currently, real-time reverse transcription polymerase chain reaction (RRT-PCR) is routinely used for the rapid detection of the H5 and H7 genes, but misidentification is frequent for emergent isolates and viruses isolated from diverse regions due to the high sequence variation among AI viruses. In this study, an RRT-PCR method was tested for the detection of matrix, H5 and H7 genes from diverse subtypes of AI viruses and from field samples obtained through AI surveillance in South Korea over the last four years. Both RRT-PCR and conventional experiment (virus isolation using egg inoculation followed by reverse transcription polymerase chain reaction) agreed on the virus-positive samples. And the comparison of the results with 174 clinical samples showed a high level of agreement without decreasing the specificity and sensitivity. This assay could be useful tool for the rapid detection of AI using the field samples from domestic poultry and wild birds in South Korea, and continuous regional updates is needed to validate primer sets as the AI virus evolves.

  12. 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.

  13. TaqMan real-time reverse transcription-PCR assay for universal detection and quantification of avian hepatitis E virus from clinical samples in the presence of a heterologous internal control RNA.

    Science.gov (United States)

    Troxler, Salome; Marek, Ana; Prokofieva, Irina; Bilic, Ivana; Hess, Michael

    2011-04-01

    Avian hepatitis E virus (HEV) isolates could be separated into at least three genotypes. In this study, the development of the first duplex TaqMan real-time reverse transcription-PCR (RT-PCR) assay for detection and quantification of avian HEV is presented. Primers and probes binding within relatively conserved open reading frame 3 (ORF3) were designed. Tenfold dilution series of in vitro-transcribed avian HEV RNA were used as the standard for quantification. A 712-bp region of the green fluorescent protein gene was transcribed in vitro and used as a heterologous internal control for both RNA isolation and real-time RT-PCR. The duplex real-time RT-PCR for avian HEV had an efficiency of 1.04, a regression squared value of 0.996, and a sensitivity of approximately 3.6 × 10(3) copies per reaction mixture when in vitro-transcribed RNA was used as the template. The presence of in vitro-transcribed heterologous internal control RNA did not affect amplification of avian HEV RNA compared to that achieved by the single assay. The sensitivity of the real-time RT-PCR assay was comparable to that of conventional RT-PCR, and it was shown to be highly specific, as tissues from uninfected chickens, mammalian HEVs, and other viral genomes did not produce positive signals. All tested field samples with virus belonging to different avian HEV genotypes were successfully detected with this new duplex TaqMan real-time RT-PCR assay.

  14. 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.

  15. Newly emerging mutations in the matrix genes of the human influenza A(H1N1)pdm09 and A(H3N2) viruses reduce the detection sensitivity of real-time reverse transcription-PCR.

    Science.gov (United States)

    Yang, Ji-Rong; Kuo, Chuan-Yi; Huang, Hsiang-Yi; Wu, Fu-Ting; Huang, Yi-Lung; Cheng, Chieh-Yu; Su, Yu-Ting; Chang, Feng-Yee; Wu, Ho-Sheng; Liu, Ming-Tsan

    2014-01-01

    New variants of the influenza A(H1N1)pdm09 and A(H3N2) viruses were detected in Taiwan between 2012 and 2013. Some of these variants were not detected in clinical specimens using a common real-time reverse transcription-PCR (RT-PCR) assay that targeted the conserved regions of the viral matrix (M) genes. An analysis of the M gene sequences of the new variants revealed that several newly emerging mutations were located in the regions where the primers or probes of the real-time RT-PCR assay bind; these included three mutations (G225A, T228C, and G238A) in the A(H1N1)pdm09 virus, as well as one mutation (C163T) in the A(H3N2) virus. These accumulated mismatch mutations, together with the previously identified C154T mutation of the A(H1N1)pdm09 virus and the C153T and G189T mutations of the A(H3N2) virus, result in a reduced detection sensitivity for the real-time RT-PCR assay. To overcome the loss of assay sensitivity due to mismatch mutations, we established a real-time RT-PCR assay using degenerate nucleotide bases in both the primers and probe and successfully increased the sensitivity of the assay to detect circulating variants of the human influenza A viruses. Our observations highlight the importance of the simultaneous use of different gene-targeting real-time RT-PCR assays for the clinical diagnosis of influenza.

  16. 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

    2017-03-21

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

  17. Quantitative Real-Time Reverse Transcription-PCR Assay for the Expression of Tob mRNA in Human Colorectal Cancer

    Institute of Scientific and Technical Information of China (English)

    Dian-chao WU

    2010-01-01

    OBJECTIVE Tob is a member of Tob/BTG antiproliferative family. To date, Tob expression in human carcinoma using clinical specimens has not been studied in depth except for lung carcinoma and thyroid carcinoma. This study is the first to investigate the expression levels of Tob gene in human colorectal cancer tissues,and their corresponding para-cancerous tissues. The correlation of expression of the Tob gene with clinicopathological characteristics of colorectal cancer was also analyzed.METHODS Quantitative real time RT-PCR was used to detect the expression of Tob mRNA in 31 colorectal cancers.RESULTS Compared with normal tissues, up-regulation of Tob mRNA was observed in 31 colorectal cancer tissues (P = 0.020).The expression level of Tob at Dukes C + D phase was higher than Dukes A + B phase, and the difference was signifi cant (P < 0.05).However, in this study, it was found that the expression of Tob mRNA was not related with age, gender, and pathological type of colorectal cancer.CONCLUSION The up-regulation of Tob may be closely associated with tumorigenesis of colorectal carcinoma.

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

    Institute of Scientific and Technical Information of China (English)

    袁瑛娜; 单潇潇; 王宗德; 石磊; 袁秀金; 谭贵良

    2011-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  20. Evaluation and application of a one-step duplex real-time reverse transcription polymerase chain reaction assay for the rapid detection of influenza A (H7N9) virus from poultry samples.

    Science.gov (United States)

    Bao, Hongmei; Ma, Yong; Shi, Jianzhong; Zeng, Xianying; Zhao, Yuhui; Wang, Xiurong; Chen, Hualan

    2015-10-01

    In China, a novel reassortant influenza A (H7N9) virus, which has caused 435 cases of human infection, has recently emerged. Most cases of human infections with the H7N9 virus are known to be associated with a poultry farm and live-poultry markets. In this study, a one-step duplex real-time reverse transcription polymerase chain reaction (RRT-PCR) assay was developed for the simultaneous detection of the hemagglutinin (HA) and neuraminidase (NA) genes of the H7N9 virus for effective surveillance and early diagnosis of cases from clinical samples collected from live-poultry markets or poultry farms. The detection limit of this assay was as low as 0.1 EID50 of H7N9 viruses, which is similar to the detection limit of the real-time RT-PCR assay released by the Word Health Organization. The coefficients of variation (CVs) of both inter-assay and intra-assay reproducibility were less than 1.55 %, showing good reproducibility. No cross-reactivity was observed with RNA of other subtypes of influenza virus or other avian respiratory viruses. The assay can effectively detect H7N9 influenza virus RNA from multiple sources, including chickens, pigeons, ducks, humans, and the environment. Furthermore, the RRT-PCR assay was evaluated with more than 700 clinical samples collected from live-poultry markets and 120 experimentally infected chicken samples. Together, these results indicate that the duplex RRT-PCR assay is a specific, sensitive, and efficient diagnostic method for the epidemiological surveillance and diagnosis of H7N9 virus from different sources, particularly poultry samples.

  1. The use of a one-step real-time reverse transcription polymerase chain reaction (rRT-PCR) for the surveillance of viral hemorrhagic septicemia virus (VHSV) in Minnesota.

    Science.gov (United States)

    Phelps, Nicholas B D; Patnayak, Devi P; Jiang, Yin; Goyal, Sagar M

    2012-12-01

    Viral hemorrhagic septicemia virus (VHSV) is a highly contagious and pathogenic virus of fish. The virus infects more than 70 fish species worldwide, in both fresh and salt water. A new viral strain (VHSV-IVb) has proven both virulent and persistent, spreading throughout the Great Lakes of North America and to inland water bodies in the region. To better understand the geographic distribution of the virus, we used a modified real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for high-throughput testing of fish for VHSV. The assay was shown to be twice as sensitive as the gold standard, virus isolation, and did not cross react with other viruses found in fish. In addition, the diagnostic turnaround time was reduced from 28 to 30 d for virus isolation to 2-4 d for rRT-PCR. To demonstrate the usefulness of the rRT-PCR assay, 115 high-priority water bodies in Minnesota were tested by both methods from April 2010 to June 2011. All survey sites tested negative for VHSV by both methods. The survey results have informed fisheries managers on the absence of VHSV in Minnesota and have better prepared them for the eventual arrival of the disease. In addition, the results demonstrate the value of this rRT-PCR as a surveillance tool to rapidly identify an outbreak so that it can be controlled in a timely manner.

  2. Considering the effect of stem-loop reverse transcription and real-time PCR analysis of blood and saliva specific microRNA markers upon mixed body fluid stains.

    Science.gov (United States)

    Uchimoto, Mari L; Beasley, Emma; Coult, Natalie; Omelia, Emma J; World, Damian; Williams, Graham

    2013-07-01

    Forensic RNA analysis is gathering pace with reports of messenger RNA analysis being used in case work, and with microRNA being increasingly researched. Such techniques address a fundamental issue in body fluid identification, namely increased specificity over existing chemical tests, and the incorporation of additional body fluids such as vaginal material. The use of RNA analysis will be of particular value to sex offences, where there can be a mixture of multiple body fluids from different people. The aim of this study was to determine whether microRNA based body fluid identification tests can be applied to mixed body fluid samples. Blood and saliva were acquired from volunteers and underwent total RNA extraction. Mixed samples were prepared using a range of ratios from 1:1 to 10:1. Each mixed sample then underwent a blood-saliva differentiation test developed in-house, which includes stem-loop reverse transcription and real-time PCR analysis. Aliquots following mixture preparation also underwent standard STR analysis, utilising Quantiplex and Next Generation Multiplex kits. Data relating to the development of an in-house blood-saliva differentiation test is presented, in which it has been demonstrated that such a test has a lower limit of detection than the enzymatic equivalent. It has been shown that not only is it possible to determine the presence of more than one body fluid, it is also possible to determine the major body fluid contributor as well as the minor contributor.

  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.

    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.

  4. 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.

  5. Detection of Echinoderm Microtubule Associated Protein Like 4-Anaplastic Lymphoma Kinase Fusion Genes in Non-small Cell Lung Cancer Clinical Samples by a Real-time Quantitative Reverse Transcription Polymerase Chain Reaction Method.

    Science.gov (United States)

    Zhao, Jing; Zhao, Jin-Yin; Chen, Zhi-Xia; Zhong, Wei; Li, Long-Yun; Liu, Li-Cheng; Hu, Xiao-Xu; Chen, Wei-Jun; Wang, Meng-Zhao

    2016-12-20

    Objective To establish a real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) for the rapid, sensitive, and specific detection of echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion genes in non-small cell lung cancer. Methods The specific primers for the four variants of EML4-ALK fusion genes (V1, V2, V3a, and V3b) and Taqman fluorescence probes for the detection of the target sequences were carefully designed by the Primer Premier 5.0 software. Then, using pseudovirus containing EML4-ALK fusion genes variants (V1, V2, V3a, and V3b) as the study objects, we further analyzed the lower limit, sensitivity, and specificity of this method. Finally, 50 clinical samples, including 3 ALK-fluorescence in situ hybridization (FISH) positive specimens, were collected and used to detect EML4-ALK fusion genes using this method. Results The lower limit of this method for the detection of EML4-ALK fusion genes was 10 copies/μl if no interference of background RNA existed. Regarding the method's sensitivity, the detection resolution was as high as 1% and 0.5% in the background of 500 and 5000 copies/μl wild-type ALK gene, respectively. Regarding the method's specificity, no non-specific amplification was found when it was used to detect EML4-ALK fusion genes in leukocyte and plasma RNA samples from healthy volunteers. Among the 50 clinical samples, 47 ALK-FISH negative samples were also negative. Among 3 ALK-FISH positive samples, 2 cases were detected positive using this method, but another was not detected because of the failure of RNA extraction. Conclusion The proposed qRT-PCR assay for the detection of EML4-ALK fusion genes is rapid, simple, sensitive, and specific, which is deserved to be validated and widely used in clinical settings.

  6. 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).

  7. Development and validation of a real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay for investigation of wild poliovirus type 1-South Asian (SOAS) strain reintroduced into Israel, 2013 to 2014.

    Science.gov (United States)

    Hindiyeh, M Y; Moran-Gilad, J; Manor, Y; Ram, D; Shulman, L M; Sofer, D; Mendelson, E

    2014-02-20

    In February 2013, wild poliovirus type 1 (WPV1) was reintroduced into southern Israel and resulted in continuous silent circulation in the highly immune population. As a part of the public health emergency response, a novel real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed, to allow for the sensitive and specific detection of the circulatingWPV1-South Asian (SOAS) strain. Specific primers and probes derived from the VP-1 region were designed, based on sequenced sewage isolates, and used to simultaneously amplify this WPV1-SOAS sequence together with bacteriophage MS-2 as internal control. High titre WPV1-SOAS stock virus was used for assay optimisation and 50 processed sewage samples collected from southern Israel and tested by reference culture based methods were used for analytical validation of the assay’s performance. The limit of detection of the multiplex qRT-PCR (SOAS/MS-2) assay was 0.1 plaque-forming unit (pfu)/reaction (20 pfu/mL) for WPV1-SOAS RNA with 100% sensitivity, specificity, positive and negative predictive values when compared to the culture based method. The turnaround time was rapid, providing results for environmental samples within 24 to 48 hours from completion of sewage processing, instead of five to seven days by culture-based analysis. Direct sewage testing by qRT-PCR assay proved to be a useful tool for rapid detection and environmental surveillance of WPV1-SOAS circulating strain during emergency response. Application of the approach for detection of WPV1-SOAS in stool samples obtained during acute flaccid paralysis (AFP) surveillance or field surveys should be further evaluated.

  8. Real-Time Reverse-Transcription Quantitative Polymerase Chain Reaction Assay Is a Feasible Method for the Relative Quantification of Heregulin Expression in Non–Small Cell Lung Cancer Tissue

    Directory of Open Access Journals (Sweden)

    Jessica Kristof

    2017-03-01

    Full Text Available In preclinical studies, heregulin ( HRG expression was shown to be the most relevant predictive biomarker for response to patritumab, a fully human anti–epidermal growth factor receptor 3 monoclonal antibody. In support of a phase 2 study of erlotinib ± patritumab in non–small cell lung cancer (NSCLC, a reverse-transcription quantitative polymerase chain reaction (RT-qPCR assay for relative quantification of HRG expression from formalin-fixed paraffin-embedded (FFPE NSCLC tissue samples was developed and validated and described herein. Test specimens included matched FFPE normal lung and NSCLC and frozen NSCLC tissue, and HRG -positive and HRG -negative cell lines. Formalin-fixed paraffin-embedded tissue was examined for functional performance. Heregulin distribution was also analyzed across 200 NSCLC commercial samples. Applied Biosystems TaqMan Gene Expression Assays were run on the Bio-Rad CFX96 real-time PCR platform. Heregulin RT-qPCR assay specificity, PCR efficiency, PCR linearity, and reproducibility were demonstrated. The final assay parameters included the Qiagen FFPE RNA Extraction Kit for RNA extraction from FFPE NSCLC tissue, 50 ng of RNA input, and 3 reference (housekeeping genes ( HMBS, IPO8 , and EIF2B1 , which had expression levels similar to HRG expression levels and were stable among FFPE NSCLC samples. Using the validated assay, unimodal HRG distribution was confirmed across 185 evaluable FFPE NSCLC commercial samples. Feasibility of an RT-qPCR assay for the quantification of HRG expression in FFPE NSCLC specimens was demonstrated.

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

    Institute of Scientific and Technical Information of China (English)

    关丽; 许松涛; 聂凯; 张丹; 李鑫娜; 许文波; 马学军

    2015-01-01

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

  10. 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.

  11. 两种实时定量RT-PCR方法检测miRNAs表达的技术分析%Technical analysis for detection and quantification of microRNAs by two real-time quantitative reverse transcription methods

    Institute of Scientific and Technical Information of China (English)

    闵自信; 杜小云; 宁启兰; 钟楠楠; 郑悦雯; 韩燕; 吕社民; 张蕊

    2013-01-01

    目的 比较两种实时定量反转录聚合酶链反应(RT-qPCR)方法检测microRNAs (miRNAs)表达含量的技术差异,优化不同实验目的和条件下研究miRNAs表达的技术方法.方法 取21d和42d两个时间点的SD大鼠关节软骨组织,采用Trizol法提取总RNA备用.选取rno-miR-15b、rno-miR-16、rno miR 195、rno-miR-497作为研究对象,分别用茎环引物和试剂公司提供的试剂盒方法反转录总RNA,并应用实时定量PCR方法检测这些miRNAs的表达量.提取人血浆中总RNA,用上述两种RT-qPCR方法实时定量检测has-miR-16的表达量.结果 两种方法检测这些miRNAs表达量,在大鼠21d和42 d这两个时间点其表达量变化趋势相同,都呈现增高的趋势,这与我们前期Solexa测序结果相同.在血浆中的结果显示,其中茎环引物反转录方法灵敏度相对较高.结论 茎环引物法在少量几个重要的miRNAs检测中具有优势,而试剂盒方法适用于大量miRNAs的筛查.%Objective To optimize the method for quantifying microRNAs in different experimental purposes and conditions by comparing two real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) methods. Methods We isolated the total RNA from the SD rat articular cartilage at postnatal day 21 and day 42 with TRIzol(R) reagent. The RT reactions were performed by stem-loop primers and the universal primer of miRNA detection kit respectively; then real time PCR was performed to test the expressions of rno-miR-15b, rno-miR-16, rno-miR-195 and rno-miR-497. In addition, the total RNAs in human plasma were isolated by using TRI Reagent BD (MRC, TR126) according to the instructions by the manufacturer with two different RT-qPCR methods to quantify the expression of has-miR-16. Results The expression change of these miRNAs was of the same increase trend by the two different RT-qPCR methods, which accorded with the results of our Solexa sequencing. The results of plasma demonstrated that

  12. Rapid detection method preliminary establishment of infectious bursal disease virus by reverse transcription loop-mediated isothermal amplification%鸡传染性法氏囊病毒反转录环媒恒温检测方法的初步建立

    Institute of Scientific and Technical Information of China (English)

    杨作丰; 石霖; 邓文超; 郭炎; 董娜; 王竹; 赵培; 张鑫; 张雅为

    2014-01-01

    The objective of this study is to develop a rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of infectious bursal disease virus (IBDV). Six primers were designed to amplify the VP1 gene of IBDV using PrimerExplor-er V4. The optimal reaction condition of the current RT-LAMP for IBDV was 65 ℃ for 60 mins. It was capable of detecting IBDV from clinical samples and differentiating IBDV from other avain vi-ruses, and does not require an additional expensive equipment. The minimum detection limit of the RT-LAMP assay was 10-6, making this assay approximately 100-fold higher than that of one-step RT-PCR. The assay were evaluated by comparison with RT-PCR, by 93.8% consistence. The specificity and simplicity of the assay could make it a useful method for the detection of IBDV infection.%本研究利用Primer Explorer V4软件设计了针对鸡传染性法氏囊病毒(IBDV)VP1基因保守区6个特异性部位的4条引物,建立了IBDV的反转录环媒恒温检测方法。该方法反应体系在恒温水浴锅中作用1 h即可得到其特有的阶梯状条带,加入荧光素后肉眼可直接观察结果。本方法特异性高,对IBDV的最低检出量为10-6稀释倍数,敏感性是普通RT-PCR的100倍。反转录环媒恒温检测方法和普通RT-PCR方法检测临床样品的符合率为93.8%。因此该方法可以作为IBDV的综合防治和疾病诊断的实用方法。

  13. Real-time fluorescence loop-mediated isothermal amplicication for the rapid detection of Mycobacterium tuberculosis%实时荧光环介导等温扩增在快速检测结核分枝杆菌中的应用

    Institute of Scientific and Technical Information of China (English)

    曹东林; 胡亮杉; 曹炜伟; 石磊; 叶蕾; 叶泽兵; 田军章

    2013-01-01

    目的:建立实时荧光环介导等温扩增快速检测结核分枝杆菌的方法。方法针对结核分枝杆菌特异序列建立实时荧光环介导等温扩增技术;对其特异性和敏感性进行评估;通过敏感性和特异性等指标验证其稳定性。结果建立的实时荧光环介导等温扩增法对结核和非结核分枝杆菌标准菌株检测的敏感性和特异性均为100%,灵敏度可达102个菌/mL;痰标本结核分歧杆菌检测的敏感性为94.64%(53/56),荧光定量聚合酶链式反应方法检测的敏感性为92.86%(52/56);检测的特异性为96.55%(28/29),荧光定量聚合酶链式反应方法检测的特异性为96.55%(28/29)。结论建立实时荧光环介导等温扩增检测结核分枝杆菌的方法,敏感性和特异性强,灵敏度高,稳定性好,可用于痰液中结核分枝杆菌的快速检测。%Objective To establish a rapid method of real-time fluorescence loop-mediated isothermal amplification (LAMP) for detection of Mycobacterium tuberculosis. Methods According to the specific sequence of IS6110 in Mycobacterium tuberculosis, 6 primers of loop-mediated isothermal amplification were designed and synthesized. With the ESE-Quant tube scanner platform, a real-time fluorescence loop-mediated isothermal amplification was established. 14 strains of non-tuberculous mycobacteria standard strains and 14 strains of Mycobacterium tuberculosis isolated from cases were used to evaluate specificity and sensitivity of the method. Sputum samples were collected from 56 positive cases for Mycobacterium tuberculosis and 29 negative cases for Mycobacterium tuberculosis. The stability of the method was validated by the sensitivity and specificity compared to the real-time fluorescent PCR method. Results Real-time fluorescence loop-mediated isothermal amplification method was established, the specificity and sensitivity of Mycobacterium tuberculosis and non-tuberculous mycobacterial

  14. 实时荧光RT-PCR方法检测香石竹环斑病毒%Detection of Carnation ringspot virus by real-time fluorescent reverse transcription polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    崔学慧; 陈舜胜; 于翠; 杨翠云

    2012-01-01

    本研究以香石竹环斑病毒(Carnation ringspot virus,CRSV)的5个分离物为研究对象,根据CRSV运动蛋白(MP)基因的保守序列设计一对特异性引物和TaqMan荧光探针,建立了检测CRSV的实时荧光RT-PCR(real-time fluorescem RTPCR)方法.该方法利用TaqMan探针水解产生的荧光信号实时监测目标基因的扩增,实现real-time fluorescent PCR扩增和检测同步进行.结果表明,本研究建立的实时荧光RT-PCR方法具有更快速、灵敏和特异的优点,与普通RT-PCR方法相比其灵敏度提高了100倍,适合于对进境种苗携带的CRSV的快速检测.%In this study, a pair of specific primers and a TaqMan probe were designed according to the conserved movement protein (MP) gene sequences of Carnation ringspot virus (CRSV), and a real-time fluorescent PCR method was applied for detection of CRSV. This method used the fluorescent signal generated from the hydrolysis of TaqMan probe to real-time monitor amplification of the target gene. It realized synchronization between real-time fluorescent RT-PCR amplification and detection. The results showed that the method was better than others in efficiency and specificity, and 100 times more sensitive than common RT-PCR. This method had potential to be applied in rapid detection of CRSV in incoming seedlings.

  15. Direct quantification of mRNA and miRNA from cell lysates using reverse transcription real time PCR: a multidimensional analysis of the performance of reagents and workflows.

    Directory of Open Access Journals (Sweden)

    Yoon Khei Ho

    Full Text Available Substantial efforts have been devoted to in vitro testing of candidate chemotherapeutics by profiling transcriptional changes across the collection of NCI-60 cell-lines. A work-flow with reagents that enable the direct quantification of RNA of different molecular sizes simultaneously in the same sample without laborious total RNA isolation will invariably increase the throughput and accuracy of the study. MicroRNAs (miRNAs are known to regulate most cellular functions, acting post-transcriptionally by repressing numerous eukaryotic mRNAs. Recent findings on the remarkable stability of miRNA prompted us to investigate the feasibility of quantifying the expression levels of both mRNA and miRNA directly from cell lysates (cell-to-Ct. Multidimensional analyses of the expressions of mRNA and miRNA across seven NCI-60 cell lines and multiple reagents were conducted to assess the performances of these reagents and workflows for cell-to-Ct measurements using reverse transcription-quantitative polymerase chain reaction (RT-qPCR. Quantification of RNA species using lysates prepared from an in-house and one of the commercial reagents demonstrated comparable performance to those prepared by the more laborious and conventional method of using guanidinium-phenol-chloroform. Additionally, miRNA was found to be highly stable in the cell lysates when incubated at room temperature for prolonged period of time and subjected to multiple freeze-thaw cycles. In summary, this study demonstrated significant differences in pre-analytical performance of a variety of commercially available reagents and described a cost-effective reagent useful for rapid, scalable, and high-throughput workflow for the detection of mRNA and miRNA from the same biological sample.

  16. The development of a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using TaqMan technology for the pan detection of bluetongue virus (BTV).

    Science.gov (United States)

    Mulholland, Catherine; McMenamy, Michael J; Hoffmann, Bernd; Earley, Bernadette; Markey, Bryan; Cassidy, Joseph; Allan, Gordon; Welsh, Michael D; McKillen, John

    2017-07-01

    Bluetongue virus (BTV) is an infectious, non-contagious viral disease of domestic and wild ruminants that is transmitted by adult females of certain Culicoides species. Since 2006, several serotypes including BTV-1, 2, 4, 6, 8, 9 and 16, have spread from the Mediterranean basin into Northern Europe for the first time. BTV-8 in particular, caused a major epidemic in northern Europe. As a result, it is evident that most European countries are at risk of BTV infection. The objective of this study was to develop and validate a real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay based on TaqMan technology for the detection of representative strains of all BTV serotypes. Primers and probes were based on genome segment 10 of the virus, the NS3 gene. The assay was tested for sensitivity, and specificity. The analytical sensitivity of the rRT-PCR assay was 200 copies of RNA per reaction. The assay did not amplify the closely related orbivirus epizootic hemorrhagic disease virus (EHDV) but successfully detected all BTV reference strains including clinical samples from animals experimentally infected with BTV-8. This real time RT-PCR assay offers a sensitive, specific and rapid alternative assay for the pan detection of BTV that could be used as part of a panel of diagnostic assays for the detection of all serotypes of BTV. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  17. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

  19. Evaluation of two singleplex reverse transcription-Insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus.

    Science.gov (United States)

    Zhang, Jianqiang; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chen, Qi; Zhang, Yan; Chiang, Cheng-Jen; Shen, Yu-Han; Li, Fu-Chun; Chang, Hsiao-Fen Grace; Gauger, Phillip C; Harmon, Karen M; Wang, Hwa-Tang Thomas

    2016-08-01

    Recent outbreaks of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) in multiple countries have caused significant economic losses and remain a serious challenge to the swine industry. Rapid diagnosis is critical for the implementation of efficient control strategies before and during PEDV and PDCoV outbreaks. Insulated isothermal PCR (iiPCR) on the portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, a singleplex PEDV RT-iiPCR, a singleplex PDCoV RT-iiPCR, and a duplex PEDV/PDCoV real-time RT-PCR (rRT-PCR) commercial reagents targeting the M gene were compared to an N gene-based PEDV rRT-PCR and an M gene-based PDCoV rRT-PCR that were previously published and used as reference PCRs. All PCR assays were highly specific and did not cross react with other porcine enteric pathogens. Analytical sensitivities of the PEDV RT-iiPCR, PDCoV RT-iiPCR and duplex PEDV/PDCoV rRT-PCR were determined using in vitro transcribed RNA as well as viral RNA extracted from ten-fold serial dilutions of PEDV and PDCoV cell culture isolates. Performance of each PCR assay was further evaluated using 170 clinical samples (86 fecal swabs, 24 feces, 19 intestines, and 41 oral fluids). Compared to the reference PEDV rRT-PCR, the sensitivity, specificity and accuracy of the PEDV RT-iiPCR were 97.73%, 98.78%, and 98.24%, respectively, and those of the duplex PEDV/PDCoV rRT-PCR were 98.86%, 96.34%, and 97.65%, respectively. Compared to the reference PDCoV rRT-PCR, the sensitivity, specificity and accuracy of the PDCoV RT-iiPCR were 100%, 100%, and 100%, respectively, and those of the PEDV/PDCoV duplex rRT-PCR were 96.34%, 100%, and 98.24%, respectively. Overall, all three new PCR assays were comparable to the reference rRT-PCRs for detection of PEDV and/or PDCoV. The PEDV and PDCoV RT-iiPCRs are potentially useful tools for on-site detection and the duplex PEDV/PDCoV rRT-PCR provides a convenient method to simultaneously detect

  20. Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses.

    Science.gov (United States)

    Waggoner, Jesse J; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Tellez, Yolanda; Sahoo, Malaya K; Balmaseda, Angel; Harris, Eva; Pinsky, Benjamin A

    2016-07-01

    Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.

  1. Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses

    OpenAIRE

    Waggoner, Jesse J.; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Tellez, Yolanda; Sahoo, Malaya K.; Balmaseda, Angel; Harris, Eva; Pinsky, Benjamin A.

    2016-01-01

    Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.

  2. Detection of Salmonella Typhi in blood with real time fluorescent quantitative reverse transcriptive polymerase chain reaction%利用实时荧光定量反转录-聚合酶链反应方法检测血液中伤寒沙门菌

    Institute of Scientific and Technical Information of China (English)

    樊粉霞; 娄静; 陈建才; 聂艳妮; 阚飙; 闫梅英

    2012-01-01

    Objective To establish an assay of real time fluorescent quantitative reverse transcriptive polymerase chain reaction (rRT-PCR) to detect Salmonella Typhi in blood. Methods Specific primers STY1631 gene of S. Typhi were designed and modified to establish rRT-PCR assay and the specificity and sensitivity of rRT-PCR with RNA as template were evaluated and verified by checking cultured Salmonella spp. in 51 serotypes, predominant non-salmonella enteric diarrheal pathogens and eight species of bacteria inducing bacteremia with fever as major symptom. The simulative blood specimens supplemented with S. Typhi were tested by the rRT-PCR assay. Results The established rRT-PCR assay successfully detected STY1631 gene of S. Typhi. Totally 48 S. Typhi isolates were amplified to be positive. Other isolates, including non-salmonella strains in 33 serotypes, predominant non-salmonella enteric diarrheal pathogens and eight species of bacteria causing bacteremia with fever, were amplified to be negative. For purified total RNA from pure cultured isolates, the detection limit of the assay was 1 pg per reaction, equal to 194 copies per reaction. The sensitivity achieved 1 × 102 cfu /ml with the purified nucleotide from simulative blood. Conclusion The rRT-PCR assay for detecting S. Typhi with high sensitivity and specificity was established, which would be suitable for the rapid diagnosis of S. Typhi infection and identification of other pathogens causing fever for the early warning, prevention and treatment of typhoid fever.%目的 建立实时荧光定量反转录-聚合酶链反应(real time fluorescent quantitative reverse transcriptionpolymerase chain reaction,rRT-PCR)方法检测伤寒沙门菌.方法 针对伤寒沙门菌STY1631基因设计特异性引物,通过优化反应条件,建立检测该靶基因的rRT-PCR方法,利用51个血清型的纯培养伤寒和非伤寒沙门菌菌株、常见非沙门致腹泻病原菌以及发热为主要症状的8种常见病原菌核糖

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

    Institute of Scientific and Technical Information of China (English)

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

    2014-01-01

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

  4. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for diagnosis of dengue

    Science.gov (United States)

    Sahni, Ajay Kumar; Grover, Naveen; Sharma, Ajay; Khan, Inam Danish; Kishore, Jugal

    2012-01-01

    Background Dengue is an emerging public health problem causing serious morbidity and mortality in tropical developing countries. Early, sensitive and specific diagnosis is paramount for clinical decision making. Currently available diagnostic tests are limited in scope and utility. This study highlights applicability of RT-LAMP in dengue diagnosis. Methods 100 dengue confirmed cases, 100 dengue negative cases and 79 healthy negative controls from dengue epidemic between Sep 2009 to Jul 2011 were included. Dengue cases were profiled using WHO guidelines 2006, haematological and biochemical parameters evaluated and diagnosed using NS1 antigen, IgM and IgG enzyme immunoassay, RT-PCR and RT-LAMP. Positive cases were serotyped, genotyped and various tests were compared. Results Mean haematocrit, PT, PTT, platelet count, activated lymphocytes, serum fibrinogen, transaminases, bilirubin, lactate dehydrogenase, protein and sodium were significantly elevated in DHF/DSS as compared to DF. NS1 antigen, RT-PCR and RT-LAMP were sensitive during 1–3 days while μ-capture IgM EIA was specific after 5–7 days of initial infection. DEN-1 genotype III was predominant. Conclusion Deranged haematocrit and liver function tests are indicators of the severity of the disease. RT-LAMP is rapid, cost effective, highly sensitive and specific qualitative and quantitative technique which can detect dengue infection in both early and intermediary stages when NS1 antigen titres are not in the detectable range and the IgM antibody titres have just started to rise. Its superiority over existing techniques, amenability for automation and promising utility in low resource healthcare setups and field conditions raise it as the new gold standard for dengue diagnosis. PMID:24600118

  5. 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...

  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......-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...... are comparable only when the same priming strategy and reaction conditions are used in all experiments and the samples contain the same total amount of RNA. Experimental accuracy is improved by running samples in (at least) duplicate starting with the reverse transcription reaction....

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

    Science.gov (United States)

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

    2015-06-01

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

  8. Rapid and real-time detection technologies for emerging viruses of biomedical importance

    Indian Academy of Sciences (India)

    M M Parida

    2008-11-01

    The development of technologies with rapid and sensitive detection capabilities and increased throughput have become crucial for responding to greater number threats posed by emerging and re-emerging viruses in the recent past. The conventional identification methods require time-consuming culturing, and/ or detection of antibodies, which are not very sensitive and specific. The recent advances in molecular biology techniques in the field of genomics and proteomics greatly facilitate the rapid identification with more accuracy. We have developed two real-time assays i.e., SYBR green I based real time reverse transcription polymerase chain reaction (RT-PCR) and RT-loop-mediated isothermal amplification (LAMP) assay for rapid detection as well as typing of some of the emerging viruses of biomedical importance viz. dengue, Japanese encephalitis, chikungunya, west Nile, severe acute respiratory syndrome virus (SARS) etc. Both these techniques are capable of detection and differentiation as well as quantifying viral load with higher sensitivity, rapidity, specificity. One of the most important advantages of LAMP is its field applicability, without requirement of any sophisticated equipments. Both these assays have been extensively evaluated and validated with clinical samples of recent epidemics from different parts of India. The establishment of these real time molecular assays will certainly facilitate the rapid detection of viruses with high degree of precision and accuracy in future.

  9. Simple, rapid and accurate molecular diagnosis of acute promyelocytic leukemia by loop mediated amplification technology.

    Science.gov (United States)

    Spinelli, Orietta; Rambaldi, Alessandro; Rigo, Francesca; Zanghì, Pamela; D'Agostini, Elena; Amicarelli, Giulia; Colotta, Francesco; Divona, Mariadomenica; Ciardi, Claudia; Coco, Francesco Lo; Minnucci, Giulia

    2015-01-01

    The diagnostic work-up of acute promyelocytic leukemia (APL) includes the cytogenetic demonstration of the t(15;17) translocation and/or the PML-RARA chimeric transcript by RQ-PCR or RT-PCR. This latter assays provide suitable results in 3-6 hours. We describe here two new, rapid and specific assays that detect PML-RARA transcripts, based on the RT-QLAMP (Reverse Transcription-Quenching Loop-mediated Isothermal Amplification) technology in which RNA retrotranscription and cDNA amplification are carried out in a single tube with one enzyme at one temperature, in fluorescence and real time format. A single tube triplex assay detects bcr1 and bcr3 PML-RARA transcripts along with GUS housekeeping gene. A single tube duplex assay detects bcr2 and GUSB. In 73 APL cases, these assays detected in 16 minutes bcr1, bcr2 and bcr3 transcripts. All 81 non-APL samples were negative by RT-QLAMP for chimeric transcripts whereas GUSB was detectable. In 11 APL patients in which RT-PCR yielded equivocal breakpoint type results, RT-QLAMP assays unequivocally and accurately defined the breakpoint type (as confirmed by sequencing). Furthermore, RT-QLAMP could amplify two bcr2 transcripts with particularly extended PML exon 6 deletions not amplified by RQ-PCR. RT-QLAMP reproducible sensitivity is 10(-3) for bcr1 and bcr3 and 10(-)2 for bcr2 thus making this assay particularly attractive at diagnosis and leaving RQ-PCR for the molecular monitoring of minimal residual disease during the follow up. In conclusion, PML-RARA RT-QLAMP compared to RT-PCR or RQ-PCR is a valid improvement to perform rapid, simple and accurate molecular diagnosis of APL.

  10. Prompt detection of influenza A and B viruses using the BD Veritor™ System Flu A+B, Quidel® Sofia® Influenza A+B FIA, and Alere BinaxNOW® Influenza A&B compared to real-time reverse transcription-polymerase chain reaction (RT-PCR).

    Science.gov (United States)

    Dunn, Jim; Obuekwe, Joy; Baun, Traci; Rogers, Justin; Patel, Twinkle; Snow, Linda

    2014-05-01

    The performance characteristics of rapid influenza diagnostic tests vary widely. This study evaluated the BD Veritor™ System Flu A+B (Veritor; BD Diagnostics, Sparks, MD, USA), Quidel® Sofia® Influenza A+B FIA (Sofia; Quidel Corp., San Diego, CA, USA), and Alere BinaxNOW® Influenza A&B (Binax; Alere Scarborough, Inc., Scarborough, ME, USA) compared to reverse transcription-polymerase chain reaction (RT-PCR) for detection of influenza viruses in nasal wash specimens from 240 pediatric patients. Positive percent agreements for influenza A and B virus detection were 93.8% and 94.2%, 95.8% and 98.1%, and 79.2% and 80.8% for Veritor, Sofia, and Binax, respectively. The Veritor and Binax tests demonstrated negative percent agreements >97.9% for detection of both influenza viruses, but the negative percent agreement of the Sofia test was 91.1% for influenza A and 70.7% for influenza B virus. Overall, the Veritor and Sofia tests were nearly as sensitive as RT-PCR and considerably more sensitive than Binax for detection of influenza viruses. However, the accuracy of the Sofia test was significantly lower than either Veritor or Binax.

  11. 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...

  12. LNA探针同时检测人副流感病毒1,2,3型多重荧光定量RT-PCR方法的建立%Simultaneous detection of human parainfluenza viruses 1, 2, 3 by multiplex real-time reverse transcription-polymerase chain reaction with LNA probes

    Institute of Scientific and Technical Information of China (English)

    姬奕昕; 毛乃颖; 王焕焕; 谢正德; 许文波

    2012-01-01

    目的 人副流感病毒1,2,3型是呼吸道感染的主要病原.本研究建立了特异、快速、灵敏的多重荧光定量RT-PCR方法用于人副流感1,2,3型病毒临床标本检测.方法 针对人副流感1,2,3型病毒设计特异性引物探针,优化荧光RT-PCR反应条件.应用体外转录方法分别制备人副流感1,2,3型病毒的标准品.验证荧光定量RT-PCR方法的特异性,敏感性和稳定性.结果 该方法对人副流感1,2,3型病毒核酸检测有高度特异性,检测的灵敏度HPIV1为10个拷贝,HPIV2为100个拷贝,HPIV3为100个拷贝.可从临床患者鼻咽吸出物标本中直接检出.结论 本研究建立的LNA探针同时检测人副流感病毒1,2,3型多重荧光定量RT-PCR方法具有较高的特异性和敏感性.适用于临床早期诊断和实验室病原谱筛查.%Objective Human parainfluenza virus (HPIV) types 1,2 and 3 are major viral pathogens responsible for upper and lower respiratory tract infections.In this study,a real-time RT-PCR was developed using multiplex primers-probe (HPIV-1,2,3) for the simultaneous detection of both HPIV1,HPIV2 and HPIV3 genomes.Methods Optimal primers and probes were designed using specialized software.The conditions for multiplex real-time RT-PCR had been optimized.The synthesis of RNA standards of HPIV1,2,3 were used a T7 RNA polymerase.Check the specificity sensitivities and stability of one step RT-PCR assay.Results Obtained in a 10-fold dilution series assay demonstrate a high sensitivity of the assay with a lowest detection limit of 10 copies for HPIV1,100 copies for HPIV2 and 100 copies for HPIV3.Conclusion The assays demonstrates an improved sensitivity and scope of detecting HPIV1,2,3 viruses relative to routine antigen detection assays while the quantitative utility may facilitate investigation of the pre-diagnosis and respiratory virus pathogenesis.

  13. 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.

  14. Reverse Transcription of Retroviruses and LTR Retrotransposons.

    Science.gov (United States)

    Hughes, Stephen H

    2015-04-01

    The enzyme reverse transcriptase (RT) was discovered in retroviruses almost 50 years ago. The demonstration that other types of viruses, and what are now called retrotransposons, also replicated using an enzyme that could copy RNA into DNA came a few years later. The intensity of the research in both the process of reverse transcription and the enzyme RT was greatly stimulated by the recognition, in the mid-1980s, that human immunodeficiency virus (HIV) was a retrovirus and by the fact that the first successful anti-HIV drug, azidothymidine (AZT), is a substrate for RT. Although AZT monotherapy is a thing of the past, the most commonly prescribed, and most successful, combination therapies still involve one or both of the two major classes of anti-RT drugs. Although the basic mechanics of reverse transcription were worked out many years ago, and the first high-resolution structures of HIV RT are now more than 20 years old, we still have much to learn, particularly about the roles played by the host and viral factors that make the process of reverse transcription much more efficient in the cell than in the test tube. Moreover, we are only now beginning to understand how various host factors that are part of the innate immunity system interact with the process of reverse transcription to protect the host-cell genome, the host cell, and the whole host, from retroviral infection, and from unwanted retrotransposition.

  15. 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.

  16. 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....

  17. 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....

  18. 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

  19. Real-time radiography

    Energy Technology Data Exchange (ETDEWEB)

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

    1981-02-26

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

  20. Real-time Cosmology

    CERN Document Server

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

    2010-01-01

    In recent years the possibility of measuring the temporal change of radial and transverse position of sources in the sky in real time have become conceivable thanks to the thoroughly improved technique applied to new astrometric and spectroscopic experiments, leading to the research domain we call Real-time cosmology. We review for the first time great part of the work done in this field, analysing both the theoretical framework and some endeavor to foresee the observational strategies and their capability to constrain models. We firstly focus on real time measurements of the overall redshift drift and angular separation shift in distant source, able to trace background cosmic expansion and large scale anisotropy, respectively. We then examine the possibility of employing the same kind of observations to probe peculiar and proper acceleration in clustered systems and therefore the gravitational potential. The last two sections are devoted to the short time future change of the cosmic microwave background, as ...

  1. Real-Time Shading

    CERN Document Server

    Olano, Marc

    2002-01-01

    This book covers real-time shading systems, their design and how they work. Procedural shading, long valued for off-line rendering and production animation is now possible on interactive graphics hardware. These developments are important for areas such as game development, product design, and scientific visualization, among others. The authors include examples of techniques for achieving common effects efficiently in a real-time shading language ranging from full procedural shading on advanced specialized hardware to limited, yet surprisingly flexible shading on unextended OpenGL, to modern P

  2. Real-Time Evaluations

    Directory of Open Access Journals (Sweden)

    UNHCR

    2002-07-01

    Full Text Available A real-time evaluation (RTE is a timely, rapid andinteractive review of a fast evolving humanitarianoperation undertaken at an early phase. Its broadobjectives are to gauge the effectiveness and impactof a given UNHCR response and to ensure that itsfindings are used as an immediate catalyst fororganisational and operational change.

  3. Comparison of fluorescent intercalating dyes for quantitative loop-mediated isothermal amplification (qLAMP).

    Science.gov (United States)

    Oscorbin, Igor P; Belousova, Ekaterina A; Zakabunin, Aleksandr I; Boyarskikh, Ulyana A; Filipenko, Maksim L

    2016-01-01

    Real-time or quantitative loop-mediated isothermal amplification (qLAMP) is a promising technique for the accurate detection of pathogens in organisms and the environment. Here we present a comparative study of the performance of six fluorescent intercalating dyes-SYTO-9, SYTO-13, SYTO-82, SYBR Green I, SYBR Gold, EvaGreen-in three different qLAMP model systems. SYTO-9 and SYTO-82, which had the best results, were used for additional enzyme and template titration studies. SYTO-82 demonstrated the best combination of time-to-threshold (Tt) and signal-to-noise ratio (SNR).

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

    OpenAIRE

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

    2017-01-01

    Background 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. Methods HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a commu...

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

    OpenAIRE

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

    2017-01-01

    Background 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. Methods HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a commu...

  6. Real Time Processing

    CERN Document Server

    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.

  7. Real Time Econometrics

    OpenAIRE

    Pesaran, M. Hashem; Timmermann, Allan

    2004-01-01

    This paper considers the problems facing decision makers using econometric models in real time. It identifies the key stages involved and highlights the role of automated systems in reducing the effect of data snooping. It sets out many choices that researchers face in construction of automated systems and discusses some of the possible ways advanced in the literature for dealing with them. The role of feedbacks from the decision maker?s actions to the data generating process is also discusse...

  8. 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.

  9. 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

  10. 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…

  11. 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…

  12. 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...

  13. 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)

  14. 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)

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

    Science.gov (United States)

    Arunrut, Narong; Suebsing, Rungkarn; Withyachumnarnkul, Boonsirm; Kiatpathomchai, Wansika

    2014-01-01

    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.

  16. 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.

  17. How Many Microorganisms Are Present? Quantitative Reverse Transcription PCR (qRT-PCR)

    Science.gov (United States)

    Price, Andy; Álvarez, Laura Acuña; Whitby, Corinne; Larsen, Jan

    Quantitative reverse transcription PCR (qRT-PCR) is a variation of conventional quantitative or real-time PCR, whereby mRNA is first converted into the complementary DNA (cDNA) by reverse transcription, the cDNA is then subsequently quantified by qPCR. The use of mRNA as the initial template allows the quantification of gene transcripts, rather than gene copy numbers. mRNA is only produced by actively metabolising cells and is produced by its corresponding gene to provide a 'blueprint' in order for a cell to manufacture a specific protein. Conventional qPCR detects not only DNA present in actively metabolising cells but also inactive and dead cells. qRT-PCR has the advantage that only actively metabolising cells are detected, hence provides a more reliable measure of microbial activity in oilfield samples. When qRT-PCR is combined with primers and probes for specific genes, the activity of microbial processes important in the oilfield, such as sulphate reduction, methanogenesis and nitrate reduction can be monitored.

  18. 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...

  19. Reference Genes for Gene Expression Analysis by Real-time Reverse Transcription Polymerase Chain Reaction of Renal Cell Carcinoma

    DEFF Research Database (Denmark)

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

    2011-01-01

    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...

  20. Real-Time Reverse Transcription PCR Assay for Detection of Senecavirus A in Swine Vesicular Diagnostic Specimens.

    Directory of Open Access Journals (Sweden)

    Alexa J Bracht

    Full Text Available Senecavirus A (SV-A, formerly, Seneca Valley virus (SVV, has been detected in swine with vesicular lesions and is thought to be associated with swine idiopathic vesicular disease (SIVD, a vesicular disease syndrome that lacks a defined causative agent. The clinical presentation of SIVD resembles that of other more contagious and economically devastating vesicular diseases, such as foot-and-mouth disease (FMD, swine vesicular disease (SVD, and vesicular stomatitis (VS, that typically require immediate rule out diagnostics to lift restrictions on animal quarantine, movement, and trade. This study presents the development of a sensitive, SYBR Green RT-qPCR assay suitable for detection of SV-A in diagnostic swine specimens. After testing 50 pigs with clinical signs consistent with vesicular disease, 44 (88% were found to be positive for SV-A by RT-qPCR as compared to none from a negative cohort of 35 animals without vesicular disease, indicating that the assay is able to successfully detect the virus in an endemic population. SV-A RNA was also detectable at a low level in sera from a subset of pigs that presented with (18% or without (6% vesicular signs. In 2015, there has been an increase in the occurrence of SV-A in the US, and over 200 specimens submitted to our laboratory for vesicular investigation have tested positive for the virus using this method. SV-A RNA was detectable in all common types of vesicular specimens including swabs and tissue from hoof lesions, oral and snout epithelium, oral swabs, scabs, and internal organ tissues such as liver and lymph node. Genome sequencing analysis from recent virus isolates was performed to confirm target amplicon specificity and was aligned to previous isolates.

  1. Field Evaluation of a Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection of Vesicular Stomatitis Virus

    Science.gov (United States)

    Sporadic outbreaks of vesicular stomatitis (VS) in the United States result in significant economic losses for the US livestock industries because VS is an OIE reportable disease and also clinically mimics foot-and-mouth disease. Rapid and accurate differentiation of these two diseases is critical ...

  2. Verification of reference genes for relative quantification of gene expression by real-time reverse transcription PCR in the pig.

    Science.gov (United States)

    Svobodová, Katerina; Bílek, Karel; Knoll, Ales

    2008-01-01

    The aim of this study was to develop a set of reliable reference genes for quantification of mRNA expression in the pig. The mRNA expression stability in pig tissues was studied for 4 genes: EEF1A1, GAPDH, HPRT1 and TOP2B. The level of expression was characterized by Ct values for each gene and each tissue. By using the geNorm algorithm, the stability of the reference genes was determined in the diaphragm, heart, kidney, liver, lungs, longissimus muscle, and spleen. On the basis of this information, suitable reference genes can be selected for mRNA expression studies in relevant pig tissues.

  3. Detection of let-7a microRNA by real-time PCR in gastric carcinoma

    Institute of Scientific and Technical Information of China (English)

    Hong-He Zhang; Xian-Jun Wang; Guo-Xiong Li; En Yang; Ning-Min Yang

    2007-01-01

    AIM:To establish an accurate and rapid stem-loop reverse transcriptional real-time PCR (RT-PCR) method to quantify human let-7a miRNA in gastric cancer.METHODS: According to the sequence of let-7a miRNA,the stem-loop reverse transcriptional primer, the primers and quantitative MGB probes of real-time PCR were designed and synthesized. The dynamic range and the sensitivity of quantitative reverse transcriptional real-time PCR were determined. The levels of let-7a miRNA were examined in 32 gastric carcinoma samples by stem-loop RT-PCR method.RESULTS: The dynamic range and sensitivity of the let-7a miRNA quantification scheme were evaluated,the result showed the assay could precisely detect 10copies of mature let-7a miRNA in as few as 0.05 ng of total RNA of gastric mucosa. The results of specificity analysis showed no fluorescence signal occurred even though 50 ng of human genomic DNA was added to the reverse transcription (RT) reaction. The expression level of let-7a miRNA in gastric tumor tissues was significantly lower compared to normal tissues in 14 samples from 32patients.CONCLUSION: The stem-loop RT-PCR is a reliable method to detect let-7a miRNA which may play an important role in the development of gastric carcinoma.

  4. [Research progress of real-time quantitative PCR method for group A rotavirus detection].

    Science.gov (United States)

    Guo, Yan-Qing; Li, Dan-Di; Duan, Zhao-Jun

    2013-11-01

    Group A rotavirus is one of the most significant etiological agents which causes acute gastroenteritis among infants and young children worldwide. So far, several method which includes electron microscopy (EM), enzyme immunoassay (EIA), reverse transcription-polymerase chain reaction (RT-PCR)and Real-time Quantitative PCR has been established for the detection of rotavirus. Compared with other methods, Real-time quantitative PCR have advantages in specificity, sensitivity, genotyping and quantitative accuracy. This article shows a overview of the application of real-time quantitative PCR technique to detecte group A rotavirus.

  5. Molecular epidemiology and a loop-mediated isothermal amplification method for diagnosis of infection with rabies virus in Zambia.

    Science.gov (United States)

    Muleya, Walter; Namangala, Boniface; Mweene, Aaron; Zulu, Luke; Fandamu, Paul; Banda, Douglas; Kimura, Takashi; Sawa, Hirofumi; Ishii, Akihiro

    2012-01-01

    The National Livestock Epidemiology and Information Center (NALEIC) in Zambia reported over 132 cases of canine rabies diagnosed by the direct fluorescent antibody test (DFAT) from 2004 to 2009. In this study, the lineage of rabies virus (RABV) in Zambia was determined by phylogenetic analysis of the nucleoprotein (N) and glycoprotein (G) gene sequences. Total RNA was extracted from 87-DFAT brain specimens out of which only 35 (40%) were positive on nested reverse transcription polymerase chain reaction (RT-PCR) for each gene, and 26 being positive for both genes. Positive specimens for the N (n=33) and G (n=35) genes were used for phylogenetic analysis. Phylogenetic analysis of the N gene showed two phylogenetic clusters in Zambia belonging to the Africa 1b lineage present in eastern and southern Africa. While one cluster exclusively comprised Zambian strains, the other was more heterogeneous regarding the RABV origins and included strains from Tanzania, Mozambique and Zambia. Phylogenetic analysis of the G gene revealed similar RABV strains in different hosts and regions of Zambia. We designed primers for reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay from the consensus sequence of the N gene in an attempt to improve the molecular diagnosis of RABV in Zambia. The specificity and reproducibility of the RT-LAMP assay was confirmed with actual clinical specimens. Therefore, the RT-LAMP assay presented in this study may prove to be useful for routine diagnosis of rabies in Zambia.

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

    Science.gov (United States)

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

    2013-11-01

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

  7. Designing Real Time Assistive Technologies

    DEFF Research Database (Denmark)

    Sonne, Tobias; Obel, Carsten; Grønbæk, Kaj

    2015-01-01

    design criteria in relation to three core components (sensing, recognizing, and assisting) for designing real time assistive technologies for children with ADHD. Based on these design criteria, we designed the Child Activity Sensing and Training Tool (CASTT), a real time assistive prototype that captures...... 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......) real time assistive technologies have potential to assist children with ADHD in regaining attention in critical school situations....

  8. Designing Real Time Assistive Technologies

    DEFF Research Database (Denmark)

    Sonne, Tobias; Obel, Carsten; Grønbæk, Kaj

    design criteria in relation to three core components (sensing, recognizing, and assisting) for designing real time assistive technologies for children with ADHD. Based on these design criteria, we designed the Child Activity Sensing and Training Tool (CASTT), a real time assistive prototype that captures...... 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......) real time assistive technologies have potential to assist children with ADHD in regaining attention in critical school situations....

  9. Concurrent Reactivation of Herpes Simplex and Varicella Zoster Viruses Confirmed by the Loop-Mediated Isothermal Amplification Assay

    Directory of Open Access Journals (Sweden)

    Tsukane Kobayashi

    2014-01-01

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

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

    National Research Council Canada - National Science Library

    Patel, Jaymin C; Oberstaller, Jenna; Xayavong, Maniphet; Narayanan, Jothikumar; DeBarry, Jeremy D; Srinivasamoorthy, Ganesh; Villegas, Leopoldo; Escalante, Ananias A; DaSilva, Alexandre; Peterson, David S; Barnwell, John W; Kissinger, Jessica C; Udhayakumar, Venkatachalam; Lucchi, Naomi W

    2013-01-01

    .... Conventional molecular diagnostic methods provide accurate results but are often resource-intensive, expensive, have a long turnaround time and are beyond the capacity of most malaria-endemic countries...

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

    National Research Council Canada - National Science Library

    Kiddle, Guy; Hardinge, Patrick; Buttigieg, Neil; Gandelman, Olga; Pereira, Clint; McElgunn, Cathal J; Rizzoli, Manuela; Jackson, Rebecca; Appleton, Nigel; Moore, Cathy; Tisi, Laurence C; Murray, James A H

    2012-01-01

    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...

  12. Real-time volume graphics

    CERN Document Server

    Engel, Klaus; Kniss, Joe; Rezk-Salama, Christof; Weiskopf, Daniel

    2006-01-01

    Based on course notes of SIGGRAPH course teaching techniques for real-time rendering of volumetric data and effects; covers both applications in scientific visualization and real-time rendering. Starts with the basics (texture-based ray casting) and then improves and expands the algorithms incrementally. Book includes source code, algorithms, diagrams, and rendered graphics.

  13. 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.

  14. Real Time PCR: Principles and Application

    Directory of Open Access Journals (Sweden)

    Safie Amini

    2005-09-01

    labeled oligonucleotide probes that bind to the PCR product in a head-to-tail fashion. When the two probes bind, their fluorophores come into close proximity, allowing energy transfer from a donor to an acceptor fluorophore. Therefore, fluorescence is detected during the annealing phase of PCR and is proportional to the amount of PCR product. FRET probes usually carry dyes that are only compatible for use on the LightCycler® machine. As the FRET system uses two primers and two probes, good design of the primers and probes is critical for successful results(9, 10.Other system: Recently, other systems such as Molecular Beacons, Scorpion or Luxprimer are also available in market and their description is beyond the scope of this review.Methods in Real-time PCRTwo-step and one-step RT-PCR cDNA synthesis uses reverse transcriptases, which are enzymes generally derived from RNAcontaining retroviruses. RT-PCR can take place in a two-step or one-step reaction. With two-step RTPCR, the RNA is first reverse-transcribed into cDNA using oligo-dT primers, random oligomers, or gene-specific primers. An aliquot of the reversetranscription reaction is then used for analysis of gene expression levels or viral load. RNA first needs to be transcribed and subsequently added to the real-time PCR. In two-step RT-PCR, it is possible to choose between different types of RT primers, depending on experimental needs. The use of oligodT primers or random oligomers for reverse transcription means that several different transcripts can be analyzed by PCR from one RT reaction. In addition, precious RNA samples can be immediately transcribed into more stable cDNA for later use and long-term storage. In one-step RT-PCR -also referred to as one-tube RT-PCR- both reverse transcription and amplification take place in the same tube, with reverse transcription preceding PCR. This is possible due to specialized reaction chemistries and cycling protocols. The fast procedure enables rapid processing of multiple

  15. Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus

    OpenAIRE

    Kai Wang; Yi Niu; Qijun Wang; Haili Liu; Yi Jin; Shenglin Zhang

    2017-01-01

    Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes. Amorphophallus is a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used a...

  16. 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.

  17. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  19. Evaluating real-time forecasts in real-time

    NARCIS (Netherlands)

    D.J.C. van Dijk (Dick); Ph.H.B.F. Franses (Philip Hans); F. Ravazzolo (Francesco)

    2007-01-01

    textabstractThe accuracy of real-time forecasts of macroeconomic variables that are subject to revisions may crucially depend on the choice of data used to compare the forecasts against. We put forward a flexible time-varying parameter regression framework to obtain early estimates of the final valu

  20. 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.

  1. Real Time Sonic Boom Display

    Science.gov (United States)

    Haering, Ed

    2014-01-01

    This presentation will provide general information about sonic boom mitigation technology to the public in order to supply information to potential partners and licensees. The technology is a combination of flight data, atmospheric data and terrain information implemented into a control room real time display for flight planning. This research is currently being performed and as such, any results and conclusions are ongoing.

  2. 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.

  3. Real time automatic scene classification

    NARCIS (Netherlands)

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

    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

  4. Decentralized Real-Time Scheduling

    Science.gov (United States)

    1990-08-01

    917-932, August, 1987. [ Daniels 86] D. C. Daniels and H. F. Wedde. Real-Time Performance of a Completely Distributed Operating System. In Proceedings...imnmediate execuoon The schedule c.-eated by the SeltctPhaseProcfj procedure is an ordere-d lis t of mocl -phnise pairs, each placed according to the

  5. 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

  6. 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.

  7. 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.

  8. Reverse Transcriptase and Cellular Factors: Regulators of HIV-1 Reverse Transcription

    Directory of Open Access Journals (Sweden)

    David Harrich

    2009-11-01

    Full Text Available There is ample evidence that synthesis of HIV-1 proviral DNA from the viral RNA genome during reverse transcription requires host factors. However, only a few cellular proteins have been described in detail that affect reverse transcription and interact with reverse transcriptase (RT. HIV-1 integrase is an RT binding protein and a number of IN-binding proteins including INI1, components of the Sin3a complex, and Gemin2 affect reverse transcription. In addition, recent studies implicate the cellular proteins HuR, AKAP149, and DNA topoisomerase I in reverse transcription through an interaction with RT. In this review we will consider interactions of reverse transcription complex with viral and cellular factors and how they affect the reverse transcription process.

  9. 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

  10. Real-Time PCR Detection of Host-Mediated Cyanophage Gene Transcripts during Infection of a Natural Microcystis aeruginosa Population

    OpenAIRE

    Yoshida, Mitsuhiro; Yoshida, Takashi; Yoshida-Takashima, Yukari; Kashima, Aki; Hiroishi, Shingo

    2010-01-01

    The aim of this study was to develop a quantitative real-time reverse transcription-PCR (real-time RT-PCR) assay to detect and quantify mRNA of cyanophages within infected Microcystis aeruginosa cells in a freshwater pond. Laboratory-based data showed that the relative abundance of the cyanophage g91 mRNA within host cells increased before cyanophage numbers increased in culture. This transcriptional pattern indicated the kinetics of the viral infection suggesting the real-time RT-PCR method ...

  11. Loop-mediated isothermal amplification for detection of nucleic acids.

    Science.gov (United States)

    Tanner, Nathan A; Evans, Thomas C

    2014-01-06

    Sequence-specific isothermal nucleic acid amplification techniques are ideally suited for use in molecular diagnostic applications because they do not require thermal cycling equipment and the reactions are typically fast. One of the most widely cited isothermal techniques is termed loop-mediated isothermal amplification (LAMP). This protocol allows amplification times as fast as 5 to 10 min. Furthermore, various methodologies to detect amplification have been applied to LAMP to increase its utility for the point-of-care market. Basic LAMP protocols are provided herein for detection of specific DNA and RNA targets, along with a method to perform multiplex LAMP reactions, permitting even greater flexibility from this powerful technique.

  12. Radiation damping in real time.

    Science.gov (United States)

    Mendes, A C; Takakura, F I

    2001-11-01

    We study the nonequilibrium dynamics of a charge interacting with its own radiation, which originates the radiation damping. The real-time equation of motion for the charge and the associated Langevin equation is found in classical limit. The equation of motion for the charge allows one to obtain the frequency-dependent coefficient of friction. In the lowest order we find that although the coefficient of static friction vanishes, there is dynamical dissipation represented by a non-Markovian dissipative kernel.

  13. The VLT Real Time Display

    Science.gov (United States)

    Herlin, T.; Brighton, A.; Biereichel, P.

    The VLT Real-Time Display (RTD) software was developed in order to support image display in real-time, providing a tool for users to display video like images from a camera or detector as fast as possible on an X-Server. The RTD software is implemented as a package providing a Tcl/Tk image widget written in C++ and an independent image handling library and can be used as a building block, adding display capabilities to dedicated VLT control applications. The RTD widget provides basic image display functionality like: panning, zooming, color scaling, colormaps, intensity changes, pixel query, overlaying of line graphics. A large set of assisting widgets, e.g., colorbar, zoom window, spectrum plot are provided to enable the building of image applications. The support for real-time is provided by an RTD image event mechanism used for camera or detector subsystems to pass images to the RTD widget. Image data are passed efficiently via shared memory. This paper describes the architecture of the RTD software and summarizes the features provided by RTD.

  14. Rapid and sensitive detection of human astrovirus in water samples by loop-mediated isothermal amplification with hydroxynaphthol blue dye

    Science.gov (United States)

    2014-01-01

    Background The aim of this paper was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, sensitive and inexpensive detection of astrovirus. Results The detection limit of LAMP using in vitro RNA transcripts was 3.6×10 copies·μL-1, which is as sensitive as the presently used PCR assays. However, the LAMP products could be identified as different colors with the naked eye following staining with hydroxynaphthol blue dye (HNB). No cross-reactivity with other gastroenteric viruses (rotavirus and norovirus) was observed, indicating the relatively high specificity of LAMP. The RT-LAMP method with HNB was used to effectively detect astrovirus in reclaimed water samples. Conclusions The LAMP technique described in this study is a cheap, sensitive, specific and rapid method for the detection of astrovirus. The RT-LAMP method can be simply applied for the specific detection of astrovirus and has the potential to be utilized in the field as a screening test. PMID:24524254

  15. 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. .

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

    Directory of Open Access Journals (Sweden)

    Hua-Wei Chen

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Joanna Kaczmarek

    2014-12-01

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

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

    Science.gov (United States)

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

    2016-03-01

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

  19. Development of Loop-Mediated Isothermal Amplification (LAMP Assays for Rapid Detection of Ehrlichia ruminantium

    Directory of Open Access Journals (Sweden)

    Geysen Dirk

    2010-11-01

    Full Text Available Abstract Background The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus Amblyomma. The disease is distributed in nearly all of sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland. This report describes the development of two different loop-mediated isothermal amplification (LAMP assays for sensitive and specific detection of E. ruminantium. Results Two sets of LAMP primers were designed from the pCS20 and sodB genes. The detection limits for each assay were 10 copies for pCS20 and 5 copies for sodB, which is at least 10 times higher than that of the conventional pCS20 PCR assay. DNA amplification was completed within 60 min. The assays detected 16 different isolates of E. ruminantium from geographically distinct countries as well as two attenuated vaccine isolates. No cross-reaction was observed with genetically related Rickettsiales, including zoonotic Ehrlichia species from the USA. LAMP detected more positive samples than conventional PCR but less than real-time PCR, when tested with field samples collected in sub-Saharan countries. Conclusions Due to its simplicity and specificity, LAMP has the potential for use in resource-poor settings and also for active screening of E. ruminantium in both heartwater-endemic areas and regions that are at risk of contracting the disease.

  20. Rapid detection of Opisthorchis viverrini copro-DNA using loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Arimatsu, Yuji; Kaewkes, Sasithorn; Laha, Thewarach; Hong, Sung-Jong; Sripa, Banchob

    2012-03-01

    Opisthorchis viverrini and other foodborne trematode infections are major health problem in Thailand, the Lao People's Democratic Republic, Vietnam and Cambodia. Differential diagnosis of O. viverrini based on the microscopic observation of parasite eggs is difficult in areas where Clonorchis sinensis and minute intestinal flukes coexist. We therefore established a rapid, sensitive and specific method for detecting O. viverrini infection from the stool samples using the loop-mediated isothermal amplification (LAMP) method. A total of five primers from seven regions were designed to target the internal transcribed spacer 1 (ITS1) in ribosomal DNA for specific amplification. Hydroxy naphthol blue (HNB) was more effective to detect the LAMP product compared to the Real-time LAMP and turbidity assay for its simple and distinct detection. The LAMP assay specifically amplified O. viverrini ITS1 but not C. sinensis and minute intestinal flukes with the limit of detection around 10(-3)ng DNA/μL. The sensitivity of the LAMP was 100% compared to egg positive samples. While all microscopically positive samples were positive by LAMP, additionally 5 of 13 (38.5%) microscopically negative samples were also LAMP positive. The technique has great potential for differential diagnosis in endemic areas with mixed O. viverrini and intestinal fluke infections. As it is an easy and simple method, the LAMP is potentially applicable for point-of-care diagnosis.

  1. Development of a loop-mediated isothermal amplification assay for detection of Phytophthora sojae.

    Science.gov (United States)

    Dai, Ting-Ting; Lu, Chen-Chen; Lu, Jing; Dong, SuoMeng; Ye, WenWu; Wang, YuanChao; Zheng, XiaoBo

    2012-09-01

    Phytophthora sojae is a devastating pathogen that causes soybean Phytophthora root rot. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay targeting the A3aPro element for visual detection of P. sojae. The A3aPro-LAMP assay efficiently amplified the target element in Phytophthora spp., Pythium spp., and true fungi isolates. Magnesium pyrophosphate resulting from the LAMP of P. sojae could be detected by real-time measurement of turbidity. Phytophthora sojae DNA products were visualized as a ladder-like banding pattern on 2% gel electrophoresis. A positive colour (sky blue) was only observed in the presence of P. sojae with the addition of hydroxynaphthol blue prior to amplification, whereas none of other isolates showed a colour change. The detection limit of the A3aPro-specific LAMP assay for P. sojae was 10 pg μL(-1) of genomic DNA per reaction. The assay also detected P. sojae from diseased soybean tissues and residues. These results suggest that the A3aPro-LAMP assay reported here can be used for the visual detection of P. sojae in plants and production fields.

  2. Development of a loop-mediated isothermal amplification method for detecting virulent Rhodococcus equi.

    Science.gov (United States)

    Kinoshita, Yuta; Niwa, Hidekazu; Higuchi, Tohru; Katayama, Yoshinari

    2016-09-01

    Rhodococcus equi is the most important causative bacterium of severe pneumonia in foals. We report herein the development of a specific loop-mediated isothermal amplification (LAMP) assay, which targets a gene encoding vapA for detecting virulent R. equi The detection limit of the LAMP assay was 10(4) colony forming units (CFU)/mL, which was equal to 10 CFU/reaction. The clinical efficacy of the LAMP assay was compared with those of 2 published PCR-based methods: nested PCR and quantitative real-time (q)PCR. Agreements between bacterial culture, which is the gold standard for detection of R. equi, and each of the 3 molecular tests were measured by calculating a kappa coefficient. The kappa coefficients of the LAMP (0.760), nested PCR (0.583), and qPCR (0.888) indicated substantial agreement, moderate agreement, and almost perfect agreement, respectively. Although the clinical efficacy of LAMP was not the best among the 3 methods tested, LAMP could be more easily introduced into less well-equipped clinics because it does not require special equipment (such as a thermocycler) for gene amplification. Veterinary practitioners could diagnose R. equi pneumonia more quickly by using LAMP and could use the results to select an appropriate initial treatment. © 2016 The Author(s).

  3. Direct detection of Marek's disease virus in poultry dust by loop-mediated isothermal amplification.

    Science.gov (United States)

    Woźniakowski, Grzegorz; Samorek-Salamonowicz, Elżbieta

    2014-11-01

    Marek's disease virus (MDV) is a serious concern for poultry production and represents a unique herpesvirus model. MDV can be shed by doubly infected chickens despite vaccination. The fully infectious MDV particles are produced in the feather follicle epithelium (FFE), and MDV remains infectious for many months in fine skin particles and feather debris. Molecular biology methods including PCR and real-time PCR have been shown to be valuable for the detection of MDV DNA in farm dust. Recently, loop-mediated isothermal amplification (LAMP) was found to be useful in the detection of MDV in feathers and internal organs of infected chickens. LAMP is also less affected by the inhibitors present in DNA samples. Taking into account the advantages of LAMP, direct detection of MDV DNA in poultry dust has been conducted in this research. The detection of MDV DNA was possible in 11 out of the 12 examined dust samples without DNA extraction. The DNA was retrieved from dust samples by dilution and incubation at 95 °C for 5 min. The direct detection of MDV DNA in the dust was possible within 30 min using a water bath and UV light. The results were confirmed by electrophoresis and melting curve analysis of the LAMP products. Our results show that LAMP may be used to test for the presence of virulent MDV in poultry farm dust without DNA extraction.

  4. Loop-mediated isothermal amplification for the detection of goose circovirus

    Directory of Open Access Journals (Sweden)

    Woźniakowski Grzegorz

    2012-06-01

    Full Text Available Abstract Background Goose circovirus (GCV presents an immunosuppressive problem in production of geese. The infection’s clinical symptoms include growth retardation or feathering disorders but the infection process may remain non-symptomatic what makes the infected birds more susceptible for secondary viral, bacterial and fungal infections. Diagnosis of GCV infection is made by histopathological examination, dot blot hybridization, polymerase chain reaction (PCR and real-time PCR. However these techniques require application of thermocyclers and qualified staff which may be cost-consuming for some diagnostic units. The aim of this study was to develop loop-mediated isothermal amplification assay (LAMP as a simple method of GCV detection. Results The presented study has shown LAMP as a rapid tool of detecting DNA of goose circovirus (GCV as soon in 30 min time. The method used three sets of primers: two outer primers (F3 and B3, two inner primers (FIP and BIP and two loop primers (FL and BL to accelerate the reaction. The optimum reaction temperature and the time were 61°C for 30 min, respectively. The results were analysed using SYBR Green dye and GelRedTM solutions. Thirty-eight isolates of GCV collected from geese flocks in Poland were examined. For comparison, real-time polymerase chain reaction with F3 and B3 primers and SYBR Green dye was conducted. The obtained results have shown GCV-LAMP as a sensitive, rapid and specific assay and alternative for PCR-based methods. Conclusions The developed technique due to its simplicity may be applied by any veterinary laboratory or even mobile diagnostics units for the routine detection of GCV.

  5. Loop-mediated isothermal amplification for the detection of goose circovirus

    Science.gov (United States)

    2012-01-01

    Background Goose circovirus (GCV) presents an immunosuppressive problem in production of geese. The infection’s clinical symptoms include growth retardation or feathering disorders but the infection process may remain non-symptomatic what makes the infected birds more susceptible for secondary viral, bacterial and fungal infections. Diagnosis of GCV infection is made by histopathological examination, dot blot hybridization, polymerase chain reaction (PCR) and real-time PCR. However these techniques require application of thermocyclers and qualified staff which may be cost-consuming for some diagnostic units. The aim of this study was to develop loop-mediated isothermal amplification assay (LAMP) as a simple method of GCV detection. Results The presented study has shown LAMP as a rapid tool of detecting DNA of goose circovirus (GCV) as soon in 30 min time. The method used three sets of primers: two outer primers (F3 and B3), two inner primers (FIP and BIP) and two loop primers (FL and BL) to accelerate the reaction. The optimum reaction temperature and the time were 61°C for 30 min, respectively. The results were analysed using SYBR Green dye and GelRedTM solutions. Thirty-eight isolates of GCV collected from geese flocks in Poland were examined. For comparison, real-time polymerase chain reaction with F3 and B3 primers and SYBR Green dye was conducted. The obtained results have shown GCV-LAMP as a sensitive, rapid and specific assay and alternative for PCR-based methods. Conclusions The developed technique due to its simplicity may be applied by any veterinary laboratory or even mobile diagnostics units for the routine detection of GCV. PMID:22695123

  6. 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.

  7. Real-time streamflow conditions

    Science.gov (United States)

    Graczyk, David J.; Gebert, Warren A.

    1996-01-01

    Would you like to know streamflow conditions before you go fishing in Wisconsin or in more distant locations? Real-time streamflow data throughout Wisconsin and the United States are available on the Internet from the U.S. Geological Survey. You can see if the stream you are interested in fishing is high due to recent rain or low because of an extended dry spell. Flow conditions at more than 100 stream-gaging stations located throughout Wisconsin can be viewed by accessing the Wisconsin District Home Page at: http://wwwdwimdn.er.usgs.gov

  8. Real-time flutter analysis

    Science.gov (United States)

    Walker, R.; Gupta, N.

    1984-01-01

    The important algorithm issues necessary to achieve a real time flutter monitoring system; namely, the guidelines for choosing appropriate model forms, reduction of the parameter convergence transient, handling multiple modes, the effect of over parameterization, and estimate accuracy predictions, both online and for experiment design are addressed. An approach for efficiently computing continuous-time flutter parameter Cramer-Rao estimate error bounds were developed. This enables a convincing comparison of theoretical and simulation results, as well as offline studies in preparation for a flight test. Theoretical predictions, simulation and flight test results from the NASA Drones for Aerodynamic and Structural Test (DAST) Program are compared.

  9. Real time analysis under EDS

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. Real-time analysis keratometer

    Science.gov (United States)

    Adachi, Iwao P. (Inventor); Adachi, Yoshifumi (Inventor); Frazer, Robert E. (Inventor)

    1987-01-01

    A computer assisted keratometer in which a fiducial line pattern reticle illuminated by CW or pulsed laser light is projected on a corneal surface through lenses, a prismoidal beamsplitter quarterwave plate, and objective optics. The reticle surface is curved as a conjugate of an ideal corneal curvature. The fiducial image reflected from the cornea undergoes a polarization shift through the quarterwave plate and beamsplitter whereby the projected and reflected beams are separated and directed orthogonally. The reflected beam fiducial pattern forms a moire pattern with a replica of the first recticle. This moire pattern contains transverse aberration due to differences in curvature between the cornea and the ideal corneal curvature. The moire pattern is analyzed in real time by computer which displays either the CW moire pattern or a pulsed mode analysis of the transverse aberration of the cornea under observation, in real time. With the eye focused on a plurality of fixation points in succession, a survey of the entire corneal topography is made and a contour map or three dimensional plot of the cornea can be made as a computer readout in addition to corneal radius and refractive power analysis.

  11. 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...

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. Real-time flood forecasting

    Science.gov (United States)

    Lai, C.; Tsay, T.-K.; Chien, C.-H.; Wu, I.-L.

    2009-01-01

    Researchers at the Hydroinformatic Research and Development Team (HIRDT) of the National Taiwan University undertook a project to create a real time flood forecasting model, with an aim to predict the current in the Tamsui River Basin. The model was designed based on deterministic approach with mathematic modeling of complex phenomenon, and specific parameter values operated to produce a discrete result. The project also devised a rainfall-stage model that relates the rate of rainfall upland directly to the change of the state of river, and is further related to another typhoon-rainfall model. The geographic information system (GIS) data, based on precise contour model of the terrain, estimate the regions that were perilous to flooding. The HIRDT, in response to the project's progress, also devoted their application of a deterministic model to unsteady flow of thermodynamics to help predict river authorities issue timely warnings and take other emergency measures.

  17. REAL TIME DATA PROCESSING FRAMEWORKS

    Directory of Open Access Journals (Sweden)

    Yash Sakaria

    2015-09-01

    Full Text Available On a business level, everyone wants to get hold of the business value and other organizational advantages that big data has to offer. Analytics has arisen as the primitive path to business value from big data. Hadoop is not just a storage platform for big data; it’s also a computational and processing platform for business analytics. Hadoop is, however, unsuccessful in fulfilling business requirements when it comes to live data streaming. The initial architecture of Apache Hadoop did not solve the problem of live stream data mining. In summary, the traditional approach of big data being co-relational to Hadoop is false; focus needs to be given on business value as well. Data Warehousing, Hadoop and stream processing complement each other very well. In this paper, we have tried reviewing a few frameworks and products which use real time data streaming by providing modifications to Hadoop.

  18. Rapid Detection/pathotyping of Newcastle disease virus isolates in clinical samples using real time polymerase chain reaction assay

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Abdul Wajid, Muhammad Wasim, Tahir Yaqub, Shafqat F Rehmani, Tasra Bibi, Nadia Mukhtar, Javed Muhammad, Umar Bacha, Suliman Qadir Afridi, Muhammad Nauman Zahid, Zia u ddin, Muhammad Zubair Shabbir, Kamran Abbas & Muneer Ahmad ### Abstract In the present protocol we describe the real time reverse transcription polymerase chain reaction (rRT-PCR) assay for the rapid detection/pathotyping of Newcastle disease virus (NDV) isoaltes in clinical samples. Fusion gene and matrix ...

  19. Comparison between Saliva and Nasopharyngeal Swab Specimens for Detection of Respiratory Viruses by Multiplex Reverse Transcription-PCR.

    Science.gov (United States)

    Kim, Young-Gon; Yun, Seung Gyu; Kim, Min Young; Park, Kwisung; Cho, Chi Hyun; Yoon, Soo Young; Nam, Myung Hyun; Lee, Chang Kyu; Cho, Yun-Jung; Lim, Chae Seung

    2017-01-01

    Nasopharyngeal swabs (NPSs) are being widely used as specimens for multiplex real-time reverse transcription (RT)-PCR for respiratory virus detection. However, it remains unclear whether NPS specimens are optimal for all viruses targeted by multiplex RT-PCR. In addition, the procedure to obtain NPS specimens causes coughing in most patients, which possibly increases the risk of nosocomial spread of viruses. In this study, paired NPS and saliva specimens were collected from 236 adult male patients with suspected acute respiratory illnesses. Specimens were tested for 16 respiratory viruses by multiplex real-time RT-PCR. Among the specimens collected from the 236 patients, at least 1 respiratory virus was detected in 183 NPS specimens (77.5%) and 180 saliva specimens (76.3%). The rates of detection of respiratory viruses were comparable for NPS and saliva specimens (P = 0.766). Nine virus species and 349 viruses were isolated, 256 from NPS specimens and 273 from saliva specimens (P = 0.1574). Adenovirus was detected more frequently in saliva samples (P saliva samples was excluded by direct sequencing. In conclusion, neither of the sampling methods was consistently more sensitive than the other. We suggest that these cost-effective methods for detecting respiratory viruses in mixed NPS-saliva specimens might be valuable for future studies. Copyright © 2016 American Society for Microbiology.

  20. Clinical virology in real time.

    Science.gov (United States)

    Niesters, Hubert G M

    2002-12-01

    The ability to detect nucleic acids has had and still has a major impact on diagnostics in clinical virology. Both quantitative and qualitative techniques, whether signal or target amplification based systems, are currently used routinely in most if not all virology laboratories. Technological improvements, from automated sample isolation to real time amplification technology, have given the ability to develop and introduce systems for most viruses of clinical interest, and to obtain clinical relevant information needed for optimal antiviral treatment options. Both polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) can currently be used together with real time detection to generate results in a short turn-around time and to determine whether variants relevant for antiviral resistance are present. These new technologies enable the introduction of an individual patient disease management concept. Within our clinical setting, we have introduced this e.g. for quantitative detection of Epstein-Barr Virus (EBV) in T-dell depleted allogeneic stem cell transplant patients. This enabled us to develop models for pre-emptive anti B-cell immunotherapy for EBV reactivation, thereby effectively reducing not the incidence of EBV-lymphoproliferative disease but the virus related mortality. Furthermore, additional clinically relevant viruses can now easily be detected simultaneously. It also becomes more feasible to introduce molecular testing for those viruses that can easily be detected using classical virological methods, like culture techniques or antigen detection. Prospective studies are needed to evaluate the clinical importance of the additional positive samples detected. It should however be made clear that a complete exchange of technologies is unlikely to occur, and that some complementary technologies should stay operational enabling the discovery of new viruses. The implementation of these molecular diagnostic technologies furthermore

  1. 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.

  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. Hendra virus detection using Loop-Mediated Isothermal Amplification.

    Science.gov (United States)

    Foord, Adam J; Middleton, Deborah; Heine, Hans G

    2012-04-01

    Hendra virus (HeV) is a zoonotic paramyxovirus endemic in Australian Pteropus bats (fruit bats or flying foxes). Although bats appear to be unaffected by the virus, HeV can spread from fruit bats to horses, causing severe disease. Human infection results from close contact with the blood, body fluids and tissues of infected horses. HeV is a biosecurity level 4 (BSL-4) pathogen, with a high case-fatality rate in humans and horses. Current assays for HeV detection require complex instrumentation and are generally time consuming. The aim of this study was to develop a Loop-Mediated Isothermal Amplification (LAMP) assay to detect nucleic acid from all known HeV strains in horses without the requirement for complex laboratory equipment. A LAMP assay targeting a conserved region of the HeV P-gene was combined with a Lateral Flow Device (LFD) for detection of amplified product. All HeV isolates, the original HeV isolated in 1994 as well as the most recent isolates from 2011 were detected. Analytical sensitivity and specificity of the HeV-LAMP assay was equal to a TaqMan assay developed previously. Significantly, these assays detected HeV in horses before clinical signs were observed. The combined LAMP-LFD procedure is a sensitive method suitable for HeV diagnosis in a resource-limited situation or where rapid test results are critical.

  4. Loop-mediated isothermal amplification of single pollen grains

    Institute of Scientific and Technical Information of China (English)

    Ali Bektaş; Ignacio Chapela

    2014-01-01

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

  5. A new visually improved and sensitive loop mediated isothermal amplification (LAMP) for diagnosis of symptomatic falciparum malaria.

    Science.gov (United States)

    Mohon, Abu Naser; Elahi, Rubayet; Khan, Wasif A; Haque, Rashidul; Sullivan, David J; Alam, Mohammad Shafiul

    2014-06-01

    Molecular diagnosis of malaria by nucleotide amplification requires sophisticated and expensive instruments, typically found only in well-established laboratories. Loop-mediated isothermal amplification (LAMP) has provided a new platform for an easily adaptable molecular technique for molecular diagnosis of malaria without the use of expensive instruments. A new primer set has been designed targeting the 18S rRNA gene for the detection of Plasmodium falciparum in whole blood samples. The efficacy of LAMP using the new primer set was assessed in this study in comparison to that of a previously described set of LAMP primers as well as with microscopy and real-time PCR as reference methods for detecting P. falciparum. Pre-addition of hydroxy napthol blue (HNB) in the LAMP reaction caused a distinct color change, thereby improving the visual detection system. The new LAMP assay was found to be 99.1% sensitive compared to microscopy and 98.1% when compared to real-time PCR. Meanwhile, its specificity was 99% and 100% in contrast to microscopy and real-time PCR, respectively. Moreover, the LAMP method was in very good agreement with microscopy and real-time PCR (0.94 and 0.98, respectively). This new LAMP method can detect at least 5parasites/μL of infected blood within 35min, while the other LAMP method tested in this study, could detect a minimum of 100parasites/μL of human blood after 60min of amplification. Thus, the new method is sensitive and specific, can be carried out in a very short time, and can substitute PCR in healthcare clinics and standard laboratories.

  6. 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

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

    Directory of Open Access Journals (Sweden)

    Zablon Kithinji Njiru

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

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

    Directory of Open Access Journals (Sweden)

    Yogesh eChander

    2014-08-01

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

  9. Colorimetric Detection of 23 Human Papillomavirus Genotypes by Loop-Mediated Isothermal Amplification.

    Science.gov (United States)

    Lin, Junxiao; Ma, Biao; Fang, Jiehong; Wang, Ye; He, Haizhen; Lin, Wei; Su, Wei; Zhang, Mingzhou

    2017-03-01

    Human papillomavirus (HPV) infection is linked to cervical cancer. With the technological development of molecular biology and epidemiology, detection and treatment of HPV has become an important mean to prevent cervical cancer. A simple, rapid, and sensitive colorimetric loop-mediated isothermal amplification (LAMP) method was established herein to detect 23 HPV genotypes. The sequences of the primers for the LAMP reaction were located in the L1 gene of the HPV genome. As it is a fluorescent dye, calcein was added before the reaction. The reaction was run under isothermal conditions at 65°C for 40 minutes. A positive reaction was indicated by a color change from yellow to fluorescent green. The fluorescence curve diagram represents the monitoring of real time quantitative instrument. 450 cervical swab samples from patients with single infections of 23 different HPV genotypes were examined to evaluate the specificity. The results revealed no cross-reaction with other HPV genotypes. A serial dilution of a cloned plasmid containing 23 HPV L1 gene sequences was employed to evaluate the sensitivity. Different HPV subtypes have different detection capability. The sensitivity of different HPV subtypes tested by LAMP assay was in the range from 1.0 x10 to 4.0 x 103 copies per reaction. The LAMP assay and the RDB (reverse dot blot) were compared for detecting and genotyping HPV among the 450 clinical samples. There were 385 (85.6%) and 375 (83.3%) HPV positive specimens detected by LAMP and RDB, respectively, as well as 306 (68.0%) and 296 (65.8%) for HR-HPV positive specimens. The agreement between the LAMP and RDB assays was 93.3% (κ = 0.75) for HPV positivity and 94.7% (κ = 0.88) for HR-HPV positivity. It was concluded that this colorimetric LAMP assay had potential application for the rapid screening of the HPV infection in resource-limited hospitals or rural clinics.

  10. Real time TaqMan RT-PCR assay for the detection of Cucumber green mottle mosaic virus.

    Science.gov (United States)

    Hongyun, Chen; Wenjun, Zhao; Qinsheng, Gu; Qing, Chen; Shiming, Lin; Shuifang, Zhu

    2008-05-01

    A real time reverse-transcription polymerase chain reaction (RT-PCR) was developed for efficient detection of Cucumber green mottle mosaic virus (CGMMV). The method was designed to use a duo-primer system with a TaqMan probe targeting the conserved sequence in 3' noncoding region (NCR) of CGMMV to detect isolates of this virus collected in China. The sensitivity of the real time RT-PCR assay was 0.13 pg of total RNA or 50 molecules of RNA transcripts. This level of sensitivity indicated that the one step real time RT-PCR developed in the present study could be used for routine testing assays. The real time RT-PCR method could assist in the implementation of quarantine measures for prevention and control of the disease caused by CGMMV.

  11. Expression Levels of RFP in Normal and Cancer Human Tissues via Real-time RT-PCR Detection

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Ret finger protein(RFP) is a member of the tripartite motif family, which is characterized by a conserved RING finger of motif, a B-box, and a coiled-coil domain(they are called RBCC generally). Although RFP was known to be an oncogene when its RBCC moiety was connected with a tyrosine kinase domain by DNA rearrangement, its biological function was not well defined. In this study, by using real-time RT-PCR, the RFP expressions in human and mouse normal tissues, and in the cervical squamous cell carcinoma, endometrial adenocarcinoma, gastric adenocarcinoma, esophageal squamous cell carcinoma, and brain cancer tissues were analyzed. The result of the study proved that the highest level of mRNA reverse transcription appeared in the normal testical tissue, whereas that in other normal tissues of human and mice were low. The mRNA reverse transcription level of RFP was higher in the endometrial adenocarcinoma tissue than in the cervical squamous cell carcinoma tissue; the mRNA reverse transcription level of RFP in the gastric adenocarcinoma tissue was significantly higher than that in the esophageal squamous cell carcinoma tissue. It was also found that the mRNA reverse transcription level of RFP in the brain cancer tissue was higher than that in the normal brain tissue. These results suggested that RFP could possibly be a useful molecular target for the development of new therapeutics for malignant tumors.

  12. Quantification of mRNA Levels Using Real-Time Polymerase Chain Reaction (PCR).

    Science.gov (United States)

    Li, Yiyi; Wang, Kai; Chen, Longhua; Zhu, Xiaoxia; Zhou, Jie

    2016-01-01

    Real-time quantitative reverse transcription PCR technique has advanced greatly over the past 20 years. Messenger RNA (mRNA) levels in cells or tissues can be quantified by this approach. It is well known that changes in mRNA expression in disease, and correlation of mRNA expression profiles with clinical parameters, serve as clinically relevant biomarkers. Hence, accurate determination of the mRNA levels is critically important in describing the biological, pathological, and clinical roles of genes in health and disease. This chapter describes a real-time PCR approach to detect and quantify mRNA expression levels, which can be used for both laboratorial and clinical studies in breast cancer research.

  13. Space Weather and Real-Time Monitoring

    OpenAIRE

    2009-01-01

    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.

  14. 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.

  15. Real-time graphics rendering engine

    CERN Document Server

    Bao, Hujun

    2011-01-01

    ""Real-Time Graphics Rendering Engine"" reveals the software architecture of the modern real-time 3D graphics rendering engine and the relevant technologies based on the authors' experience developing this high-performance, real-time system. The relevant knowledge about real-time graphics rendering such as the rendering pipeline, the visual appearance and shading and lighting models are also introduced. This book is intended to offer well-founded guidance for researchers and developers who are interested in building their own rendering engines. Hujun Bao is a professor at the State Key Lab of

  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 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...

  18. 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...

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

    Science.gov (United States)

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

    2016-07-01

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

  20. Quantification of vitellogenin mRNA induction in mosquitofish (Gambusia affinis) by reverse transcription real-time polymerase chain reaction (RT-PCR).

    Science.gov (United States)

    Leusch, F D L; Van den Heuvel, M R; Laurie, A D; Chapman, H F; Gooneratne, S Ravi; Tremblay, L A

    2005-01-01

    A method to quantify induction of vitellogenin (Vtg) mRNA in adult male mosquitofish was developed. Male mosquitofish were exposed to 0, 1, 20 and 250 ng l(-1) 17beta-oestradiol (E(2)) for 4 and 8 days in static exposures, and liver Vtg mRNA and 18S rRNA expression were quantified in duplex RT-PCR. Liver 18S rRNA expression was very consistent among individuals, and there was a highly significant increase in Vtg mRNA expression after exposure of mosquitofish for just 4 days at 250 ng l(-1) E(2). Lower doses did not induce Vtg mRNA expression even at 4 or 8 days. This method could be used as a rapid test to detect exposure of mosquitofish to oestrogenic chemicals. Further work is needed to determine if increased Vtg mRNA levels in male mosquitofish induce Vtg synthesis, and to determine the usefulness of the method in field sampling.

  1. 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

    control genes was used to correct five genes of interest. These genes were three genes involved in iron acquisition (tbpA, exbB, and fhuD), the heat shock protein gene groEL, and a putative quorum-sensing gene (luxS). The level of tbpA, exbB, and fhuD expression in A. pleuropneumoniae showed significant...

  2. Clearance of human-pathogenic viruses from sludge: study of four stabilization processes by real-time reverse transcription-PCR and cell culture.

    Science.gov (United States)

    Monpoeho, S; Maul, A; Bonnin, C; Patria, L; Ranarijaona, S; Billaudel, S; Ferré, V

    2004-09-01

    Sludges derived from wastewater treatment are foul-smelling, biologically unstable substances. As well as containing numerous pathogenic microorganisms, they also consist of organic matter that can be used as agricultural fertilizer. Legislation nevertheless requires sludges to be virologically tested prior to spreading by the counting of infectious enterovirus particles. This method, based on culture of enterovirus on BGM cells, is lengthy and not very sensitive. The aim of this study was to propose an alternative method of genome quantification for all enteroviruses that is applicable to verifying the elimination of viruses in complex samples such as sludges. Our complete protocol was compared to the official method, consisting of enterovirus enumeration with the most probable number of cythopathic unit (MPNCU) assay through the study of four stabilization procedures: liming, composting, heat treatment, and mesophile anaerobic digestion. Enterovirus quantities at the start of the stabilization procedures were between 37 and 288 MPNCU/g on the one scale and between 4 and 5 log genome copies/g on the other. It was shown that all procedures except mesophile anaerobic digestion were highly effective in the elimination of enterovirus particles and genomes in wastewater sludges. Reduction of viruses by mesophile anaerobic digestion was by only 1 log (infectious particles and genomes). In conclusion, stabilization processes can indeed be checked by virological quality control of sludges with gene amplification. However, the infectivity of genomes needs to be confirmed with cell culture or a correlation model if the virological risk inherent in the agricultural use of such sludges is to be fully addressed.

  3. 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.

  4. Expression analysis of ETS1 gene in peripheral blood mononuclear cells with systemic lupus erythematosus by real-time reverse transcription PCR

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    @@ To the editor: Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with complex genetic inheritance.l In recent years, genome-wide association studies (GWAS) had further provided novel insights into the genetics background of SLE by identifying multiple susceptibility genes in different ethnic populations.

  5. Rapid detection and quantification of Ebola Zaire virus by one-step real-time quantitative reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Ro, Young-Tae; Ticer, Anysha; Carrion, Ricardo; Patterson, Jean L

    2017-04-01

    Given that Ebola virus causes severe hemorrhagic fever in humans with mortality rates as high as 90%, rapid and accurate detection of this virus is essential both for controlling infection and preventing further transmission. Here, a one-step qRT-PCR assay for rapid and quantitative detection of an Ebola Zaire strain using GP, VP24 or VP40 genes as a target is introduced. Routine assay conditions for hydrolysis probe detection were established from the manufacturer's protocol used in the assays. The analytical specificity and sensitivity of each assay was evaluated using in vitro synthesized viral RNA transcripts. The assays were highly specific for the RNA transcripts, no cross-reactivity being observed among them. The limits of detection of the assays ranged from 10(2) to 10(3) copies per reaction. The assays were also evaluated using viral RNAs extracted from cell culture-propagated viruses (Ebola Zaire, Sudan and Reston strains), confirming that they are gene- and strain-specific. The RT-PCR assays detected viral RNAs in blood samples from virus-infected animal, suggesting that they can be also a useful method for identifying Ebola virus in clinical samples. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  6. DNA detection of Clostridium difficile infection based on real-time resistance measurement.

    Science.gov (United States)

    Liu, C; Jiang, D N; Xiang, G M; Luo, F K; Liu, L L; Yu, J C; Pu, X Y

    2013-09-03

    We used a newly developed electrochemical method, real-time resistance measurement, based on loop-mediated isothermal amplification (LAMP), with real-time resistance monitoring and derivative analysis. DNA extracted from specimens was amplified through LAMP reaction. The 2 products of LAMP, DNA and pyrophosphate, both are negative ions; they combine with positive dye (crystal violet) and positive ions (Mg(2+)), which leads to an increase in the resistivity of the reaction liquid. The changes of resistivity were measured in real-time with a specially designed resistance electrode, to detect Clostridium difficile DNA. We found that electrochemical detection of C. difficile could be completed in 0.5-1 h, with a detection limit of 10(2) CFU/mL, with high accuracy (95.0%), sensitivity (91.1%), and specificity (97.3%) compared to PCR methods. C. difficile is commonly associated with antibiotic-induced diarrhea. Due to the difficulty in performing anaerobic culture and cytotoxicity neutralization assays, a simple, rapid, sensitive, and accurate method is preferred. We conclude that real-time resistance measurement is a rapid, sensitive, and stable method for the diagnosis of C. difficile infection that could be applied to gene chips and pocket instruments.

  7. 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....

  8. 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.

  9. Interactive Real-time Magnetic Resonance Imaging

    DEFF Research Database (Denmark)

    Brix, Lau

    Real-time acquisition, reconstruction and interactively changing the slice position using magnetic resonance imaging (MRI) have been possible for years. However, the current clinical use of interactive real-time MRI is limited due to an inherent low spatial and temporal resolution. This PhD proje...

  10. 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 veri...

  11. Real-time communication protocols: an overview

    NARCIS (Netherlands)

    Hanssen, Ferdy; Jansen, Pierre 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

  12. Real-time communication protocols: an overview

    OpenAIRE

    Hanssen, Ferdy; Jansen, Pierre 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 the pros and cons regarding the At-Home Anywhere project are discussed.

  13. Real-time Pricing in Power Markets

    DEFF Research Database (Denmark)

    Boom, Anette; Schwenen, Sebastian

    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...

  14. Interactive Real-time Magnetic Resonance Imaging

    DEFF Research Database (Denmark)

    Brix, Lau

    Real-time acquisition, reconstruction and interactively changing the slice position using magnetic resonance imaging (MRI) have been possible for years. However, the current clinical use of interactive real-time MRI is limited due to an inherent low spatial and temporal resolution. This PhD proje...

  15. 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.

  16. 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

  17. 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.

  18. Validation of Reference Genes for Transcriptional Analyses in Pleurotus ostreatus by Using Reverse Transcription-Quantitative PCR.

    Science.gov (United States)

    Castanera, Raúl; López-Varas, Leticia; Pisabarro, Antonio G; Ramírez, Lucía

    2015-06-15

    Recently, the lignin-degrading basidiomycete Pleurotus ostreatus has become a widely used model organism for fungal genomic and transcriptomic analyses. The increasing interest in this species has led to an increasing number of studies analyzing the transcriptional regulation of multigene families that encode extracellular enzymes. Reverse transcription (RT) followed by real-time PCR is the most suitable technique for analyzing the expression of gene sets under multiple culture conditions. In this work, we tested the suitability of 13 candidate genes for their use as reference genes in P. ostreatus time course cultures for enzyme production. We applied three different statistical algorithms and obtained a combination of stable reference genes for optimal normalization of RT-quantitative PCR assays. This reference index can be used for future transcriptomic analyses and validation of transcriptome sequencing or microarray data. Moreover, we analyzed the expression patterns of a laccase and a manganese peroxidase (lacc10 and mnp3, respectively) in lignocellulose and glucose-based media using submerged, semisolid, and solid-state fermentation. By testing different normalization strategies, we demonstrate that the use of nonvalidated reference genes as internal controls leads to biased results and misinterpretations of the biological responses underlying expression changes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. Reverse transcription-polymerase chain reaction molecular testing of cytology specimens: Pre-analytic and analytic factors.

    Science.gov (United States)

    Bridge, Julia A

    2017-01-01

    The introduction of molecular testing into cytopathology laboratory practice has expanded the types of samples considered feasible for identifying genetic alterations that play an essential role in cancer diagnosis and treatment. Reverse transcription-polymerase chain reaction (RT-PCR), a sensitive and specific technical approach for amplifying a defined segment of RNA after it has been reverse-transcribed into its DNA complement, is commonly used in clinical practice for the identification of recurrent or tumor-specific fusion gene events. Real-time RT-PCR (quantitative RT-PCR), a technical variation, also permits the quantitation of products generated during each cycle of the polymerase chain reaction process. This review addresses qualitative and quantitative pre-analytic and analytic considerations of RT-PCR as they relate to various cytologic specimens. An understanding of these aspects of genetic testing is central to attaining optimal results in the face of the challenges that cytology specimens may present. Cancer Cytopathol 2017;125:11-19. © 2016 American Cancer Society.

  20. 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.

  1. The ALMA Real Time Control System

    Science.gov (United States)

    Kern, Jeffrey S.; Juerges, Thomas A.; Marson, Ralph G.

    2009-01-01

    The Atacama Large Millimeter Array (ALMA) is a revolutionary millimeter and submillimeter array being developed on the Atacama plateau of northern Chile. An international partnership lead by NRAO, ESO, and NAOJ this powerful and flexible telescope will provide unprecedented observations of this relatively unexplored frequency range. The control subsystem for the Atacama Large Millimeter Array must coordinate the monitor and control of at least sixty six antennas (in four different styles), two correlators, and all of the ancillary equipment (samplers, local oscillators, front ends, etc.). This equipment will be spread over tens of kilometers and operated remotely. Operation of the array requires a robust, scalable, and maintainable real time control system. The real time control system is responsible for monitoring and control of any devices where there are fixed deadlines. Examples in the ALMA context are antenna pointing and fringe tracking. Traditionally the real time portion of a large software system is an intricate and error prone portion of the software. As a result the real time portion is very expensive in terms of effort expended both during construction and during maintenance phases of a project. The ALMA real time control system uses a Linux based real time operating system to interact with the hardware and the CORBA based ALMA Common Software to communicate in the distributed computing environment. Mixing the requirements of real time computing and the non-deterministic CORBA middleware has produced an interesting design. We discuss the architecture, design, and implementation of the ALMA real time control system. Highlight some lessons learned along the way, and justify our assertion that this should be the last large scale real time control system in radio astronomy.

  2. Achieving real-time performance in FIESTA

    Science.gov (United States)

    Wilkinson, William; Happell, Nadine; Miksell, Steve; Quillin, Robert; Carlisle, Candace

    1988-01-01

    The Fault Isolation Expert System for TDRSS Applications (FIESTA) is targeted for operation in a real-time online environment. Initial stages of the prototype development concentrated on acquisition and representation of the knowledge necessary to isolate faults in the TDRSS Network. Recent efforts focused on achieving real-time performance including: a discussion of the meaning of FIESTA real-time requirements, determination of performance levels (benchmarking) and techniques for optimization. Optimization techniques presented include redesign of critical relations, filtering of redundant data and optimization of patterns used in rules. Results are summarized.

  3. Real-time medical applications and telecommunications.

    Science.gov (United States)

    Stravs, M

    1999-01-01

    Telecommunications play an important role in telemedicine. Many forms of telecommunication services based on different telecommunication technologies are developed for various needs. The paper deals with complex real-time applications which demand high telecommunication requirements. At the beginning, medical applications are categorised and real-time applications qualified as multimedia applications. Requirements for multimedia elements are listed separately. Later on, short introduction of related telecommunication protocols is given. Real-time medical applications can show their ability in case of guaranteed quality of services delivered by telecommunication network as it is explained in the end.

  4. Real time detecting system for turning force

    CERN Document Server

    Yue Xiao Bin

    2001-01-01

    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

  5. Complementary assays reveal a relationship between HIV-1 uncoating and reverse transcription.

    Science.gov (United States)

    Hulme, Amy E; Perez, Omar; Hope, Thomas J

    2011-06-14

    During the early stages of HIV-1 replication the conical capsid composed of p24(CA) protein dissociates from the rest of the cytoplasmic viral complex by a process called uncoating. Although proper uncoating is known to be required for HIV-1 infection, many questions remain about the timing and factors involved in the process. Here we have used two complementary assays to study the process of uncoating in HIV-1-infected cells, specifically looking at the timing of uncoating and its relationship to reverse transcription. We developed a fluorescent microscopy-based uncoating assay that detects the association of p24(CA) with HIV-1 viral complexes in cells. We also used an owl monkey kidney (OMK) cell assay that is based on timed TRIM-CypA-mediated restriction of HIV-1 replication. Results from both assays indicate that uncoating is initiated within 1 h of viral fusion. In addition, treatment with the reverse transcriptase inhibitor nevirapine delayed uncoating in both assays. Analysis of reverse transcription products in OMK cells revealed that the generation of early reverse transcription products coincides with the timing of uncoating in these assays. Collectively, these results suggest that some aspect of reverse transcription has the ability to influence the kinetics of uncoating.

  6. 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...

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

    Science.gov (United States)

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

    2008-01-01

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

  8. Visualization in Real-Time Experiment Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The objective of this project will be to migrate some of the outputs from the WFF Mission Planning Lab (MPL) into a real-time visualization system.  The MPL is...

  9. Real Time Study and Related Variables

    Institute of Scientific and Technical Information of China (English)

    WU Xiao-qing

    2016-01-01

    This paper first, illustrates the advantages of applying real time study to linguistic researches. Second, this paper also compares linguistic variables with linguistic variant; nasality, stronger constraint and weaker constraint have been clearly de-fined as well.

  10. 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.

  11. Real-Time Sensor-Actuator Networks

    OpenAIRE

    Sastry, Shivakumar; S. S. Iyengar

    2005-01-01

    Emerging technologies offer new paradigms for computation, control, collaboration, and communication. To realize the full potential of these technologies in industry, defense, and homeland security applications, it is necessary to exploit the real-time distributed computing capabilities of sensor-actuator networks. To reliably design and develop such networks, it is necessary to develop deeper insight into the underlying model for real-time computation and the infrastructure at the node level...

  12. Real Time Information Fusion in Military Systems

    Directory of Open Access Journals (Sweden)

    E. Bhagiratharao

    1990-01-01

    Full Text Available With the proliferation of sensors on platforms like battle ships and aircraft, the information to be handled by the battlefield commanders has significantly increased in the recent time. From a deluge of information flowing from sensors, the battlefield commander is required to make situation assessment in real-time and take appropriate action. Recent studies by cognitive scientists have indicated that decision making by individuals as well as a team suffer from several biases. For these two reasons, the battlefield commanders need assistance of real-time information fusion systems to take objective assessment of highly dynamic battle situation in real-time information fusion systems to take objective assessment of a highly dynamic battle situation in real-time. The real-time information fusion systems at a single platform level as well as that applicable for geographically distributed platforms is discussed in detail in this paper. It was concluded that by carrying out these activities at the platform level as well as at 'global' level involving several platforms, the limitations in performance of any sensor due to propagation effects or due to enemy counter measures can be significantly minimised or totally eliminated. At the same time the functional effectiveness of each sensor onboard different platforms, becomes better than when it had to operate autonomously within the real-time information fusion facility. By carrying out global real-time information fusion activity in a theatre of war, all the platforms operating in the area will have the benefit of the best sensor in that area on each aspect of the capability. A few examples of real-time information fusion system are also discussed.

  13. 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....

  14. The LAA real-time benchmarks

    Energy Technology Data Exchange (ETDEWEB)

    Block, R.K.; Krischer, W.; Lone, S. [CERN, Geneva (Switzerland)

    1989-04-01

    In the context of the LAA detector development program a subgroup Real Time Data Processing has tackled the problem of intelligent triggering. The main goal of this group is to show how fast digital devices, implemented as custom-made or commercial processors, can execute some basic algorithms, and how they can be embedded in the data flow between detector readout components and fully programmable commercial processors, which are expected to be the final data processing filter in real time.

  15. Analysis of real-time vibration data

    Science.gov (United States)

    Safak, E.

    2005-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yi Wang

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

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

    Directory of Open Access Journals (Sweden)

    Jinhua Liu

    2017-01-01

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

  18. Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of lymphocystis disease virus.

    Science.gov (United States)

    Li, Qiong; Yue, Zhiqin; Liu, Hong; Liang, Chengzhu; Zheng, Xiaolong; Zhao, Yuran; Chen, Xiao; Xiao, Xizhi; Chen, Changfu

    2010-02-01

    A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of lymphocystis disease virus (LCDV). A set of five specific primers, two inner and two outer primers and a loop primer, were designed on the basis of the major capsid protein gene of LCDV. The reaction time and temperatures were optimized for 60 min at 63 degrees C, respectively. LAMP amplification products were detected by a ladder-like appearance on agarose gel electrophoresis or a naked-eye inspection of a color change in the reaction tube by addition of SYBR Green I. The assay was specific for LCDV, and there was no cross-reactivity with white spot syndrome virus (WSSV) or six other Iridoviridae viruses (epizootic hematopoietic necrosis virus, EHNV; tiger frog virus, TFV; Bohle iridovirus, BIV; soft-shelled turtle iridovirus, STIV; infectious spleen and kidney necrosis virus, ISKNV; red sea bream iridovirus, RSIV). The detection limit of the LAMP assay was 15 fg, which was similar to that of real-time quantitative polymerase chain reaction (PCR) and 10-fold higher than the conventional PCR. The LAMP assay was evaluated using 109 clinical samples, and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for detection of LCDV. The LCDV LAMP assay has potential for early diagnosis of LCDV infection.

  19. Development of a multiplex loop-mediated isothermal amplification method for the simultaneous detection of Salmonella spp. and Vibrio parahaemolyticus

    Science.gov (United States)

    Liu, Ningwei; Zou, Dayang; Dong, Derong; Yang, Zhan; Ao, Da; Liu, Wei; Huang, Liuyu

    2017-01-01

    Rapid detection of food-borne pathogens is important in the food industry, to monitor and prevent the spread of these pathogens through contaminated food products. We therefore established a multiplex real-time loop-mediated isothermal amplification (LAMP) assay to simultaneously detect and distinguish Salmonella spp. and Vibrio parahaemolyticus DNA in a single reaction. Two target sequences, one specific for Salmonella and the other specific for Vibrio parahaemolyticus, were amplified by specific LAMP primers in the same reaction tube. After amplification at 65 °C for 60 min, the amplified products were subjected to melting curve analysis and thus could be distinguished based on the different melting temperatures (Tm values) of the two specifically amplified products. The specificity of the multiplex LAMP assay was evaluated using 19 known bacterial strains, including one V. parahaemolyticus and seven Salmonella spp. strains. The multiplex LAMP showed 100% inclusivity and exclusivity, and a detection limit similar to that of multiplex PCR. In addition, we observed and corrected preferential amplification induced by what we call LAMP selection in the multiplex LAMP reaction. In conclusion, our assay was rapid, specific, and quantitative, making it a useful tool for the food industry. PMID:28349967

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

    Directory of Open Access Journals (Sweden)

    SA Faggion

    2013-01-01

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

  1. Development of Au-Nanoprobes Combined with Loop-Mediated Isothermal Amplification for Detection of Isoniazid Resistance in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Jutturong Ckumdee

    2016-01-01

    Full Text Available Multidrug resistant tuberculosis (MDR-TB is Mycobacterium tuberculosis that does not respond to isoniazid and rifampicin, so the condition worsens continuously and creates difficulties for treatment by public health control programmes, especially in developing countries. The real time polymerase chain reaction (PCR combined with agarose gel electrophoresis or strip tests is useful molecular tools for diagnosis of MDR-TB. Novel loop-mediated isothermal amplification (LAMP can also detect drug resistance, which is a one-point mutation, by designing inner primers of 5′ end specific with the mutant. Au-nanoprobes on hybridisation with LAMP products containing target-specific sequences remain red, whereas test samples without specific sequences in the probe turn purple due to salt-induced aggregation of the Au-nanoprobes. In this study, a strategy was designed based on the LAMP of a DNA sample coupled to specific Au-nanoprobes, which showed the potential to provide a rapid and sensitive method for detecting isoniazid resistance at katG gene position 315 (G→C. 46 clinical samples were tested and showed 100% specificity and sensitivity compared with Genotype® MDR-TB Plus. This method was advantageous because it is rapid, cheap, specific, and sensitive. Further, it does not require thermal cycles for MDR-TB detection.

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

    Science.gov (United States)

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

    2017-01-01

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

  3. 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.

  4. VEGF system expression by immunohistochemistry and real-time RT-PCR study on collared peccary placenta

    DEFF Research Database (Denmark)

    Santos, Tatiana C.; Oliveira, Moacir F.; Papa, Paula C.;

    2014-01-01

    in the placenta and uterus of the collared peccary in nonpregnant females in the luteal phase and throughout pregnancy (>35, 75, 115, and 135 days). The material was examined by immunohistochemistry and by real-time reverse transcription polymerase chain reaction. Intense positive immunolabeling was observed...... in late pregnancy. In the collared peccary, the expression of the VEGF-ligand receptor system was similar to that in porcine and ruminant placentas, suggesting that an epitheliochorial placenta has the same physiological and interhemal barrier during vascular gestational development. The expression...

  5. Visualization of Real-Time Data

    Science.gov (United States)

    Stansifer, Ryan; Engrand, Peter

    1996-01-01

    In this project we explored various approaches to presenting real-time data from the numerous systems monitored on the space shuttle to computer users. We examined the approach that several projects at the Kennedy Space Center (KSC) used to accomplish this. We undertook to build a prototype system to demonstrate that the Internet and the Java programming language could be used to present the real-time data conveniently. Several Java programs were developed that presented real-time data in different forms including one form that emulated the display screens of the PC GOAL system which is familiar to many at KSC. Also, we developed several communications programs to supply the data continuously. Furthermore, a framework was created using the World Wide Web (WWW) to organize the collection and presentation of the real-time data. We believe our demonstration project shows the great flexibility of the approach. We had no particular use of the data in mind, instead we wanted the most general and the least complex framework possible. People who wish to view data need only know how to use a WWW browser and the address (the URL). People wanting to build WWW documents containing real-time data need only know the values of a few parameters, they do not need to program in Java or any other language. These are stunning advantages over more monolithic systems.

  6. 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......-line, but interactive, slice alignment tool was used to support the notion that 3D blood flow quantification in the heart possesses the ability to obtain curves and volumes which are not statistical different from standard 2D flow. Secondly, the feasibility of an interactive real-time MRI system was exploited...... with regard to optimal sampling strategy for detecting motion in four different anatomies on two different MRI scanner brands. A fully implemented interactive real-time MRI system was exploited in a group of healthy fetuses and proved its eligibility as an alternative diagnostic tool for fetal imaging...

  7. Deterministic Real-time Thread Scheduling

    CERN Document Server

    Yun, Heechul; Sha, Lui

    2011-01-01

    Race condition is a timing sensitive problem. A significant source of timing variation comes from nondeterministic hardware interactions such as cache misses. While data race detectors and model checkers can check races, the enormous state space of complex software makes it difficult to identify all of the races and those residual implementation errors still remain a big challenge. In this paper, we propose deterministic real-time scheduling methods to address scheduling nondeterminism in uniprocessor systems. The main idea is to use timing insensitive deterministic events, e.g, an instruction counter, in conjunction with a real-time clock to schedule threads. By introducing the concept of Worst Case Executable Instructions (WCEI), we guarantee both determinism and real-time performance.

  8. Continuous, real time microwave plasma element sensor

    Science.gov (United States)

    Woskov, Paul P.; Smatlak, Donna L.; Cohn, Daniel R.; Wittle, J. Kenneth; Titus, Charles H.; Surma, Jeffrey E.

    1995-01-01

    Microwave-induced plasma for continuous, real time trace element monitoring under harsh and variable conditions. The sensor includes a source of high power microwave energy and a shorted waveguide made of a microwave conductive, refractory material communicating with the source of the microwave energy to generate a plasma. The high power waveguide is constructed to be robust in a hot, hostile environment. It includes an aperture for the passage of gases to be analyzed and a spectrometer is connected to receive light from the plasma. Provision is made for real time in situ calibration. The spectrometer disperses the light, which is then analyzed by a computer. The sensor is capable of making continuous, real time quantitative measurements of desired elements, such as the heavy metals lead and mercury.

  9. Durham adaptive optics real-time controller.

    Science.gov (United States)

    Basden, Alastair; Geng, Deli; Myers, Richard; Younger, Eddy

    2010-11-10

    The Durham adaptive optics (AO) real-time controller was initially a proof of concept design for a generic AO control system. It has since been developed into a modern and powerful central-processing-unit-based real-time control system, capable of using hardware acceleration (including field programmable gate arrays and graphical processing units), based primarily around commercial off-the-shelf hardware. It is powerful enough to be used as the real-time controller for all currently planned 8 m class telescope AO systems. Here we give details of this controller and the concepts behind it, and report on performance, including latency and jitter, which is less than 10 μs for small AO systems.

  10. Real-Time Visualization of Tissue Ischemia

    Science.gov (United States)

    Bearman, Gregory H. (Inventor); Chrien, Thomas D. (Inventor); Eastwood, Michael L. (Inventor)

    2000-01-01

    A real-time display of tissue ischemia which comprises three CCD video cameras, each with a narrow bandwidth filter at the correct wavelength is discussed. The cameras simultaneously view an area of tissue suspected of having ischemic areas through beamsplitters. The output from each camera is adjusted to give the correct signal intensity for combining with, the others into an image for display. If necessary a digital signal processor (DSP) can implement algorithms for image enhancement prior to display. Current DSP engines are fast enough to give real-time display. Measurement at three, wavelengths, combined into a real-time Red-Green-Blue (RGB) video display with a digital signal processing (DSP) board to implement image algorithms, provides direct visualization of ischemic areas.

  11. Quantification of llama inflammatory cytokine mRNAs by real-time RT-PCR.

    Science.gov (United States)

    Odbileg, Raadan; Konnai, Satoru; Usui, Tatsufumi; Ohashi, Kazuhiko; Onuma, Misao

    2005-02-01

    We have developed a method by which llama cytokine mRNAs can be quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of llama, reverse transcribed to cDNA, and cytokine profiles for interleukin (IL)-1alpha, IL-1beta, IL-6 and tumor necrosis factor (TNF) alpha were quantified by real-time PCR. The expressions of mRNAs of inflammatory cytokines IL-1alpha, IL-1beta, IL-6 and TNFalpha were upregulated upon stimulation with LPS in a dose- and time-dependent manner. Incubation of PBMCs with 100 and 1,000 pg/ml of LPS for 3 to 6 hr resulted in the acceleration of the mRNA levels of inflammatory cytokines. Here, we describe a highly sensitive and reproducible method to quantify the transcription of llama cytokine mRNAs by real-time RT-PCR with the double-stranded DNA-binding dye SYBR Green I.

  12. RT real-time PCR-based quantification of Uromyces fabae in planta.

    Science.gov (United States)

    Voegele, Ralf T; Schmid, Annette

    2011-09-01

    Quantification of obligate biotrophic parasites has been a long-standing problem in plant pathology. Many attempts have been made to determine how much of a pathogen is present in infected plant tissue. Methods of quantification included scoring disease symptoms, microscopic evaluation, determination of specific compounds like Ergosterol, and lately nucleic acid-based technologies. All of these methods have their drawbacks, and even real-time PCR may not be quantitative if for example the organism of interest has specific and differing numbers of nuclei in different infection structures. We applied reverse transcription (RT) real-time PCR to quantify Uromyces fabae within its host plant Vicia faba. We used three different genes, which have been shown to be constitutively expressed. Our analyses show an exponential increase of fungal material between 4 and 9 days post inoculation and thereafter reaching a steady state of around 45% of total RNA. We also used haustorium-specific genes to determine the amount of haustoria present at each time point. These analyses parallel the development of the whole fungus with the exception of the steady-state level, which is only around 5% of the total RNA. This indicates that RT real-time PCR is a suitable method for quantification of obligate biotrophic parasites, and also for the differentiation of developmental stages. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  13. [Rapid detection of rotavirus in water samples using immunomagnetic separation combined with real time PCR].

    Science.gov (United States)

    Yang, Wan; He, Miao; Li, Dan; Shi, Han-Chang; Liu, Li

    2009-05-15

    A quantitative and rapid detection method for rotavirus in water samples was developed, by using immunomagnetic separation combined with reverse transcription and real time polymerase chain reaction (IMS-RT-real time PCR). Magnetic beads coated with antibodies directed against group A rotavirus were used to capture and purify the virus in water samples. The experimental results showed that IMS was optimized when 1 mL samples were supplemented with 10 microL of immunomagnetic beads, 2.5 microL of Tween 20 and incubated for 2 h. The IMS method was employed in the detection of rotavirus in seeded virus eluant such as 3% beef extract successfully and thus manifested its compatibility with established virus concentration methods. The IMS-RT-real time PCR method could yield quantitative results within about 5 h with a detection limit at 1 x 10(4) copies/mL (equivalent to 3-4 PFU/mL). The method exhibited a high level correlation (R2 = 0.9816) with cell culture assay, indicating that it could perform as well as cell culture assay does in infection tests. And the method functioned satisfactorily in seeded concentrate of secondary waste water treatment plant effluent, reclaimed water, surface water and tap water.

  14. 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.

  15. 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.

  16. Real-time Shakemap implementation in Austria

    Science.gov (United States)

    Weginger, Stefan; Jia, Yan; Papi Isaba, Maria; Horn, Nikolaus

    2017-04-01

    ShakeMaps provide near-real-time maps of ground motion and shaking intensity following significant earthquakes. They are automatically generated within a few minutes after occurrence of an earthquake. We tested and included the USGS ShakeMap 4.0 (experimental code) based on python in the Antelope real-time system with local modified GMPE and Site Effects based on the conditions in Austria. The ShakeMaps are provided in terms of Intensity, PGA, PGV and PSA. Future presentation of ShakeMap contour lines and Ground Motion Parameter with interactive maps and data exchange over Web-Services are shown.

  17. Real Time Implementation Of Face Recognition System

    Directory of Open Access Journals (Sweden)

    Megha Manchanda

    2014-10-01

    Full Text Available This paper proposes face recognition method using PCA for real time implementation. Nowadays security is gaining importance as it is becoming necessary for people to keep passwords in their mind and carry cards. Such implementations however, are becoming less secure and practical, also is becoming more problematic thus leading to an increasing interest in techniques related to biometrics systems. Face recognition system is amongst important subjects in biometrics systems. This system is very useful for security in particular and has been widely used and developed in many countries. This study aims to achieve face recognition successfully by detecting human face in real time, based on Principal Component Analysis (PCA algorithm.

  18. 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.

  19. 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

  20. Real-time elastography of the prostate.

    Science.gov (United States)

    Junker, D; De Zordo, T; Quentin, M; Ladurner, M; Bektic, J; Horniger, W; Jaschke, W; Aigner, F

    2014-01-01

    Palpation of organs is one of the oldest clinical examination techniques, for instance, if you think of the palpation of the breast or the digital rectal examination of the prostate, where hard palpable regions are suspicious for cancer. This is the basic principle of real-time elastography, an ultrasound technique, which is able to visualise tissue elasticity. Since prostate cancer features an increased stiffness due to the higher cell and vessel density than the normal surrounding tissue, real-time elastography has been used for several years for prostate cancer detection. This review introduces the different techniques of ultrasound elastography and furthermore summarises its limitations and potentials.

  1. Real-Time Elastography of the Prostate

    Directory of Open Access Journals (Sweden)

    D. Junker

    2014-01-01

    Full Text Available Palpation of organs is one of the oldest clinical examination techniques, for instance, if you think of the palpation of the breast or the digital rectal examination of the prostate, where hard palpable regions are suspicious for cancer. This is the basic principle of real-time elastography, an ultrasound technique, which is able to visualise tissue elasticity. Since prostate cancer features an increased stiffness due to the higher cell and vessel density than the normal surrounding tissue, real-time elastography has been used for several years for prostate cancer detection. This review introduces the different techniques of ultrasound elastography and furthermore summarises its limitations and potentials.

  2. Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification

    Science.gov (United States)

    The technique of loop-mediated isothermal amplification (LAMP) utilizes 4 (or 6) primers targeting 6 (or 8) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an in...

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

    Science.gov (United States)

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

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

    Science.gov (United States)

    Red rot, caused by Colletotrichum falcatum, is a destructive disease prevalent in most sugarcane-producing countries. Disease-free sugarcane planting materials are essential as the pathogen spreads primarily through infected setts. The present study was undertaken to develop loop-mediated isothermal...

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

    Institute of Scientific and Technical Information of China (English)

    Xue-en FANG; Jian LI; Qin CHEN

    2008-01-01

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

  6. LAMP (Loop-mediated isothermal amplification of DNA) - A technique for biotype discrimination in Bemisia tabaci

    Science.gov (United States)

    Loop-mediated isothermal amplification of DNA (LAMP) can amplify a target DNA sequence at a constant temperature in about 1 hour. LAMP technology has great potential for agricultural applications because of the need for rapid and inexpensive diagnoses. Assays based on LAMP technology are well suited...

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

    NARCIS (Netherlands)

    Kong, X.; Qin, W.; Xiaoqing, X.; Kong, F.; Schoen, C.D.; Feng, J.; Wang, Z.; Zhang, H.

    2016-01-01

    A rapid LAMP (loop-mediated isothermal amplification) detection method was developed on the basis of the ITS sequence of P. viticola, the major causal agent of grape downy mildew. Among the 38 fungal and oomycete species tested, DNA isolated exclusively from P. viticola resulted in a specific

  8. Evaluation of a commercial loop-mediated isothermal amplification assay for diagnosis of Bordetella pertussis infection.

    Science.gov (United States)

    Kamachi, Kazunari; Moriuchi, Takumi; Hiramatsu, Yukihiro; Otsuka, Nao; Shibayama, Keigo

    2017-02-01

    We evaluated a commercial loop-mediated isothermal amplification (LAMP) assay kit for Bordetella pertussis detection. The LAMP primers were designed to target the ptxP1 allele of the pertussis toxin promoter, but the assay could detect B. pertussis ptxP3 and ptxP8 strains in addition to ptxP1 strains, with high analytical sensitivity.

  9. 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.

  10. Evaluation of Altona Diagnostics RealStar Zika Virus Reverse Transcription-PCR Test Kit for Zika Virus PCR Testing.

    Science.gov (United States)

    L'Huillier, Arnaud G; Lombos, Ernesto; Tang, Elaine; Perusini, Stephen; Eshaghi, Alireza; Nagra, Sandeep; Frantz, Christine; Olsha, Romy; Kristjanson, Erik; Dimitrova, Kristina; Safronetz, David; Drebot, Mike; Gubbay, Jonathan B

    2017-05-01

    With the emerging Zika virus (ZIKV) epidemic, accessible real-time reverse transcription-PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR test kit (Altona PCR) has been approved for emergency use authorization by the U.S. FDA. Our aim was to verify the Altona PCR by comparing it to the CDC-designed dual-target ZIKV rRT-PCR reference assay (reference PCR) and describe the demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada. A large set of clinical specimens was tested for ZIKV by the Altona PCR and the reference PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting the ZIKV NS5 gene. A total of 671 serum specimens were tested by the reference PCR: 58 (8.6%) were positive, 193 (28.8%) were equivocal, and 420 (62.6%) were negative. Ninety percent of the reference PCR-positive patients were tested in the first 5 days after symptom onset. The Altona PCR was performed on 284/671 specimens tested by the reference PCR. The Altona PCR was positive for 53/58 (91%) reference PCR-positive specimens and 16/193 (8%) reference PCR-equivocal specimens; the ZIKV NS5 PCR was positive for all 68 Altona PCR-positive specimens and negative for all 181 Altona PCR-negative specimens that underwent the NS5 PCR. The Altona PCR has very good sensitivity (91%) and specificity (97%) compared to the reference PCR. The Altona PCR can be used for ZIKV diagnostic testing and has less extensive verification requirements than a laboratory-developed test. Copyright © 2017 American Society for Microbiology.

  11. Selection of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in Brassica napus under various stress conditions.

    Science.gov (United States)

    Wang, Zheng; Chen, Yu; Fang, Hedi; Shi, Haifeng; Chen, Keping; Zhang, Zhiyan; Tan, Xiaoli

    2014-10-01

    Data normalization is essential for reliable output of quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) assays, as the unsuitable choice of reference gene(s), whose expression might be influenced by exogenous treatments in plant tissues, could cause misinterpretation of results. To date, no systematic studies on reference genes have been performed in stressed Brassica napus. In this study, we investigated the expression variations of nine candidate reference genes in 40 samples of B. napus leaves subjected to various exogenous treatments. Parallel analyses by geNorm and NormFinder revealed that optimal reference genes differed across the different sets of samples. The best-ranked reference genes were PP2A and TIP41 for salt stress, TIP41 and ACT7 for heavy metal (Cr(6+)) stress, PP2A and UBC21 for drought stress, F-box and SAND for cold stress, F-box and ZNF for salicylic acid stress, TIP41, ACT7, and PP2A for methyl jasmonate stress, TIP41 and ACT7 for abscisic acid stress, and TIP41, UBC21, and PP2A for Sclerotinia sclerotiorum stress. Two newly employed reference genes, TIP41 and PP2A, showed better performances, suggesting their suitability in multiple conditions. To further validate the suitability of the reference genes, the expression patterns of BnWRKY40 and BnMKS1 were studied in parallel. This study is the first systematic analysis of reference gene selection for qRT-PCR normalization in B. napus, an agriculturally important crop, under different stress conditions. The results will contribute toward more accurate and widespread use of qRT-PCR in gene analysis of the genus Brassica.

  12. Detection of Babesia microti parasites by highly sensitive 18S rRNA reverse transcription PCR.

    Science.gov (United States)

    Hanron, Amelia E; Billman, Zachary P; Seilie, Annette M; Chang, Ming; Murphy, Sean C

    2017-03-01

    Babesia are increasingly appreciated as a cause of transfusion-transmitted infection. Sensitive methods are needed to screen blood products. We report herein that B. microti 18S rRNA is over 1,000-fold more abundant than its coding genes, making reverse transcription PCR (RT-PCR) much more sensitive than PCR. Babesia 18S rRNA may be useful for screening the blood supply.

  13. Detection of All Species of the Genus Alphavirus by Reverse Transcription-PCR with Diagnostic Sensitivity▿

    OpenAIRE

    Grywna, K.; Kupfer, B.; Panning, M.; Drexler, J. F.; Emmerich, P.; Drosten, C.; Kummerer, B. M.

    2010-01-01

    Clinical arbovirus screening requires exclusion of a broad range of viruses with as few assays as possible. We present a reverse transcription-PCR (RT-PCR) for the detection of all species of the genus Alphavirus qualified for exclusion screening (limit of detection [LOD], 5 to 100 RNA copies per reaction across all Alphavirus species; detection of viremia down to ca. 10,000 copies per ml).

  14. A reverse transcription-polymerase chain reaction assay for the detection of avian pneumovirus (Colorado strain).

    Science.gov (United States)

    Ali, A; Reynolds, D L

    1999-01-01

    A reverse transcription-polymerase chain reaction assay was developed for the detection of avian pneumovirus (Colorado strain) (APV-Col). The specific primers were designed from the published sequence of the matrix protein gene of APV-Col. The primers amplified a product of 631 nucleotides from APV-Col. The assay identified only APV-Col and did not react with Newcastle disease virus and infectious bronchitis virus.

  15. Detection of Human Picornaviruses by Multiplex Reverse Transcription-PCR and Liquid Hybridization

    OpenAIRE

    Jokela, Pia; Joki-Korpela, Päivi; Maaronen, Marita; Glumoff, Virpi; Hyypiä, Timo

    2005-01-01

    A qualitative multiplex reverse transcription (RT)-PCR and liquid hybridization assay for the detection of human enteroviruses, rhinoviruses, parechoviruses, and Aichi virus was developed. Furthermore, a separate assay for the recognition of hepatitis A virus was established to complement the test pattern so that all human picornaviruses were covered. The amplicons, which represented the 5′ untranslated regions of the viral RNA genomes, were identified in liquid hybridization reactions with g...

  16. 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.

  17. Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

    Science.gov (United States)

    Murgha, Yusuf; Beliveau, Brian; Semrau, Kassandra; Schwartz, Donald; Wu, Chao-Ting; Gulari, Erdogan; Rouillard, Jean-Marie

    2015-06-01

    Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

  18. Virion-incorporated alpha-enolase suppresses the early stage of HIV-1 reverse transcription.

    Science.gov (United States)

    Kishimoto, Naoki; Iga, Nozomi; Yamamoto, Kengo; Takamune, Nobutoki; Misumi, Shogo

    2017-03-04

    Human immunodeficiency virus type-1 (HIV-1) particles contain not only viral-encoded but also host-encoded proteins. Interestingly, several studies showed that host proteins play a critical role in viral infectivity, replication and/or immunoreactivity in the next target cells. Here, we show that alpha-enolase (ENO1) is incorporated into HIV-1 virions and the virion-incorporated ENO1 prevents the early stage of HIV-1 reverse transcription. We found that viral particles contain two isoforms of ENO1 with different isoelectric points by two-dimensional electrophoresis. Suppression of ENO1 expression by RNA interference in the HIV-1 producer cells decreased ENO1 incorporation into virions without altering the packaging of viral structural proteins and viral production but increased viral infectivity. Although the low-level-ENO1-packaging virus maintained comparable levels of reverse transcriptase activity, viral genomic RNA and tRNA(Lys3) packaging to the control virus, its levels of early cDNA products of reverse transcription were higher than those of the control virus. In contrast, the high-level-ENO1-packaging virus, which was produced from ENO1-overexpressing cells, showed decreased infectivity and the levels of early cDNA products. Taken together, these findings reveal a novel function of ENO1 as a negative regulation factor targeting HIV-1 reverse transcription.

  19. 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…

  20. 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.

  1. Quantitative real-time imaging of glutathione

    Science.gov (United States)

    Glutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown. Here, we report a reversible reaction-based fluorescent probe—designated as RealThiol (RT)—that can quantitatively monitor the real-time ...

  2. The Power of Real-Time PCR

    Science.gov (United States)

    Valasek, Mark A.; Repa, Joyce J.

    2005-01-01

    In recent years, real-time polymerase chain reaction (PCR) has emerged as a robust and widely used methodology for biological investigation because it can detect and quantify very small amounts of specific nucleic acid sequences. As a research tool, a major application of this technology is the rapid and accurate assessment of changes in gene…

  3. Real-time dynamics of proton decay

    CERN Document Server

    Grigoriev, D

    2005-01-01

    Substituting Skyrmion for nucleon, one can potentially see -- in real time -- how the monopole is catalysing the proton (or neutron) decay, and even obtain a plausible estimate for catalysis cross-section. Here we discuss the key aspects of a practical implementation of such approach and demonstrate how one can overcome the main technical problems: Gauss constraint violation and reflections at the boundaries.

  4. 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...

  5. Studying Complex Interactions in Real Time

    DEFF Research Database (Denmark)

    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 such behavio...

  6. 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…

  7. 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.

  8. Scene independent real-time indirect illumination

    DEFF Research Database (Denmark)

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

    2005-01-01

    -time rendering of arbitrary dynamic environments and for interactive preview of feature animations. Through DRM we simulate two diffuse reflections of light, but can also, in combination with traditional real-time methods for specular reflections, simulate more complex light paths. DRM is a GPU-based method...

  9. Real time estimates of GDP growth

    NARCIS (Netherlands)

    Groot, de E.A. (Bert); Franses, P.H.P.H.

    2005-01-01

    This paper describes the components of the EICIE, the Econometric Institute Current Indicator of the Economy. This measure concerns quarterly and annual growth of Dutch real Gross Domestic Product. The key component of our real-time forecasting model for Dutch quarterly GDP is weekly staffing servic

  10. Real-time Texture Error Detection

    Directory of Open Access Journals (Sweden)

    Dan Laurentiu Lacrama

    2008-01-01

    Full Text Available This paper advocates an improved solution for the real-time error detection of texture errors that occurs in the production process in textile industry. The research is focused on the mono-color products with 3D texture model (Jacquard fabrics. This is a more difficult task than, for example, 2D multicolor textures.

  11. 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...

  12. Real time estimates of GDP growth

    NARCIS (Netherlands)

    E.A. de Groot (Bert); Ph.H.B.F. Franses (Philip Hans)

    2005-01-01

    textabstractThis paper describes the components of the EICIE, the Econometric Institute Current Indicator of the Economy. This measure concerns quarterly and annual growth of Dutch real Gross Domestic Product. The key component of our real-time forecasting model for Dutch quarterly GDP is weekly sta

  13. Real-time analysis of telemetry data

    Science.gov (United States)

    Kao, Simon A.; Laffey, Thomas J.; Schmidt, James L.; Read, Jackson Y.; Dunham, Larry L.

    1987-01-01

    This paper descibes a knowledge-based system for performing real-time monitoring and analysis of telemetry data from the NASA Hubble Space Telescope (HST). In order to handle asynchronous inputs and perform in real time the system consists of three or more separate processes, which run concurrently and communicate via a message passing scheme. The data management process gathers, compresses, and scales the incoming telemetry data befoe sending it to the other tasks. The inferencing process uses the incoming data to perform a real-time analysis of the state and health of the Space Telescope. The I/O process receives telemetry monitors from the data management process, updates its graphical displays in real time, and acts as the interface to the console operator. The three processes may run on the same or different computers. This system is currently under development and is being used to monitor testcases produced by the Bass Telemetry System in the Hardware/Software Integration Facility at Lockheed Missile and Space Co. in Sunnyvale, California.

  14. Real time estimates of GDP growth

    NARCIS (Netherlands)

    Groot, de E.A. (Bert); Franses, P.H.P.H.

    2005-01-01

    This paper describes the components of the EICIE, the Econometric Institute Current Indicator of the Economy. This measure concerns quarterly and annual growth of Dutch real Gross Domestic Product. The key component of our real-time forecasting model for Dutch quarterly GDP is weekly staffing

  15. Real time estimates of GDP growth

    NARCIS (Netherlands)

    E.A. de Groot (Bert); Ph.H.B.F. Franses (Philip Hans)

    2005-01-01

    textabstractThis paper describes the components of the EICIE, the Econometric Institute Current Indicator of the Economy. This measure concerns quarterly and annual growth of Dutch real Gross Domestic Product. The key component of our real-time forecasting model for Dutch quarterly GDP is weekly

  16. Evaluation of real-time RT-PCR assays for detection and quantification of norovirus genogroups I and II.

    Science.gov (United States)

    Rupprom, Kitwadee; Chavalitshewinkoon-Petmitr, Porntip; Diraphat, Pornphan; Kittigul, Leera

    2017-02-20

    Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three TaqMan real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit (assay A: Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays (assay B: LightCycler RNA Master Hybprobe and assay C: RealTime ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity (10(3) DNA copies/mL) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups. No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle (Cq) value of assay B for GII was lower than assays A and C with statistical significance (P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17, and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.

  17. Development of a reverse transcription loop-mediated isothermal amplification assay for the detection of vesicular stomatitis New Jersey virus: use of rapid molecular assay to differentiate between vesicular disease viruses

    Science.gov (United States)

    Vesicular stomatitis (VS) is endemic in Central America and northern regions of South America. Sporadic outbreaks of VS can occur in cattle and pigs where the clinical presentation can be similar to foot-and-mouth disease (FMD). There is therefore a pressing need for rapid, sensitive and specific d...

  18. 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

    and quantification of viable Campylobacter jejuni directly from chicken faecal samples. The results of this method anda DNA-based quantitative real-time PCR (qPCR) method were compared with those of a bacterial culture method. Using bacterial culture andRT-qPCR methods, viable C. jejuni cells could be detected......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...... for up to 5 days in both the C. jejuni spiked and the naturally contaminated faecalsamples. We found that no RT-qPCR signals were obtained when viable C. jejuni cells could not be counted by the culture method. In contrast,using a DNA-based qPCR method, dead or non-viable Campylobacter cells were...

  19. System Equivalent for Real Time Digital Simulator

    Science.gov (United States)

    Lin, Xi

    2011-07-01

    The purpose of this research is to develop a method of making system equivalents for the Real Time Digital Simulator (RTDS), which should enhance its capability of simulating large power systems. The proposed equivalent combines a Frequency Dependent Network Equivalent (FDNE) for the high frequency electromagnetic transients and a Transient Stability Analysis (TSA) type simulation block for the electromechanical transients. The frequency dependent characteristic for FDNE is obtained by curve-fitting frequency domain admittance characteristics using the Vector Fitting method. An approach for approximating the frequency dependent characteristic of large power networks from readily available typical power-flow data is also introduced. A new scheme of incorporating TSA solution in RTDS is proposed. This report shows how the TSA algorithm can be adapted to a real time platform. The validity of this method is confirmed with examples, including the study of a multi in-feed HVDC system based network.

  20. Real time processor for array speckle interferometry

    Science.gov (United States)

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

    1989-01-01

    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.

  1. Real Time Radiation Exposure And Health Risks

    Science.gov (United States)

    Hu, Shaowen; Barzilla, Janet E.; Semones, Edward J.

    2015-01-01

    Radiation from solar particle events (SPEs) poses a serious threat to future manned missions outside of low Earth orbit (LEO). Accurate characterization of the radiation environment in the inner heliosphere and timely monitoring the health risks to crew are essential steps to ensure the safety of future Mars missions. In this project we plan to develop an approach that can use the particle data from multiple satellites and perform near real-time simulations of radiation exposure and health risks for various exposure scenarios. Time-course profiles of dose rates will be calculated with HZETRN and PDOSE from the energy spectrum and compositions of the particles archived from satellites, and will be validated from recent radiation exposure measurements in space. Real-time estimation of radiation risks will be investigated using ARRBOD. This cross discipline integrated approach can improve risk mitigation by providing critical information for risk assessment and medical guidance to crew during SPEs.

  2. Real-Time Watercolor for Animation

    Institute of Scientific and Technical Information of China (English)

    Thomas Luft; Oliver Deussen

    2006-01-01

    We present algorithms that allow for real-time rendering of 3D-scenes with a watercolor painting appearance. Our approach provides an appropriate simplification of the visual complexity, imitates characteristic natural effects of watercolor, and provides two essential painting techniques: the wet-on-wet and the wet-on-dry painting. We concentrate on efficient algorithms based on image space processing rather than on an exact simulation. This allows for the real-time rendering of 3D-scenes. During an animation a high frame-to-frame coherence can be achieved due to a stable segmentation scheme. Finally, we seamlessly integrate a smooth illumination into the watercolor renderings using information from the 3D-scene.

  3. Monte Carlo study of real time dynamics

    CERN Document Server

    Alexandru, Andrei; Bedaque, Paulo F; Vartak, Sohan; Warrington, Neill C

    2016-01-01

    Monte Carlo studies involving real time dynamics are severely restricted by the sign problem that emerges from highly oscillatory phase of the path integral. In this letter, we present a new method to compute real time quantities on the lattice using the Schwinger-Keldysh formalism via Monte Carlo simulations. The key idea is to deform the path integration domain to a complex manifold where the phase oscillations are mild and the sign problem is manageable. We use the previously introduced "contraction algorithm" to create a Markov chain on this alternative manifold. We substantiate our approach by analyzing the quantum mechanical anharmonic oscillator. Our results are in agreement with the exact ones obtained by diagonalization of the Hamiltonian. The method we introduce is generic and in principle applicable to quantum field theory albeit very slow. We discuss some possible improvements that should speed up the algorithm.

  4. Real time PCR. Application in dengue studies

    Directory of Open Access Journals (Sweden)

    Jeanette Prada-Arismendy

    2011-06-01

    Full Text Available PCR (polymerase chain reaction is a routinely used tool in every diagnostic and research laboratory. This technique has been used in detection of mutations and pathogens, forensic investigation, and even is the base tool for human genome sequencing. A modification of PCR technique, real time PCR, allows the quantification of nucleic acids with higher sensibility, specificity and reproducibility. This article is intended to clarify the foundations of real-time PCR, using an application model for virology. In the actual work, it was quantified the viral load of dengue virus serotype 2 produced from infected murine macrophages; the obtained results in this work established that murine strain BALB/c presents a greater susceptibility to dengue virus infection, which establishes BALB/c murine strain as a best model of study for investigation of dengue virus infection physiopathology.

  5. AMON: Transition to real-time operations

    Science.gov (United States)

    Cowen, D. F.; Keivani, A.; Tešić, G.

    2016-04-01

    The Astrophysical Multimessenger Observatory Network (AMON) will link the world's leading high-energy neutrino, cosmic-ray, gamma-ray and gravitational wave observatories by performing real-time coincidence searches for multimessenger sources from observatories' subthreshold data streams. The resulting coincidences will be distributed to interested parties in the form of electronic alerts for real-time follow-up observation. We will present the science case, design elements, current and projected partner observatories, status of the AMON project, and an initial AMON-enabled analysis. The prototype of the AMON server has been online since August 2014 and processing archival data. Currently, we are deploying new high-uptime servers and will be ready to start issuing alerts as early as winter 2015/16.

  6. Collecting data in real time with postcards

    DEFF Research Database (Denmark)

    2013-01-01

    The success of information technology (IT) in transforming healthcare is often limited by the lack of clear understanding of the context at which the technology is used. Various methods have been proposed to understand healthcare context better in designing and implementing Health Information...... 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...... and Australia. The benefits of this technique are illustrated. There are limitations in using postcard techniques and this paper provides a detail discussion about these limitations. Postcard techniques provide unique advantages in understanding real time healthcare context and it is an important technique...

  7. Real Time Radiation Monitoring Using Nanotechnology

    Science.gov (United States)

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

  8. 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.

  9. "Fast" Is Not "Real-Time": Designing Effective Real-Time AI Systems

    Science.gov (United States)

    O'Reilly, Cindy A.; Cromarty, Andrew S.

    1985-04-01

    Realistic practical problem domains (such as robotics, process control, and certain kinds of signal processing) stand to benefit greatly from the application of artificial intelligence techniques. These problem domains are of special interest because they are typified by complex dynamic environments in which the ability to select and initiate a proper response to environmental events in real time is a strict prerequisite to effective environmental interaction. Artificial intelligence systems developed to date have been sheltered from this real-time requirement, however, largely by virtue of their use of simplified problem domains or problem representations. The plethora of colloquial and (in general) mutually inconsistent interpretations of the term "real-time" employed by workers in each of these domains further exacerbates the difficul-ties in effectively applying state-of-the-art problem solving tech-niques to time-critical problems. Indeed, the intellectual waters are by now sufficiently muddied that the pursuit of a rigorous treatment of intelligent real-time performance mandates the redevelopment of proper problem perspective on what "real-time" means, starting from first principles. We present a simple but nonetheless formal definition of real-time performance. We then undertake an analysis of both conventional techniques and AI technology with respect to their ability to meet substantive real-time performance criteria. This analysis provides a basis for specification of problem-independent design requirements for systems that would claim real-time performance. Finally, we discuss the application of these design principles to a pragmatic problem in real-time signal understanding.

  10. Real-time optical information processing

    CERN Document Server

    Javidi, Bahram

    1994-01-01

    Real-Time Optical Information Processing covers the most recent developments in optical information processing, pattern recognition, neural computing, and materials for devices in optical computing. Intended for researchers and graduate students in signal and information processing with some elementary background in optics, the book provides both theoretical and practical information on the latest in information processing in all its aspects. Leading researchers in the field describe the significant signal processing algorithms architectures in optics as well as basic hardware concepts,

  11. Real-Time Neutron Radiography at CARR

    Institute of Scientific and Technical Information of China (English)

    HE; Lin-feng; HAN; Song-bai; WANG; Hong-li; WU; Mei-mei; WEI; Guo-hai; WANG; Yu

    2012-01-01

    <正>A real-time detector system for neutron radiography based on CMOS camera has been designed for the thermal neutron imaging facility under construction at China Advanced Research Reactor (CARR). This system is equipped with a new scientific CMOS camera with 5.5 million pixels and speed up to 100 fps at full frame. The readout noise is less than 2.4 electron per pixel. It is capable of providing

  12. Robust synthesis for real-time systems

    DEFF Research Database (Denmark)

    Larsen, Kim Guldstrand; Legay, Axel; Traonouez, Louis-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 ...... strategies in timed games. Finally, we consider the parametric robustness problem and propose a counter-example refinement heuristic for computing safe perturbation values....

  13. Real-time RGBD SLAM system

    Science.gov (United States)

    Czupryński, BłaŻej; Strupczewski, Adam

    2015-09-01

    A real-time tracking and mapping SLAM system is presented. The developed system uses input from an RGBD sensor and tracks the camera pose from frame to frame. The tracking is based on matched feature points and is performed with respect to selected keyframes. The system is robust and scalable, as an arbitrary number of keyframes can be chosen for visualization and tracking depending on the desired accuracy and speed. The presented system is also a good platform for further research.

  14. 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....

  15. Real Time Route for Dynamic Road Congestions

    Directory of Open Access Journals (Sweden)

    A. M. Riad

    2012-05-01

    Full Text Available Minimizing service delivery and travel time during rush hours downtown is strategic target for several organizations, especially the emergency organizations. This paper presents an On-line and Real-time Dynamic Route System (ORDRS which benefits from the advantages and integration between information system and communications technology. It utilizes Global Positioning System (GPS, Geographical Information Systems (GIS, and Global System for Mobile communications (GSM; for producing the real time routes for vehicles. GPS-Tracker is the main input device for ORDRS. It is fixated in a vehicle, sends vehicle's movement data (Geo-info to the control center wirelessly through either Short Message Service (SMS or General Packet Radio Service (GPRS. Geo-info includes time, date, longitude, latitude, speed, and etc., these data is classified over time during weekdays into interval time slices, each slice is 30 minutes. Speeds are treated by GIS tools to determine historical and real time speeds for each street segment in the road network which is being used for calculating time impedance (cost matrix for each street segment dynamically. ORDRS uses a cost matrix of the current time slice for determining the best route to each vehicle in duty attached. Several algorithms was used to calculate the shortest route, a comparison between Dijekstra and Yen algorithms was studied.

  16. The IGS Real-Time Service

    Science.gov (United States)

    Caissy, Mark; Agrotis, Loukis; Weber, Georg; Fisher, Steven

    2013-04-01

    The IGS Real-Time Service (RTS) is being rolled out in 2013 following the successful completion of the IGS Real-Time Pilot Project. The RTS has recently completed beta testing and is now operating at the level of initial operating capability. The service will reach full operating capability by the end of 2013. RTS products include GNSS data streams and GNSS orbit and clock correction streams. These products are available in real-time in accordance with the IGS open-data policy using RTCM standard formats and the NTRIP transportation protocol. The RTS is key to IGS's support of the GGOS Natural Hazards theme. Of particular importance in this context is the high degree of redundancy that is build into the RTS in order to reliably support public-good scientific applications commonly associated with natural hazards; for example, precise-point positioning applications requiring high accuracy and low latency related to earthquakes and tsunamis . This presentation will illustrate the data gathering through product generation to user distribution design of the RTS, highlighting built-in robustness at various stages. The presentation will also present an assessment of the performance of the service to date.

  17. Real-time Interactive Tree Animation.

    Science.gov (United States)

    Quigley, Ed; Yu, Yue; Huang, Jingwei; Lin, Winnie; Fedkiw, Ronald

    2017-01-30

    We present a novel method for posing and animating botanical tree models interactively in real time. Unlike other state of the art methods which tend to produce trees that are overly flexible, bending and deforming as if they were underwater plants, our approach allows for arbitrarily high stiffness while still maintaining real-time frame rates without spurious artifacts, even on quite large trees with over ten thousand branches. This is accomplished by using an articulated rigid body model with as-stiff-as-desired rotational springs in conjunction with our newly proposed simulation technique, which is motivated both by position based dynamics and the typical O(N) algorithms for articulated rigid bodies. The efficiency of our algorithm allows us to pose and animate trees with millions of branches or alternatively simulate a small forest comprised of many highly detailed trees. Even using only a single CPU core, we can simulate ten thousand branches in real time while still maintaining quite crisp user interactivity. This has allowed us to incorporate our framework into a commodity game engine to run interactively even on a low-budget tablet. We show that our method is amenable to the incorporation of a large variety of desirable effects such as wind, leaves, fictitious forces, collisions, fracture, etc.

  18. 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.

  19. Evaluation of loop-mediated isothermal amplification for the rapid, reliable, and robust detection of Salmonella in produce.

    Science.gov (United States)

    Yang, Qianru; Wang, Fei; Jones, Kelly L; Meng, Jianghong; Prinyawiwatkul, Witoon; Ge, Beilei

    2015-04-01

    Rapid, reliable, and robust detection of Salmonella in produce remains a challenge. In this study, loop-mediated isothermal amplification (LAMP) was comprehensively evaluated against real-time quantitative PCR (qPCR) for detecting diverse Salmonella serovars in various produce items (cantaloupe, pepper, and several varieties of lettuce, sprouts, and tomato). To mimic real-world contamination events, produce samples were surface-inoculated with low concentrations (1.1-2.9 CFU/25 g) of individual Salmonella strains representing ten serovars and tested after aging at 4 °C for 48 h. Four DNA extraction methods were also compared using produce enrichment broths. False-positive or false-negative results were not observed among 178 strains (151 Salmonella and 27 non-Salmonella) used to evaluate assay specificity. The detection limits for LAMP were 1.8-4 CFU per reaction in pure culture and 10(4)-10(6) CFU per 25 g (i.e., 10(2)-10(4) CFU per g) in produce without enrichment, comparable to those obtained by qPCR. After 6-8 h of enrichment, both LAMP and qPCR consistently detected these low concentrations of Salmonella of diverse serovars in all produce items except sprouts. The PrepMan Ultra sample preparation reagent yielded the best results among the four DNA extraction methods. Upon further validation, LAMP may be a valuable tool for routine Salmonella testing in produce. The difficulty of detecting Salmonella in sprouts, whether using LAMP or qPCR, warrants further study.

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

    Science.gov (United States)

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

    2017-01-01

    Background 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. Methods 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. Results 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. Conclusions 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. PMID:28095434

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

    Directory of Open Access Journals (Sweden)

    Masahiro Itonaga

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

  2. Evaluation of loop-mediated isothermal amplification for the rapid identification of bacteria and resistance determinants in positive blood cultures.

    Science.gov (United States)

    Rödel, J; Bohnert, J A; Stoll, S; Wassill, L; Edel, B; Karrasch, M; Löffler, B; Pfister, W

    2017-01-06

    The use of molecular assays to rapidly identify pathogens and resistance genes directly from positive blood cultures (BCs) contribute to shortening the time required for the diagnosis of bloodstream infections. In this work, loop-mediated isothermal amplification (LAMP) assays have been examined for their potential use in BC diagnosis. Three different assays were applied. The commercially available eazyplex® MRSA test detects Staphylococcus aureus, S. epidermidis, mecA, and mecC. Two in-house assays [Gram-positive (GP) and Gram-negative (GN)] have been developed for the detection of streptococci, enterococci, vanA, vanB, Pseudomonas spp., Enterobacteriaceae, and the bla CTX-M family. A total of 370 positive BCs were analyzed. LAMP test results were obtained within 30 min, including sample preparation. Amplification was measured by real-time fluorescence detection. The threshold time for fluorescence intensity values ranged from 6.25 to 13.75 min. The specificity and sensitivity of the assays varied depending on the target. Overall, from 87.7% of BCs, true-positive results were obtained, compared to routine standard diagnosis. Twenty-one tests were true-negative because of the lack of an appropriate target (5.7%). The concordance of positive test results for resistance genes with subsequent antibiotic susceptibility testing was 100%. From 15 BC bottles with mixed cultures, eazyplex® assays produced correct results in 73% of the cases. This study shows that LAMP assays are fast and cost-saving tools for rapid BC testing in order to expedite the diagnostic report and improve the antibiotic stewardship for sepsis patients.

  3. Most probable number - loop mediated isothermal amplification (MPN-LAMP) for quantifying waterborne pathogens in <25min.

    Science.gov (United States)

    Ahmad, Farhan; Stedtfeld, Robert D; Waseem, Hassan; Williams, Maggie R; Cupples, Alison M; Tiedje, James M; Hashsham, Syed A

    2017-01-01

    We are reporting a most probable number approach integrated to loop mediated isothermal technique (MPN-LAMP) focusing on Gram-negative Escherichia coli and Gram-positive Enterococcus faecalis bacterial cells without nucleic acids extraction. LAMP assays for uidA from E. coli and gelE from E. faecalis were successfully performed directly on cells up to single digit concentration using a commercial real time PCR instrument. Threshold time values of LAMP assays of bacterial cells, heat treated bacterial cells (95°C for 5min), and their purified genomic DNA templates were similar, implying that amplification could be achieved directly from bacterial cells at 63°C. Viability of bacterial cells was confirmed by using propidium monoazide in a LAMP assay with E. faecalis. To check its functionality on a microfluidic platform, MPN-LAMP assays targeting <10CFU of bacteria were also translated onto polymeric microchips and monitored by a low-cost fluorescence imaging system. The overall system provided signal-to-noise (SNR) ratios up to 800, analytical sensitivity of <10CFU, and time to positivity of about 20min. MPN-LAMP assays were performed for cell concentrations in the range of 10(5)CFU to <10CFU. MPN values from LAMP assays confirmed that the amplifications were from <10CFU. The method described here, applicable directly on cells at 63°C, eliminates the requirement of complex nucleic acids extraction steps, facilitating the development of sensitive, rapid, low-cost, and field-deployable systems. This rapid MPN-LAMP approach has the potential to replace conventional MPN method for waterborne pathogens.

  4. Specific diagnosis of Opisthorchis viverrini using loop mediated isothermal amplification (LAMP) targeting parasite microsatellites

    OpenAIRE

    Arimatsu, Yuji; Kaewkes, Sasithorn; Laha, Thewarach; Sripa, Banchob

    2014-01-01

    Opisthorchis viverrini and other food-borne trematode infections are major health problems in Thailand, the Lao People's Democratic Republic, Vietnam and Cambodia. Differential diagnosis of O. viverrini based on the microscopic observation of parasite eggs is difficult in areas where Clonorchis sinensis and minute intestinal flukes coexist. Recently, Loop-mediated isothermal amplification (LAMP) has been widely used for detection and identification of trematode for its simple method that is u...

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

    OpenAIRE

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

    2016-01-01

    Ascaris lumbricoides is a nematode parasite that causes the common tropical infection ascariasis in humans. It is also considered among the neglected tropical diseases. Diagnosis relies mainly on microscopy-based methods which are laborious, are limited by low sensitivity, and require high expertise. We have developed a loop mediated isothermal amplification (LAMP) for diagnosis of ascariasis in fecal samples, based on the first internal transcribed (ITS-1) spacer region of the ribosomal DNA....

  6. Real-time electrochemical LAMP: a rational comparative study of different DNA intercalating and non-intercalating redox probes.

    Science.gov (United States)

    Martin, Alexandra; Bouffier, Laurent; Grant, Kathryn B; Limoges, Benoît; Marchal, Damien

    2016-06-20

    We present a comparative study of ten redox-active probes for use in real-time electrochemical loop-mediated isothermal amplification (LAMP). Our main objectives were to establish the criteria that need to be fulfilled for minimizing some of the current limitations of the technique and to provide future guidelines in the search for ideal redox reporters. To ensure a reliable comparative study, each redox probe was tested under similar conditions using the same LAMP reaction and the same entirely automatized custom-made real-time electrochemical device (designed for electrochemically monitoring in real-time and in parallel up to 48 LAMP samples). Electrochemical melt curve analyses were recorded immediately at the end of each LAMP reaction. Our results show that there are a number of intercalating and non-intercalating redox compounds suitable for real-time electrochemical LAMP and that the best candidates are those able to intercalate strongly into ds-DNA but not too much to avoid inhibition of the LAMP reaction. The strongest intercalating redox probes were finally shown to provide higher LAMP sensitivity, speed, greater signal amplitude, and cleaner-cut DNA melting curves than the non-intercalating molecules.

  7. Reverse Transcription-PCR Analysis of the Regulation of the Manganese Peroxidase Gene Family

    OpenAIRE

    Gettemy, Jessica M.; Ma, Biao; Alic, Margaret; Gold, Michael H.

    1998-01-01

    Manganese peroxidase (MnP) gene expression in the lignin-degrading fungus Phanerochaete chrysosporium is regulated by nutrient nitrogen levels and by Mn(II), the substrate for the enzyme, as well as by heat shock and other factors. Reverse transcription-PCR (RT-PCR) of total RNA can distinguish the mRNAs of each of the three sequenced P. chrysosporium mnp genes, i.e., mnp1, mnp2, and mnp3. Quantitative RT-PCR demonstrates that each of the three transcripts is present at a similar low basal le...

  8. EMA-real-time PCR as a reliable method for detection of viable Salmonella in chicken and eggs.

    Science.gov (United States)

    Wang, Luxin; Mustapha, Azlin

    2010-04-01

    Culture-based Salmonella detection takes at least 4 d to complete. The use of TaqMan probes allows the real-time PCR technique to be a rapid and sensitive way to detect foodborne pathogens. However, unlike RNA-based PCR, DNA-based PCR techniques cannot differentiate between DNA from live and dead cells. Ethidium bromide monoazide (EMA) is a dye that can bind to DNA of dead cells and prevent its amplification by PCR. An EMA staining step prior to PCR allows for the effective inhibition of false positive results from DNA contamination by dead cells. The aim of this study was to design an accurate detection method that can detect only viable Salmonella cells from poultry products. The sensitivity of EMA staining coupled with real-time PCR was compared to that of an RNA-based reverse transcription (RT)-real-time PCR. To prevent false negative results, an internal amplification control was added to the same reaction mixture as the target Salmonella sequences. With an optimized EMA staining step, the detection range of a subsequent real-time PCR was determined to be 10(3) to 10(9) CFU/mL for pure cultures and 10(5) to 10(9) CFU/mL for food samples, which was a wider detection range than for RT-real-time PCR. After a 12-h enrichment step, EMA staining combined with real-time PCR could detect as low as 10 CFU/mL Salmonella from chicken rinses and egg broth. The use of EMA with a DNA-based real-time PCR can successfully prevent false positive results and represents a simple, yet accurate detection tool for enhancing the safety of food.

  9. Establishment of loop-mediated isothermal amplification method for detection of milk allergen in foods%食品中牛奶过敏原成分LAMP检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    程晋霞; 曾静; 马丹; 魏海燕; 张海予

    2014-01-01

    Objective The loop-mediated isothermal amplification (LAMP) detection method was established for milk allergen testing and compared with the real-time PCR detection methods. Methods The cyt-b gene of bovine was used to design LAMP primers and then establish reaction system. The spe-cificity and sensitivity of LAMP were compared with real-time PCR detection method. Results The specificity of LAMP method was tested by 9 different milk and goat milk products. The results showed that the LAMP method was highly specific to milk. No cross-reaction was founded. The detection limit of LAMP reached 0.5%in base-material addition test which was consistent with the real-time PCR method. Through 69 practical food samples testing, the LAMP results were consistent with real-time PCR results. Conclusion The LAMP detection method of milk allergen established in this study is simple, economi-cal and reliable, which can effectively reduce the detection time and apply to the detection of milk aller-gen with good application prospects.%目的:建立食品过敏原牛奶成分LAMP(loop-mediated isothermal amplification)检测方法,并与实时荧光PCR(real-time PCR)检测方法比对。方法针对牛线粒体细胞色素b(cyt-b)基因设计LAMP引物并建立反应体系,在特异性和灵敏度方面与real-time PCR检测方法比对。结果本研究建立的LAMP方法检测9份不同品牌的牛奶和羊奶及其加工制品,没有出现交叉反应,具有良好的特异性。该方法的检测灵敏度为0.5%,与real-time PCR方法检测灵敏度相当。检测了69份实际样品,检测结果与real-time PCR检测结果一致。结论本研究建立的食品过敏原牛奶成分LAMP检测方法简单经济,检测结果可靠,可有效缩短检测时间,适用于过敏原牛奶成分的检测,具有良好的应用前景。

  10. 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.

  11. Real-time PCR probe optimization using design of experiments approach.

    Science.gov (United States)

    Wadle, S; Lehnert, M; Rubenwolf, S; Zengerle, R; von Stetten, F

    2016-03-01

    Primer and probe sequence designs are among the most critical input factors in real-time polymerase chain reaction (PCR) assay optimization. In this study, we present the use of statistical design of experiments (DOE) approach as a general guideline for probe optimization and more specifically focus on design optimization of label-free hydrolysis probes that are designated as mediator probes (MPs), which are used in reverse transcription MP PCR (RT-MP PCR). The effect of three input factors on assay performance was investigated: distance between primer and mediator probe cleavage site; dimer stability of MP and target sequence (influenza B virus); and dimer stability of the mediator and universal reporter (UR). The results indicated that the latter dimer stability had the greatest influence on assay performance, with RT-MP PCR efficiency increased by up to 10% with changes to this input factor. With an optimal design configuration, a detection limit of 3-14 target copies/10 μl reaction could be achieved. This improved detection limit was confirmed for another UR design and for a second target sequence, human metapneumovirus, with 7-11 copies/10 μl reaction detected in an optimum case. The DOE approach for improving oligonucleotide designs for real-time PCR not only produces excellent results but may also reduce the number of experiments that need to be performed, thus reducing costs and experimental times.

  12. Real-time image and video processing

    CERN Document Server

    Kehtarnavaz, Nasser

    2006-01-01

    This book presents an overview of the guidelines and strategies for transitioning an image or video processing algorithm from a research environment into a real-time constrained environment. Such guidelines and strategies are scattered in the literature of various disciplines including image processing, computer engineering, and software engineering, and thus have not previously appeared in one place. By bringing these strategies into one place, the book is intended to serve the greater community of researchers, practicing engineers, industrial professionals, who are interested in taking an im

  13. 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.

  14. Object detection in real-time

    Science.gov (United States)

    Solder, Ulrich; Graefe, Volker

    1991-03-01

    An algorithm working on monocular gray-scale image sequences for object detection combined with a road tracker is presented. This algorithm appropriate for the real-time demands of an autonomous car driving with speeds over 40 km/h may be used for triggering obstacle avoidance maneuvers such as coming to a safe stop automatically in front of an obstacle or following another car. Moving and static objects have been detected in real-world experiments on various types of roads even under unfavorable weather conditions. . Morgenthaler and

  15. 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.

  16. Real-time inclinometer using accelerometer MEMS

    CERN Document Server

    Hanto, D; Hermanto, B; Puranto, P; Handoko, L T

    2011-01-01

    A preliminary design of inclinometer for real-time monitoring system of soil displacement is proposed. The system is developed using accelerometer sensor with microelectromechanical system (MEMS) device. The main apparatus consists of a single MEMS sensor attached to a solid pipe and stucked pependicularly far away below the soil surface. The system utilizes small fractions of electrical signals from MEMS sensor induced by the pipe inclination due to soil displacements below the surface. It is argued that the system is accurate enough to detect soil displacements responsible for landslides, and then realizes a simple and low cost landslide early warning system.

  17. 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.

  18. 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....../output conformance serves as the notion of implementation correctness, essentially timed trace inclusion taking environment assumptions into account. Test cases can be generated offline and later executed, or they can be generated and executed online. For both approaches this chapter discusses how to specify test...... objectives, derive test sequences, apply these to the system under test, and assign a verdict....

  19. Real-time detection of gravitational microlensing

    CERN Document Server

    Pratt, M R; Axelrod, T S; Becker, A; Bennett, D P; Cook, K H; Freeman, K C; Griest, K; Guern, J A; Lehner, M; Marshall, S L; Peterson, B A; Quinn, P J; Reiss, D; Rodgers, A W; Stubbs, C W; Sutherland, W; Welch, D

    1995-01-01

    Real-time detection of microlensing has moved from proof of concept in 1994 to a steady stream of events this year. Global dissemination of these events by the MACHO and OGLE collaborations has made possible intensive photometric and spectroscopic followup from widely dispersed sites confirming the microlensing hypothesis. Improved photometry and increased temporal resolution from followup observations greatly increases the possibility of detecting deviations from the standard point-source, point-lens, inertial motion microlensing model. These deviations are crucial in understanding individual lensing systems by breaking the degeneracy between lens mass, position and velocity. We report here on GMAN (Global Microlensing Alert Network), the coordinated followup of MACHO alerts.

  20. Low cost real time interactive analysis system

    Science.gov (United States)

    Stetina, F.

    1988-01-01

    Efforts continue to develop a low cost real time interactive analysis system for the reception of satellite data. A multi-purpose ingest hardware software frame formatter was demonstrated for GOES and TIROS data and work is proceeding on extending the capability to receive GMS data. A similar system was proposed as an archival and analysis system for use with INSAT data and studies are underway to modify the system to receive the planned SeaWiFS (ocean color) data. This system was proposed as the core of a number of international programs in support of U.S. AID activities. Systems delivered or nearing final testing are listed.

  1. Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification.

    Science.gov (United States)

    Silva, Gonçalo; Bömer, Moritz; Nkere, Chukwuemeka; Kumar, P Lava; Seal, Susan E

    2015-09-15

    Yam mosaic virus (YMV; genus Potyvirus) is considered to cause the most economically important viral disease of yams (Dioscorea spp.) in West Africa which is the dominant region for yam production globally. Yams are a vegetatively propagated crop and the use of virus-free planting material forms an essential component of disease control. Current serological and PCR-based diagnostic methods for YMV are time consuming involving a succession of target detection steps. In this study, a novel assay for specific YMV detection is described that is based on isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA). This test has been shown to be reproducible and able to detect as little as 14 pg/μl of purified RNA obtained from an YMV-infected plant, a sensitivity equivalent to that obtained with the reverse transcription-polymerase chain reaction (RT-PCR) in current general use. The RT-exoRPA assay has, however, several advantages over the RT-PCR; positive samples can be detected in less than 30 min, and amplification only requires a single incubation temperature (optimum 37°C). These features make the RT-exoRPA assay a promising candidate for adapting into a field test format to be used by yam breeding programmes or certification laboratories. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. A new high-speed droplet-real-time polymerase chain reaction method can detect bovine respiratory syncytial virus in less than 10 min.

    Science.gov (United States)

    Uehara, Masayuki; Matsuda, Kazuyuki; Sugano, Mitsutoshi; Honda, Takayuki

    2014-03-01

    The polymerase chain reaction (PCR) has been widely used for diagnosis of infectious diseases of domestic animals. Rapid detection of respiratory pathogens of cattle is useful for making therapeutic decisions. Therefore, we developed a new genetic-based method called droplet-real-time PCR, which can detect bovine respiratory syncytial virus (BRSV) within 10 min. Our droplet-real-time PCR markedly reduced the reaction time of reverse transcription-PCR while maintaining the same sensitivity as conventional real-time PCR, and it can be used as a rapid assay for detection of BRSV. Furthermore, our method is potentially applicable for rapid diagnosis of almost all infectious diseases, including highly pathogenic avian influenza virus.

  3. Real time water chemistry monitoring and diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Gaudreau, T.M.; Choi, S.S. [EPRIsolutions, Palo Alto, CA (United States)

    2002-07-01

    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)

  4. Video Surveillance for Real Time Objects

    Directory of Open Access Journals (Sweden)

    N.R. Raajan

    2012-12-01

    Full Text Available In this study, we describe an integrated solution for video surveillance in a fortified environment. The focus of this study is on identification of real time objects on different environments. The system is composed of robust object detection module, which normally detects the presence of abandoned objects, concealed objects hidden inside the human clothing, objects in dark environment and performs image segmentation with the intention of facilitating human operator’s task of retrieving the cause of a buzzer. The abandoned objects are detected by image segmentation based on temporal rank order filtering. Image fusion technique which fuses a color visual image and a corresponding IR image for concealed objects in guarded environment and in some cases like dark environment heat signature can be used for detecting real time objects etc. In the clips of interest, the key frame is the one depicting a person leaving a dangerous object and is determined on the basis of a feature indicating the movement around the dangerous region.

  5. Real-time PCR in microfluidic devices

    Science.gov (United States)

    Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Hansen-Hagge, Thomas; Gärtner, Claudia

    2014-03-01

    A central method in a standard biochemical laboratory is represented by the polymerase chain reaction (PCR), therefore many attempts have been performed so far to implement this technique in lab-on-a-chip (LOC) devices. PCR is an ideal candidate for miniaturization because of a reduction of assay time and decreased costs for expensive bio-chemicals. In case of the "classical" PCR, detection is done by identification of DNA fragments electrophoretically separated in agarose gels. This method is meanwhile frequently replaced by the so-called Real-Time-PCR because here the exponential increase of amplificates can be observed directly by measurement of DNA interacting fluorescent dyes. Two main methods for on-chip PCRs are available: traditional "batch" PCR in chambers on a chip using thermal cycling, requiring about 30 minutes for a typical PCR protocol and continuous-flow PCR, where the liquid is guided over stationary temperature zones. In the latter case, the PCR protocol can be as fast as 5 minutes. In the presented work, a proof of concept is demonstrated for a real-time-detection of PCR products in microfluidic systems.

  6. Integrated real-time roof monitoring

    Institute of Scientific and Technical Information of China (English)

    SHEN Bao-tang; GUO Hua; KING Andrew

    2009-01-01

    CSIRO has recently developed a real-time roof monitoring system for under-ground coal mines and successfully tried the system in gate roads at Ulan Mine. The sys-tem integrated displacement monitoring, stress monitoring and seismic monitoring in one package. It included GEL multianchor extensometers, vibrating wire uniaxial stress meters, ESG seismic monitoring system with microseismic sensors and high-frequency AE sen-sors. The monitoring system automated and the data can be automatically collected by a central computer located in an underground nonhazardous area. The data are then trans-ferred to the surface via an optical fiber cable. The real-time data were accessed at any location with an Internet connection. The trials of the system in two tailgates at Ulan Mine demonstrate that the system is effective for monitoring the behavior and stability of read-ways during Iongwall mining. The continuous roof displacement/stress data show clear precursors of roof falls. The seismic data (event count and locations) provide insights into the roof failure process during roof fall.

  7. An efficient real time superresolution ASIC system

    Science.gov (United States)

    Reddy, Dikpal; Yue, Zhanfeng; Topiwala, Pankaj

    2008-04-01

    Superresolution of images is an important step in many applications like target recognition where the input images are often grainy and of low quality due to bandwidth constraints. In this paper, we present a real-time superresolution application implemented in ASIC/FPGA hardware, and capable of 30 fps of superresolution by 16X in total pixels. Consecutive frames from the video sequence are grouped and the registered values between them are used to fill the pixels in the higher resolution image. The registration between consecutive frames is evaluated using the algorithm proposed by Schaum et al. The pixels are filled by averaging a fixed number of frames associated with the smallest error distances. The number of frames (the number of nearest neighbors) is a user defined parameter whereas the weights in the averaging process are decided by inverting the corresponding smallest error distances. Wiener filter is used to post process the image. Different 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 the hardware, which gives us a fine balance between the number of bits and performance. The algorithm performs with real time speed with very impressive superresolution results.

  8. 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.

  9. ROBUST MEMORY MANAGEMENT USING REAL TIME CONCEPTS

    Directory of Open Access Journals (Sweden)

    V. Karthikeyan

    2014-01-01

    Full Text Available Memory fragmentation is the development of a large number of separate free areas. Memory management in embedded systems demand effective implementation schemes to avoid fragmentation problem. Existing dynamic memory allocation methods fail to suit real time system requirements. Execution times need to be deterministic and this motivates the need for allocation and deallocation to be done in constant time with the help of API’s. In µC/OS-II, memory allocation is semi-dynamic and a buddy allocator dynamic memory allocation algorithm is commonly used. Programmer must statically allocate a memory and partition the region using µC/OS-II Kernel API. Tasks can only request pre-partitioned fixed-size memory space from µC/OS-II. Memory allocation times are influenced by the ratio of memory allocation to the stack size of the task. In this research work memory management in LPC 1768 environment using RTOS µC/OS-II is proposed. Effective sharing of memory blocks among tasks co exists with partition. The captured results shows that the memory allocation and deallocation suits real time. The implication of the work is that, the necessity to reserve a static set of locations ahead of time is eliminated so that memory can be allocated at compile or design time.

  10. Using Quantitative Real-Time PCR to Detect MicroRNA Expression Profile During Embryonic Stem Cell Differentiation.

    Science.gov (United States)

    Pan, Xiaoping; Murashov, Alexander K; Stellwag, Edmund J; Zhang, Baohong

    2017-01-01

    Quantitative real-time PCR (qRT-PCR) is a reliable method to determine and monitor microRNA (miRNA) expression profiles in different cells, tissues, and organisms. Although there are several different strategies in performing qRT-PCR to determine miRNA expression, all of them have two steps in common: reverse transcription for obtaining cDNA from mature miRNA sequencing and standard real-time PCR for amplification of cDNA. This chapter demonstrates the application of quantitative real-time PCR for determining miRNA expression profiles during mouse embryonic stem cell differentiation. In this method, a mature miRNA sequence is first reverse transcribed into a long cDNA with a 40-50 nt miRNA-specific stem-loop primer; then, a standard real-time PCR reaction is performed for determining miRNA expression using a forward miRNA-specific primer and a universal reverse primer.

  11. Real-Time and Near Real-Time Data for Space Weather Applications and Services

    Science.gov (United States)

    Singer, H. J.; Balch, C. C.; Biesecker, D. A.; Matsuo, T.; Onsager, T. G.

    2015-12-01

    Space weather can be defined as conditions in the vicinity of Earth and in the interplanetary environment that are caused primarily by solar processes and influenced by conditions on Earth and its atmosphere. Examples of space weather are the conditions that result from geomagnetic storms, solar particle events, and bursts of intense solar flare radiation. These conditions can have impacts on modern-day technologies such as GPS or electric power grids and on human activities such as astronauts living on the International Space Station or explorers traveling to the moon or Mars. While the ultimate space weather goal is accurate prediction of future space weather conditions, for many applications and services, we rely on real-time and near-real time observations and model results for the specification of current conditions. In this presentation, we will describe the space weather system and the need for real-time and near-real time data that drive the system, characterize conditions in the space environment, and are used by models for assimilation and validation. Currently available data will be assessed and a vision for future needs will be given. The challenges for establishing real-time data requirements, as well as acquiring, processing, and disseminating the data will be described, including national and international collaborations. In addition to describing how the data are used for official government products, we will also give examples of how these data are used by both the public and private sector for new applications that serve the public.

  12. 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...

  13. Rapid screening of innate immune gene expression in zebrafish using reverse transcription - multiplex ligation-dependent probe amplification

    Directory of Open Access Journals (Sweden)

    Spaink Herman P

    2011-06-01

    Full Text Available Abstract Background With the zebrafish increasingly being used in immunology and infectious disease research, there is a need for efficient molecular tools to evaluate immune gene expression in this model species. RT-MLPA (reverse transcription - multiplex ligation-dependent probe amplification provides a sensitive and reproducible method, in which fluorescently labelled amplification products of unique lengths are produced for a defined set of target transcripts. The method employs oligonucleotide probes that anneal to adjacent sites on a target sequence and are then joined by a heat-stable ligase. Subsequently, multiplex PCR with universal primers gives rise to amplicons that can be analyzed with standard sequencing equipment and relative quantification software. Allowing the simultaneous quantification of around 40 selected markers in a one-tube assay, RT-MLPA is highly useful for high-throughput screening applications. Findings We employed a dual-colour RT-MLPA probe design for chemical synthesis of probe pairs for 34 genes involved in Toll-like receptor signalling, transcriptional activation of the immune response, cytokine and chemokine production, and antimicrobial defence. In addition, six probe pairs were included for reference genes unaffected by infections in zebrafish. First, we established assay conditions for adult zebrafish infected with different strains of Mycobacterium marinum causing acute and chronic disease. Addition of competitor oligonucleotides was required to achieve peak heights in a similar range for genes with different expression levels. For subsequent analysis of embryonic samples it was necessary to adjust the amounts of competitor oligonucleotides, as the expression levels of several genes differed to a large extent between adult and embryonic tissues. Assay conditions established for one-day-old Salmonella typhimurium-infected embryos could be transferred without further adjustment to five-day-old M. marinum

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

    Science.gov (United States)

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

    2016-01-08

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

  15. Feedback as real-time constructions

    DEFF Research Database (Denmark)

    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...... instant it takes place. This article argues for a clear distinction between the timing of communicative events, such as responses that are provided as help for feedback constructions, and the feedback construction itself as an event in a psychic system. Although feedback is described as an internal......, system-relative construction, different teaching environments offer diverse conditions for feedback constructions. The final section of this article explores this idea with the help of examples from both synchronous oral interaction and asynchronous text-based interaction mediated by digital media....

  16. 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...... 3D sensor ubiquitous and have resulted in a vast amount of new applications and methods. However, such low-cost sensors are generally limited in their accuracy and precision, making them unsuitable for e.g. accurate tracking and pose estimation. With recent improvements in projector technology......]. A high performance flexible open source software toolkit is presented [Contribution C], which makes real time scanning possible on commodity hardware. Further, an approach is presented to correct for motion artifacts in dynamic scenes [Contribution E]. An application for such systems is presented...

  17. Real-time, face recognition technology

    Energy Technology Data Exchange (ETDEWEB)

    Brady, S.

    1995-11-01

    The Institute for Scientific Computing Research (ISCR) at Lawrence Livermore National Laboratory recently developed the real-time, face recognition technology KEN. KEN uses novel imaging devices such as silicon retinas developed at Caltech or off-the-shelf CCD cameras to acquire images of a face and to compare them to a database of known faces in a robust fashion. The KEN-Online project makes that recognition technology accessible through the World Wide Web (WWW), an internet service that has recently seen explosive growth. A WWW client can submit face images, add them to the database of known faces and submit other pictures that the system tries to recognize. KEN-Online serves to evaluate the recognition technology and grow a large face database. KEN-Online includes the use of public domain tools such as mSQL for its name-database and perl scripts to assist the uploading of images.

  18. Near real-time stereo vision system

    Science.gov (United States)

    Anderson, Charles H. (Inventor); Matthies, Larry H. (Inventor)

    1993-01-01

    The apparatus for a near real-time stereo vision system for use with a robotic vehicle is described. The system is comprised of two cameras mounted on three-axis rotation platforms, image-processing boards, a CPU, and specialized stereo vision algorithms. Bandpass-filtered image pyramids are computed, stereo matching is performed by least-squares correlation, and confidence ranges are estimated by means of Bayes' theorem. In particular, Laplacian image pyramids are built and disparity maps are produced from the 60 x 64 level of the pyramids at rates of up to 2 seconds per image pair. The first autonomous cross-country robotic traverses (of up to 100 meters) have been achieved using the stereo vision system of the present invention with all computing done onboard the vehicle. The overall approach disclosed herein provides a unifying paradigm for practical domain-independent stereo ranging.

  19. 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.

  20. Real time speech formant analyzer and display

    Energy Technology Data Exchange (ETDEWEB)

    Holland, George E. (Ames, IA); Struve, Walter S. (Ames, IA); Homer, John F. (Ames, IA)

    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.

  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. 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.

  3. 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, introdu......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...

  4. Real-time forecasts of dengue epidemics

    Science.gov (United States)

    Yamana, T. K.; Shaman, J. L.

    2015-12-01

    Dengue is a mosquito-borne viral disease prevalent in the tropics and subtropics, with an estimated 2.5 billion people at risk of transmission. In many areas with endemic dengue, disease transmission is seasonal but prone to high inter-annual variability with occasional severe epidemics. Predicting and preparing for periods of higher than average transmission is a significant public health challenge. Here we present a model of dengue transmission and a framework for optimizing model simulations with real-time observational data of dengue cases and environmental variables in order to generate ensemble-based forecasts of the timing and severity of disease outbreaks. The model-inference system is validated using synthetic data and dengue outbreak records. Retrospective forecasts are generated for a number of locations and the accuracy of these forecasts is quantified.

  5. Terrestrial Real-Time Volcano Monitoring

    Science.gov (United States)

    Franke, M.

    2013-12-01

    As volcano monitoring involves more and different sensors from seismic to GPS receivers, from video and thermal cameras to multi-parameter probes measuring temperature, ph values and humidity in the ground and the air, it becomes important to design real-time networks that integrate and leverage the multitude of available parameters. In order to do so some simple principles need to be observed: a) a common time base for all measurements, b) a packetized general data communication protocol for acquisition and distribution, c) an open and well documented interface to the data permitting standard and emerging innovative processing, and d) an intuitive visualization platform for scientists and civil defense personnel. Although mentioned as simple principles, the list above does not necessarily lead to obvious solutions or integrated systems, which is, however, required to take advantage of the available data. Only once the different data streams are put into context to each other in terms of time and location can a broader view be obtained and additional information extracted. The presentation is a summary of currently available technologies and how they can achieve the goal of an integrated real-time volcano monitoring system. A common time base are standard for seismic and GPS networks. In different projects we extended this to video feeds and time-lapse photography. Other probes have been integrated with vault interface enclosures (VIE) as used in the Transportable Array (TA) of the USArray. The VIE can accommodate the sensors employed in volcano monitoring. The TA has shown that Antelope is a versatile and robust middleware. It provides the required packetized general communication protocol that is independent from the actual physical communication link leaving the network design to adopt appropriate and possible hybrid solutions. This applies for the data acquisition and the data/information dissemination providing both a much needed collaboration platform, as

  6. 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.

  7. A Flexible Real-Time Architecture

    Energy Technology Data Exchange (ETDEWEB)

    WICKSTROM,GREGORY L.

    2000-08-17

    Assuring hard real-time characteristics of I/O associated with embedded software is often a difficult task. Input-Output related statements are often intermixed with the computational code, resulting in I/O timing that is dependent on the execution path and computational load. One way to mitigate this problem is through the use of interrupts. However, the non-determinism that is introduced by interrupt driven I/O may be so difficult to analyze that it is prohibited in some high consequence systems. This paper describes a balanced hardware/software solution to obtain consistent interrupt-free I/O timing, and results in software that is much more amenable to analysis.

  8. Real-time color holographic interferometry

    Science.gov (United States)

    Desse, Jean-Michel; Albe, Felix; Tribillon, Jean-Louis

    2002-09-01

    A new optical technique based on real-time color holographic interferometry has been developed for analyzing unsteady aerodynamic wakes in fluid mechanics or for measuring displacements and deformations in solid mechanics. The technique's feasibility is demonstrated here. It uses three coherent wavelengths produced simultaneously by a cw laser (mixed argon and krypton). Holograms are recorded on single-layer panchromatic silver halide (Slavich PFG 03C) plates. Results show the optical setup can be adjusted to obtain a uniform background color. The interference fringe pattern visualized is large and colored and exhibits a single central white fringe, which makes the zero order of the interferogram easy to identify. An application in a subsonic wind tunnel is presented, in which the unsteady wake past a cylinder is recorded at high rate.

  9. Wi-Fi real time location systems

    Science.gov (United States)

    Doll, Benjamin A.

    This thesis objective was to determine the viability of utilizing an untrained Wi-Fi. real time location system as a GPS alternative for indoor environments. Background. research showed that GPS is rarely able to penetrate buildings to provide reliable. location data. The benefit of having location information in a facility and how they might. be used for disaster or emergency relief personnel and their resources motivated this. research. A building was selected with a well-deployed Wi-Fi infrastructure and its. untrained location feature was used to determine the distance between the specified. test points and the system identified location. It was found that the average distance. from the test point throughout the facility was 14.3 feet 80% of the time. This fell within. the defined viable range and supported that an untrained Wi-Fi RTLS system could be a. viable solution for GPS's lack of availability indoors.

  10. Real-Time Imaging of Quantum Entanglement

    CERN Document Server

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

    2013-01-01

    Quantum Entanglement - correlations between at least two systems that are stronger than classically explainable - is widely regarded as one of the most prominent features of quantum mechanics and quantum information science. Although, the creation of entanglement between two systems has become possible in laboratories, it has been out of the grasp of one of the most natural ways to investigate nature: direct visual observation. Here we show that modern imaging technology, namely a triggered intensified charge coupled device (ICCD) camera, is fast and sensitive enough to image in real-time the influence of the measurement of one photon on its entangled partner. To demonstrate the non-classicality of the measurements quantitatively from the registered intensity we develop a novel method to statistically analyze the image and precisely quantify the number of photons within a certain region. In addition, we show the high flexibility of our experimental setup in creating any desired spatial-mode entanglement, even...

  11. 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.

  12. Real Time Eye Template Detection and Tracking

    Directory of Open Access Journals (Sweden)

    Richa Mehta

    2012-06-01

    Full Text Available There has been a growing interest in the field of facial expression recognition especially in the last two decades. An example of such a system is the improvement of driver carefulness and accident reduction. The driver’s face is tracked while he is driving and he is warned if there seems to be an alerting fact that can result in an accident such as sleepy eyes, or looking out of the road. Furthermore, with a facial feature tracker, it becomes possible to play a synthesized avatar so that it imitates the expressions of the performer. Human-Computer Interaction (HCI systems may also be enriched by a facial feature tracker. For a user who is incapable of using her hands, a facial expression controller may be a solution to send limited commands to a computer. Eye blinking is one of the prominent areas to solve many real world problems. The process of blink detection consists of two phases. These are eye tracking followed by detection of blink. The work that has been carried out for eye tracking only is not suitable for eye blink detection. Therefore some approaches had been proposed for eye tracking along with eyes blink detection. In this thesis, real time implementation is done to count number of eye blinks in an image sequence. At last after analyzing all these approaches some of the parameters we obtained on which better performance of eye blink detection algorithm depend. This project focuses on automatic eye blink detection in real time. The aim of this thesis is to count the number of eye blinks in a video. This project will be performed on a video database of the facial expressions.

  13. Towards real time speckle controlled retinal photocoagulation

    Science.gov (United States)

    Bliedtner, Katharina; Seifert, Eric; Stockmann, Leoni; Effe, Lisa; Brinkmann, Ralf

    2016-03-01

    Photocoagulation is a laser treatment widely used for the therapy of several retinal diseases. Intra- and inter-individual variations of the ocular transmission, light scattering and the retinal absorption makes it impossible to achieve a uniform effective exposure and hence a uniform damage throughout the therapy. A real-time monitoring and control of the induced damage is highly requested. Here, an approach to realize a real time optical feedback using dynamic speckle analysis is presented. A 532 nm continuous wave Nd:YAG laser is used for coagulation. During coagulation, speckle dynamics are monitored by a coherent object illumination using a 633nm HeNe laser and analyzed by a CMOS camera with a frame rate up to 1 kHz. It is obvious that a control system needs to determine whether the desired damage is achieved to shut down the system in a fraction of the exposure time. Here we use a fast and simple adaption of the generalized difference algorithm to analyze the speckle movements. This algorithm runs on a FPGA and is able to calculate a feedback value which is correlated to the thermal and coagulation induced tissue motion and thus the achieved damage. For different spot sizes (50-200 μm) and different exposure times (50-500 ms) the algorithm shows the ability to discriminate between different categories of retinal pigment epithelial damage ex-vivo in enucleated porcine eyes. Furthermore in-vivo experiments in rabbits show the ability of the system to determine tissue changes in living tissue during coagulation.

  14. DDX3 DEAD-Box RNA helicase inhibits hepatitis B virus reverse transcription by incorporation into nucleocapsids.

    Science.gov (United States)

    Wang, Haifeng; Kim, Seahee; Ryu, Wang-Shick

    2009-06-01

    Viruses utilize host factors in many steps of their life cycles. Yet, little is known about host factors that contribute to the life cycle of hepatitis B virus (HBV), which replicates its genome by reverse transcription. To identify host factors that contribute to viral reverse transcription, we sought to identify cellular proteins that interact with HBV polymerase (Pol) by using affinity purification coupled with mass spectrometry. One of the HBV Pol-interacting host factors identified was DDX3 DEAD-box RNA helicase, which unwinds RNA in an ATPase-dependent manner. Recently, it was shown that DDX3 is essential for both human immunodeficiency virus and hepatitis C virus infection. In contrast, we found that the ectopic expression of DDX3 led to significantly reduced viral DNA synthesis. The DDX3-mediated inhibition of viral DNA synthesis did not affect RNA encapsidation, a step prior to reverse transcription, and indicated that DDX3 inhibits HBV reverse transcription. Mutational analysis revealed that mutant DDX3 with an inactive ATPase motif, but not that with an inactive RNA helicase motif, failed to inhibit viral DNA synthesis. Our interpretation is that DDX3 inhibits viral DNA synthesis at a step following ATP hydrolysis but prior to RNA unwinding. Finally, OptiPrep density gradient analysis revealed that DDX3 was incorporated into nucleocapsids, suggesting that DDX3 inhibits viral reverse transcription following nucleocapsid assembly. Thus, DDX3 represents a novel host restriction factor that limits HBV infection.

  15. An Efficient Secure Real-Time Concurrency Control Protocol

    Institute of Scientific and Technical Information of China (English)

    XIAO Yingyuan; LIU Yunsheng; CHEN Xiangyang

    2006-01-01

    Secure real-time databases must simultaneously satisfy two requirements in guaranteeing data security and minimizing the missing deadlines ratio of transactions. However, these two requirements can conflict with each other and achieve one requirement is to sacrifice the other. This paper presents a secure real-time concurrency control protocol based on optimistic method. The concurrency control protocol incorporates security constraints in a real-time optimistic concurrency control protocol and makes a suitable tradeoff between security and real-time requirements by introducing secure influence factor and real-time influence factor. The experimental results show the concurrency control protocol achieves data security without degrading real-time performance significantly.

  16. Counterselection of prokaryotic ribosomal RNA during reverse transcription using non-random hexameric oligonucleotides.

    Science.gov (United States)

    Gonzalez, J M; Robb, F T

    2007-12-01

    Ribosomal RNA (rRNA) is the major component in total RNA extracts, interfering with the synthesis of cDNA corresponding to messenger RNA (mRNA). In this study, we present a novel strategy for selectively discriminating against rRNA and favoring mRNA from prokaryotes during synthesis of cDNA by reverse transcriptase. Our technique is based on the fact that rRNA sequences, in many species, are G+C rich relative to the genome at large, and highly conserved among prokaryotes. The sequence TTTT is therefore rarely found in rRNA sequences. However, TTTT priming sites are found at a much higher frequency in protein-encoding gene sequences. We designed specific hexamers (HD/DHTTTT) to prime reverse transcription reactions resulting in a selective synthesis of cDNA corresponding to mRNA from prokaryotic total RNA extractions.

  17. 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.

  18. 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.

  19. Rapid,sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification

    Institute of Scientific and Technical Information of China (English)

    高宏伟; 李富花; 张晓军; 王兵; 相建海

    2010-01-01

    Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming.The rapid detection and identification of V.anguillarum,and other pathogens that infect marine organisms,is crucial to effective disease management.In this study,we developed a loop-mediated amplification (LAMP) assay to detect V.anguillarum in an hour in a single tube without the need for thermal cycling.Conserved regions of the metalloproteinase (empA) gene of V.anguillarum served as the...

  20. 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

  1. 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 Mg(2+)), 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 (Mg(2+)) 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.

  2. 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

  3. CRANS - CONFIGURABLE REAL-TIME ANALYSIS SYSTEM

    Science.gov (United States)

    Mccluney, K.

    1994-01-01

    In a real-time environment, the results of changes or failures in a complex, interconnected system need evaluation quickly. Tabulations showing the effects of changes and/or failures of a given item in the system are generally only useful for a single input, and only with regard to that item. Subsequent changes become harder to evaluate as combinations of failures produce a cascade effect. When confronted by multiple indicated failures in the system, it becomes necessary to determine a single cause. In this case, failure tables are not very helpful. CRANS, the Configurable Real-time ANalysis System, can interpret a logic tree, constructed by the user, describing a complex system and determine the effects of changes and failures in it. Items in the tree are related to each other by Boolean operators. The user is then able to change the state of these items (ON/OFF FAILED/UNFAILED). The program then evaluates the logic tree based on these changes and determines any resultant changes to other items in the tree. CRANS can also search for a common cause for multiple item failures, and allow the user to explore the logic tree from within the program. A "help" mode and a reference check provide the user with a means of exploring an item's underlying logic from within the program. A commonality check determines single point failures for an item or group of items. Output is in the form of a user-defined matrix or matrices of colored boxes, each box representing an item or set of items from the logic tree. Input is via mouse selection of the matrix boxes, using the mouse buttons to toggle the state of the item. CRANS is written in C-language and requires the MIT X Window System, Version 11 Revision 4 or Revision 5. It requires 78K of RAM for execution and a three button mouse. It has been successfully implemented on Sun4 workstations running SunOS, HP9000 workstations running HP-UX, and DECstations running ULTRIX. No executable is provided on the distribution medium; however

  4. RTMOD: Real-Time MODel evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Graziani, G; Galmarini, S. [Joint Research centre, Ispra (Italy); Mikkelsen, T. [Risoe National Lab., Wind Energy and Atmospheric Physics Dept. (Denmark)

    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

  5. HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells

    Directory of Open Access Journals (Sweden)

    Ennifar Eric

    2008-06-01

    Full Text Available Abstract Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1 is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT, is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex – the reverse transcription complex (RTC – consisting of viral genomic RNA associated with viral proteins (including RT and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein – the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF. We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the C-terminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT.

  6. 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

    2017-09-04

    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. Published by Elsevier Inc.

  7. Use of Real-time RT-PCR Analysis for mRNA Expression of Tobacco Ferritin Gene (NtFer1)

    Institute of Scientific and Technical Information of China (English)

    JIANG Tingbo; LI Fengjuan; YANG Chuanping

    2006-01-01

    To understand the use of real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) for detecting the relative abundance of mRNA, the expression of a tobacco ferritin gene (NtFer1) was detected by Northern blot and real-time RT-PCR. The results indicated that both of the two methods were able to detect mRNA expression of NtFer1 clearly and similarly, namely NtFer1 expression was responsive to iron-overload, and the abundance of NtFer1 mRNA was greatly increased after iron loaded for 6 h. To compare the effect and sensitivity of two methods, results revealed that Northern blot need 30 μg of total RNA and at least 3 days for the total protocol performance, whereas real-time RT-PCR only need 2 μg of total RNA and 1.5 h. The real-time RT-PCR is rather sensitive and effective than Northern blot. Real-time RT-PCR analysis can be used to rapidly detect the relative abundance of mRNA expression instead of Northern blot analysis.

  8. Strategic Approach To Produce Low-Cost, Efficient, and Stable Competitive Internal Controls for Detection of RNA Viruses by Use of Reverse Transcription-PCR▿

    Science.gov (United States)

    Villanova, Gabriela V.; Gardiol, Daniela; Taborda, Miguel A.; Reggiardo, Virginia; Tanno, Hugo; Rivadeneira, Emilia D.; Perez, Germán R.; Giri, Adriana A.

    2007-01-01

    Molecular diagnostics based on reverse transcription (RT)-PCR are routinely complicated by the lack of stable internal controls, leading to falsely negative results. We describe a strategy to produce a stable competitive internal control (CIC) based on a Qβ phage derivative (recombinant Qβ [rQβ]) bearing primers KY78 and KY80, which are widely used in the detection of hepatitis C virus (HCV). rQβ was RNase resistant and stable at 4°C for 452 days in SM medium (0.1 M NaCl, 8 mM MgSO4·7H2O, 50 mM Tris HCl [pH 7.5], 2% gelatin) and for 125 days after lyophilization and reconstitution. rQβ performance as a CIC was evaluated. rQβ was added to HCV-positive samples, followed by RNA extraction and a CIC-HCV RT-PCR assay. This method combines RT-PCR, liquid hybridization with nonradioactive probes, and enzyme immunoanalysis. No influence of the CIC on qualitative HCV detection was observed independently of viral load, and results had high concordance with those of commercial kits. In conclusion, we describe a versatile, low-cost alternative strategy to armored RNA technology that can be adapted for detection or real-time applications of any RNA target. Moreover, the CIC reported here is an essential reagent for HCV screening in blood banks in resource-limited settings. PMID:17699653

  9. Molecular detection of the clostridia in an anaerobic biohydrogen fermentation system by hydrogenase mRNA-targeted reverse transcription-PCR.

    Science.gov (United States)

    Chang, Jui-Jen; Chen, Wei-En; Shih, Shiou-Yun; Yu, Sian-Jhong; Lay, Jiunn-Jyi; Wen, Fu-Shyan; Huang, Chieh-Chen

    2006-05-01

    Molecular biological approaches were developed to monitor the potential biohydrogen-producing clostridia in an anaerobic semisolid fermentation system that used brewery yeast waste as the fermentation substrate. The denaturing gradient gel electrophoresis with 16S rDNA gene-targeted polymerase chain reaction (PCR) analysis was employed to confirm the existence of clostridia in the system. Remarkably, reproducible nucleotide sequences of clostridia were obtained from different hydrogen production stages by using hydrogenase gene-targeted reverse transcription (RT)-PCR. These RNA-based information suggested that the predominant hydrogen-producing strains possess either a specific Clostridium pasteurianum-like or a specific Clostridium saccharobutylicum-like hydrogenase sequence. Comparison of the hydrogenase gene-targeted sequence profiles between PCR and RT-PCR revealed that the specific C. pasteurianum-like hydrogenase harboring bacterial strains were dominant in both mRNA and bacterial population level. On the other hand, the specific C. saccharobutylicum-like hydrogenase harboring strains expressed high level of hydrogenase mRNA but may not be dominant in population. Furthermore, quantitative real-time RT-PCR analysis showed the expression pattern of the clostridial hydrogenase mRNA and may serve as an activity index for the system.

  10. Simultaneous detection of five enteric viruses associated with gastroenteritis by use of a PCR assay: a single real-time multiplex reaction and its clinical application.

    Science.gov (United States)

    Jiang, Yixiang; Fang, Lin; Shi, Xiaolu; Zhang, Hailong; Li, Yinghui; Lin, Yiman; Qiu, Yaqun; Chen, Qingliang; Li, Hui; Zhou, Li; Hu, Qinghua

    2014-04-01

    We developed a highly sensitive reverse transcription and multiplex real-time PCR (rtPCR) assay that can identify five viruses, including six genogroups, in a single reaction: norovirus genogroups I and II; sapovirus genogroups I, II, IV, and V; human rotavirus A; adenovirus serotypes 40 and 41; and human astrovirus. In comparison to monoplex rtPCR assays, the sensitivities and specificities of the multiplex rtPCR ranged from 75% to 100% and from 99% to 100%, respectively, evaluated on 812 clinical stool specimens.

  11. Application of real-time GPS to earthquake early warning

    National Research Council Canada - National Science Library

    Richard M. Allen; Alon Ziv

    2011-01-01

      Real-time GPS can provide static-offset observations during an earthquake Real-time GPS provides a robust constrain on magnitude for warnings GPS networks should be used as a companion to seismic...

  12. 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...

  13. 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.

  14. Real-time flexible preventive maintenance scheduling.

    Science.gov (United States)

    Kendall, E B; Cronk, J W; White, R N

    1993-01-01

    There are still obstacles to overcome as we enter the programming phase of this project. We envision an automated system, similar to an expert system, that performs the interval/history analysis and makes the changes. Initially a field will need to be added to the inventory to denote whether a device belongs to one of the previously described groups that are exempt from interval changes. An intermediate step will be the formatting of a periodic report showing equipment that meets the change criteria as described in the two rules. For now, the actual changes would be reviewed and made by our management and technical staff. This report would be retained as documentation of the basis for each change, for our own benefit and to meet JCAHO requirements. We are still discussing whether the repair count should include all repairs (user error, abuse, unpredictable failure, etc.) or just those that are "significant and preventable" and could have been averted by PM. This is perhaps a question whose answer might vary from hospital to hospital, depending upon size and patient mix. With more emphasis being placed on process outcomes, on quality of work, and on getting the most benefit for our efforts, we believe our flexible, real-time PM scheduling program is a major step in the right direction. It is outcome-driven and it focuses resources where they are needed the most.

  15. Real time model for public transportation management

    Directory of Open Access Journals (Sweden)

    Ireneusz Celiński

    2014-03-01

    Full Text Available Background: The article outlines managing a public transportation fleet in the dynamic aspect. There are currently many technical possibilities of identifying demand in the transportation network. It is also possible to indicate legitimate basis of estimating and steering demand. The article describes a general public transportation fleet management concept based on balancing demand and supply. Material and methods: The presented method utilizes a matrix description of demand for transportation based on telemetric and telecommunication data. Emphasis was placed mainly on a general concept and not the manner in which data was collected by other researchers.  Results: The above model gave results in the form of a system for managing a fleet in real-time. The objective of the system is also to optimally utilize means of transportation at the disposal of service providers. Conclusions: The presented concept enables a new perspective on managing public transportation fleets. In case of implementation, the project would facilitate, among others, designing dynamic timetables, updated based on observed demand, and even designing dynamic points of access to public transportation lines. Further research should encompass so-called rerouting based on dynamic measurements of the characteristics of the transportation system.

  16. Recommendations for Real-Time Speech MRI

    Science.gov (United States)

    Lingala, Sajan Goud; Sutton, Brad P.; Miquel, Marc E.; Nayak, Krishna S.

    2016-01-01

    Real-time magnetic resonance imaging (RT-MRI) is being increasingly used for speech and vocal production research studies. Several imaging protocols have emerged based on advances in RT-MRI acquisition, reconstruction, and audio-processing methods. This review summarizes the state-of-the-art, discusses technical considerations, and provides specific guidance for new groups entering this field. We provide recommendations for performing RT-MRI of the upper airway. This is a consensus statement stemming from the ISMRM-endorsed Speech MRI summit held in Los Angeles, February 2014. A major unmet need identified at the summit was the need for consensus on protocols that can be easily adapted by researchers equipped with conventional MRI systems. To this end, we provide a discussion of tradeoffs in RT-MRI in terms of acquisition requirements, a priori assumptions, artifacts, computational load, and performance for different speech tasks. We provide four recommended protocols and identify appropriate acquisition and reconstruction tools. We list pointers to open-source software that facilitate implementation. We conclude by discussing current open challenges in the methodological aspects of RT-MRI of speech. PMID:26174802

  17. Real-time optoacoustic monitoring of stroke

    Science.gov (United States)

    Kneipp, Moritz; Turner, Jake; Hambauer, Sebastian; Krieg, Sandro M.; Lehmberg, Jens; Lindauer, Ute; Razansky, Daniel

    2014-03-01

    Characterizing disease progression and identifying possible therapeutic interventions in stroke is greatly aided by the use of longitudinal function imaging studies. In this study, we investigate the applicability of real-time multispectral optoacoustic tomography (MSOT) as a tool for non-invasive monitoring of the progression of stroke in the whole brain. The middle cerebral artery occlusion (MCAO) method was used to induce stroke. Mice were imaged under isoflurane anesthesia preoperatively and at several time points during and after the 60-minute occlusion. The animals were sacrificed after 24 hours and their excised brains frozen at -80°C for sectioning. The cryosection were stained using H&E staining to identify the ischemic lesion. Major vessels are readily identifiable in the whole mouse head in the in vivo optoacoustic scans. During ischemia, a reduction in cerebral blood volume is detectable in the cortex. Post ischemia, spectral unmixing of the optoacoustic signals shows an asymmetry of the deoxygenated hemoglobin in the hemisphere affected by MCAO. This hypoxic area was mainly located around the boundary of the ischemic lesion and was therefore identified as the ischemic penumbra. Non-invasive functional MSOT imaging is able to visualize the hypoxic penumbra in brains affected by stroke. Stopping the spread of the infarct area and revitalizing the penumbra is central in stroke research, this new imaging technique may therefore prove to be a valuable tool in the monitoring and developing new treatments.

  18. Real-time Detection of Locked Modes

    Science.gov (United States)

    Angelini, S.; Granetz, R. S.; Wolfe, S. M.

    2007-11-01

    Disruptions are one of the largest problems facing tokamaks. In a large-scale experiment such as ITER, disruptions would cause crippling damage and severe setbacks in experimentation. One method for disruption mitigation involves the use of a gas jet which has been tested on both normally running plasmas and vertical displacement events (VDEs) on Alcator C-Mod. In both cases, the jet was successful in mitigating disruption effects. The gas jet has not yet been tested on other types of disruptions. Locked-mode major disruptions are easily created in C-Mod and could be used to test the effectiveness of the gas jet as a mitigation method if the jet could be fired early enough. It has been empirically observed that the electron cyclotron emissions (ECE) signal displays a flattening of the normally-present sawteeth before the current quench occurs in certain locked-mode major disruptions. A procedure is being written which will detect the ECE flattening by reading changes in the standard deviation of the signal. This procedure will be programmed into the digital plasma control system (DPCS) for real-time testing.

  19. Real-time DIRCM system modeling

    Science.gov (United States)

    Petersson, Mikael

    2004-12-01

    Directed infrared countermeasures (DIRCM) play an increasingly important role in electronic warfare to counteract threats posed by infrared seekers. The usefulness and performance of such countermeasures depend, for example, on atmospheric conditions (attenuation and turbulence) and platform vibrations, causing pointing and tracking errors for the laser beam and reducing the power transferred to the seeker aperture. These problems make it interesting to simulate the performance of a DIRCM system in order to understand how easy or difficult it is to counteract an approaching threat and evaluate limiting factors in various situations. This paper describes a DIRCM model that has been developed, including atmospheric effects such as attenuation and turbulence as well as closed loop tracking algorithms, where the retro reflex of the laser is used for the pointing control of the beam. The DIRCM model is part of a large simulation framework (EWSim), which also incorporates several descriptions of different seekers (e.g. reticle, rosette, centroid, nutating cross) and models of robot dynamics. Effects of a jamming laser on a specific threat can be readily verified by simulations within this framework. The duel between missile and countermeasure is simulated in near real-time and visualized graphically in 3D. A typical simulation with a reticle seeker jammed by a modulated laser is included in the paper.

  20. Real-Time Principal-Component Analysis

    Science.gov (United States)

    Duong, Vu; Duong, Tuan

    2005-01-01

    A recently written computer program implements dominant-element-based gradient descent and dynamic initial learning rate (DOGEDYN), which was described in Method of Real-Time Principal-Component Analysis (NPO-40034) NASA Tech Briefs, Vol. 29, No. 1 (January 2005), page 59. To recapitulate: DOGEDYN is a method of sequential principal-component analysis (PCA) suitable for such applications as data compression and extraction of features from sets of data. In DOGEDYN, input data are represented as a sequence of vectors acquired at sampling times. The learning algorithm in DOGEDYN involves sequential extraction of principal vectors by means of a gradient descent in which only the dominant element is used at each iteration. Each iteration includes updating of elements of a weight matrix by amounts proportional to a dynamic initial learning rate chosen to increase the rate of convergence by compensating for the energy lost through the previous extraction of principal components. In comparison with a prior method of gradient-descent-based sequential PCA, DOGEDYN involves less computation and offers a greater rate of learning convergence. The sequential DOGEDYN computations require less memory than would parallel computations for the same purpose. The DOGEDYN software can be executed on a personal computer.

  1. 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.

  2. 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.

  3. Execution of a High Level Real-Time Language

    OpenAIRE

    Luqi; Berzins, Valdis

    1988-01-01

    Prototype System Description Language (PSDL) is a high level real-time language with special features for hard real-time system specification and design. It can be used to firm up requirements through execution of its software prototypes The language is designed based on a real-time model merging data and control flow and its implementation is beyond conventional compiler technology because of the need to meet real-time constraints. In this paper we describe and illustrate our research result...

  4. A Real-Time Linux for Multicore Platforms

    Science.gov (United States)

    2013-12-20

    Multicore Platforms, 25th Euromicro Conference on Real-TimeSystems. 09-JUL-13, . : , Jeremy Erickson, James Anderson. Reducing Tardiness Under Global...Baruah. "Improved tardiness bounds for Global EDF," Proceedings of the EuroMicro Conference on Real-Time Systems, Brussels, Belgium, IEEE Computer...missed. In a soft real-time application, some deadline tardiness is permissible. In the definition of "soft real-time" that we have focused on

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

    Science.gov (United States)

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

    2014-09-01

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

  6. Survey of real-time processing systems for big data

    DEFF Research Database (Denmark)

    Liu, Xiufeng; Lftikhar, Nadeem; Xie, Xike

    2014-01-01

    for big data; Its open-source implementation such as Hadoop has become the de-facto standard for processing big data, however, Hadoop has the limitation of supporting real-time updates. The improvements in Hadoop for the real-time capability, and the other alternative real-time frameworks have been...

  7. Real-time PCR detection of host-mediated cyanophage gene transcripts during infection of a natural Microcystis aeruginosa population.

    Science.gov (United States)

    Yoshida, Mitsuhiro; Yoshida, Takashi; Yoshida-Takashima, Yukari; Kashima, Aki; Hiroishi, Shingo

    2010-01-01

    The aim of this study was to develop a quantitative real-time reverse transcription-PCR (real-time RT-PCR) assay to detect and quantify mRNA of cyanophages within infected Microcystis aeruginosa cells in a freshwater pond. Laboratory-based data showed that the relative abundance of the cyanophage g91 mRNA within host cells increased before cyanophage numbers increased in culture. This transcriptional pattern indicated the kinetics of the viral infection suggesting the real-time RT-PCR method to be a potential tool for environmental monitoring of cyanophage infections. In this field survey, the numbers of infected M. aeruginosa cell populations estimated from cyanophage numbers were low at 0.01-2.9 cells mL(-1). The highest relative abundance of phage g91 RNA (10(-2) per rnpB transcript) was at about the same levels of expression as laboratory-based growth data for Ma-LMM01 (estimated density of infected host cells: 10(5) cells mL(-1)); and was observed when cyanophage numbers rapidly increased (as well as a decrease in host cell numbers). Quantification of cyanophage numbers is important to understand ecological relationships between the phage and its hosts. Our data suggest the quantification of phage gene transcripts within a natural host cell population to be a strong tool for investigating the quantitative effects of phage lysis during infection of the host population.

  8. Quantitative detection of Cucumber vein yellowing virus in susceptible and partially resistant plants using real-time PCR.

    Science.gov (United States)

    Picó, Belén; Sifres, Alicia; Nuez, Fernando

    2005-09-01

    A method for the detection of Cucumber vein yellowing virus (CVYV) that combines reverse transcription with real-time PCR (SYBR((R)) Green chemistry) was developed using specific primers designed from a nucleotide sequence of the RNA polymerase gene (NIb) conserved among all the available CVYV strains. This method provided a linear assay over five to six orders of magnitude and reproducibly detected titres as low as 10(3) molecules of the target CVYV cDNA. Real-time PCR gave reproducible results for the quantification of CVYV in young leaves of susceptible and resistant cucumber landraces after mechanical inoculation. Significant differences in the starting amount of target cDNA were found between the analyzed genotypes, indicating differences in viral accumulation that correlated to their different levels of resistance. Real-time PCR results validated our previous findings using slot-blot hybridization, the dominance of the strong resistance to CVYV displayed by C.sat 10, and provided improved reliability and sensitivity of detection. This method has great potential in resistance breeding for germplasm screening, characterization of resistance mechanisms and genetic studies.

  9. 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.

  10. Real-Time Gender Classification by Face

    Directory of Open Access Journals (Sweden)

    Eman Fares Al Mashagba

    2016-03-01

    Full Text Available The identification of human beings based on their biometric body parts, such as face, fingerprint, gait, iris, and voice, plays an important role in electronic applications and has become a popular area of research in image processing. It is also one of the most successful applications of computer–human interaction and understanding. Out of all the abovementioned body parts,the face is one of most popular traits because of its unique features.In fact, individuals can process a face in a variety of ways to classify it by its identity, along with a number of other characteristics, such as gender, ethnicity, and age. Specifically, recognizing human gender is important because people respond differently according to gender. In this paper, we present a robust method that uses global geometry-based features to classify gender and identify age and human beings from video sequences. The features are extracted based on face detection using skin color segmentation and the computed geometric features of the face ellipse region. These geometric features are then used to form the face vector trajectories, which are inputted to a time delay neural network and are trained using the Broyden–Fletcher–Goldfarb–Shanno (BFGS function. Results show that using the suggested method with our own dataset under an unconstrained condition achieves a 100% classification rate in the training set for all application, as well as 91.2% for gender classification, 88% for age identification, and 83% for human identification in the testing set. In addition, the proposed method establishes the real-time system to be used in three applications with a simple computation for feature extraction.

  11. Variational optical flow computation in real time.

    Science.gov (United States)

    Bruhn, Andrés; Weickert, Joachim; Feddern, Christian; Kohlberger, Timo; Schnörr, Christoph

    2005-05-01

    This paper investigates the usefulness of bidirectional multigrid methods for variational optical flow computations. Although these numerical schemes are among the fastest methods for solving equation systems, they are rarely applied in the field of computer vision. We demonstrate how to employ those numerical methods for the treatment of variational optical flow formulations and show that the efficiency of this approach even allows for real-time performance on standard PCs. As a representative for variational optic flow methods, we consider the recently introduced combined local-global method. It can be considered as a noise-robust generalization of the Horn and Schunck technique. We present a decoupled, as well as a coupled, version of the classical Gauss-Seidel solver, and we develop several multgrid implementations based on a discretization coarse grid approximation. In contrast, with standard bidirectional multigrid algorithms, we take advantage of intergrid transfer operators that allow for nondyadic grid hierarchies. As a consequence, no restrictions concerning the image size or the number of traversed levels have to be imposed. In the experimental section, we juxtapose the developed multigrid schemes and demonstrate their superior performance when compared to unidirectional multgrid methods and nonhierachical solvers. For the well-known 316 x 252 Yosemite sequence, we succeeded in computing the complete set of dense flow fields in three quarters of a second on a 3.06-GHz Pentium4 PC. This corresponds to a frame rate of 18 flow fields per second which outperforms the widely-used Gauss-Seidel method by almost three orders of magnitude.

  12. 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

    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......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...

  13. Application of real-time RT-PCR quantification to evaluate differential expres sion of Arabidopsis Aux/IAAgenes

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The molecular techniques including Northern blot, dot blot, in situ hybridization, etc. have been success fully used to estimate semi-quantitatively mRNA levels in plant samples. In this study, we employed a real-time reverse transcription-PCR (RT-PCR) assay using SYBR Green Ifluorescence methodology to evaluate accurate quant itation and sequence specific detection of Aux/IAA mRNA levels in Arabidopsis. Results obtained indicate a linear dynamic range of 102-106 Aux/IAA mRNA copies with standard de viations of generally less than 15%. As a model experiment,the outcome of analysis of expression patterns of five Aux/IAA genes in Arabidopsis under various chemical and temperature treatments is presented. The method presented here provides a sensitive and rapid technique to evaluate plant Aux/IAA mRNA expression levels in nanogram order.

  14. ControlShell - A real-time software framework

    Science.gov (United States)

    Schneider, Stanley A.; Ullman, Marc A.; Chen, Vincent W.

    1991-01-01

    ControlShell is designed to enable modular design and impplementation of real-time software. It is an object-oriented tool-set for real-time software system programming. It provides a series of execution and data interchange mechansims that form a framework for building real-time applications. These mechanisms allow a component-based approach to real-time software generation and mangement. By defining a set of interface specifications for intermodule interaction, ControlShell provides a common platform that is the basis for real-time code development and exchange.

  15. Hyd5 gene based analysis of cereals and malt for gushing-inducing Fusarium spp. by real-time LAMP using fluorescence and turbidity measurements.

    Science.gov (United States)

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

    2013-04-01

    The surface active class 2 hydrophobin Hyd5p (GenBank accession number DQ449530) has been identified as a causative agent for over foaming (gushing) of beer. In order to estimate the potential of brewing cereals and malt to induce gushing in beer we used a previously described set of primers to amplify a partial sequence of the hyd5 gene in Fusarium culmorum and closely related species in a real-time loop-mediated isothermal amplification assay. Real-time LAMP was optimized and performed on two different platforms using a turbidimeter and a fluorescence reader to monitor the reaction on line. Serial dilutions of purified target DNA were used to set up a calibration curve for quantitative estimation of DNA concentrations. Analysis of model barley samples prepared by mixing infected with non-infected material in different ratios demonstrated a positive correlation between the real-time LAMP results and respective infection levels. In order to investigate the performance of the newly established methods, samples of barley were analyzed for their gushing potential using the Hyd5 real-time LAMP assay and a reference in vitro test for gushing prediction (Modified Carlsberg Test, MCT) in parallel experiments. Results showed that the real-time LAMP assay was in accordance with the reference test in 50% of cases with both platforms used. It predicted a lower number of gushing-positive samples as compared to the current reference test.

  16. Satellite clock corrections estimation to accomplish real time ppp: experiments for brazilian real time network

    Science.gov (United States)

    Marques, Haroldo; Monico, João; Aquino, Marcio; Melo, Weyller

    2014-05-01

    The real time PPP method requires the availability of real time precise orbits and satellites clocks corrections. Currently, it is possible to apply the solutions of clocks and orbits available by BKG within the context of IGS Pilot project or by using the operational predicted IGU ephemeris. The accuracy of the satellite position available in the IGU is enough for several applications requiring good quality. However, the satellites clocks corrections do not provide enough accuracy (3 ns ~ 0.9 m) to accomplish real time PPP with the same level of accuracy. Therefore, for real time PPP application it is necessary to further research and develop appropriated methodologies for estimating the satellite clock corrections in real time with better accuracy. Currently, it is possible to apply the real time solutions of clocks and orbits available by Federal Agency for Cartography and Geodesy (BKG) within the context of IGS Pilot project. The BKG corrections are disseminated by a new proposed format of the RTCM 3.x and can be applied in the broadcasted orbits and clocks. Some investigations have been proposed for the estimation of the satellite clock corrections using GNSS code and phase observable at the double difference level between satellites and epochs (MERVAT, DOUSA, 2007). Another possibility consists of applying a Kalman Filter in the PPP network mode (HAUSCHILD, 2010) and it is also possible the integration of both methods, using network PPP and observables at double difference level in specific time intervals (ZHANG; LI; GUO, 2010). For this work the methodology adopted consists in the estimation of the satellite clock corrections based on the data adjustment in the PPP mode, but for a network of GNSS stations. The clock solution can be solved by using two types of observables: code smoothed by carrier phase or undifferenced code together with carrier phase. In the former, we estimate receiver clock error; satellite clock correction and troposphere, considering

  17. Rapid and specific detection of Lassa virus by reverse transcription-PCR coupled with oligonucleotide array hybridization.

    Science.gov (United States)

    Olschläger, Stephan; Günther, Stephan

    2012-07-01

    To facilitate sequence-specific detection of DNA amplified in a diagnostic reverse transcription (RT)-PCR for Lassa virus, we developed an array featuring 47 oligonucleotide probes for post-PCR hybridization of the amplicons. The array procedure may be performed with low-tech equipment and does not take longer than agarose gel detection.

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

    Directory of Open Access Journals (Sweden)

    Pan Lei

    2012-10-01

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

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

    Science.gov (United States)

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

    2015-12-03

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

  20. 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.

    Science.gov (United States)

    Sun, Yi; Quyen, Than Linh; Hung, Tran Quang; Chin, Wai Hoe; Wolff, Anders; Bang, Dang Duong

    2015-04-21

    Foodborne disease is a major public health threat worldwide. Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture or molecular-based methods are time consuming and usually take a few hours to days to complete. In response to the demand for rapid on line or on 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 bead-based sample preparation and loop-mediated isothermal amplification (LAMP) for rapid and quantitative detection of Salmonella spp. in food samples. The whole diagnostic procedures including DNA isolation, isothermal amplification, and real-time detection were accomplished in a single chamber. Up to eight samples could be handled simultaneously and the system was capable to detect Salmonella at concentration of 50 cells per test within 40 min. The simple design, together with high level of integration, isothermal amplification, and quantitative analysis of multiple samples in short time, will greatly enhance the practical applicability of the LOC system for rapid on-site screening of Salmonella for applications in food safety control, environmental surveillance, and clinical diagnostics.