Quantitation of RHD by real-time polymerase chain reaction for determination of RHD zygosity and RHD mosaicism/chimerism

DEFF Research Database (Denmark)

Krog, Grethe Risum; Clausen, Frederik Banch; Dziegiel, Morten Hanefeld

2007-01-01

Determination of RHD zygosity of the spouse is crucial in preconception counseling of families with history of hemolytic disease of the fetus and newborn caused by anti-D. RHD zygosity can be determined by quantitative real-time polymerase chain reaction (PCR) basically by determining RHD dosage,......, and this feature is relevant in investigating RHD mosaicism and chimerism....

Monitoring Acidophilic Microbes with Real-Time Polymerase Chain Reaction (PCR) Assays

Energy Technology Data Exchange (ETDEWEB)

Frank F. Roberto

2008-08-01

Many techniques that are used to characterize and monitor microbial populations associated with sulfide mineral bioleaching require the cultivation of the organisms on solid or liquid media. Chemolithotrophic species, such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, or thermophilic chemolithotrophs, such as Acidianus brierleyi and Sulfolobus solfataricus can grow quite slowly, requiring weeks to complete efforts to identify and quantify these microbes associated with bioleach samples. Real-time PCR (polymerase chain reaction) assays in which DNA targets are amplified in the presence of fluorescent oligonucleotide primers, allowing the monitoring and quantification of the amplification reactions as they progress, provide a means of rapidly detecting the presence of microbial species of interest, and their relative abundance in a sample. This presentation will describe the design and use of such assays to monitor acidophilic microbes in the environment and in bioleaching operations. These assays provide results within 2-3 hours, and can detect less than 100 individual microbial cells.

A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus

Directory of Open Access Journals (Sweden)

Aline Lavado Tolardo

2016-06-01

Full Text Available Vesiculoviruses (VSV are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections.

Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA

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Ana Lisa do Vale Gomes

2006-10-01

Full Text Available This report describes the development of a SYBR Green I based real time polymerase chain reaction (PCR protocol for detection on the ABI Prism 7000 instrument. Primers targeting the gene encoding the SSU rRNA were designed to amplify with high specificity DNA from Schistosoma mansoni, in a real time quantitative PCR system. The limit of detection of parasite DNA for the system was 10 fg of purified genomic DNA, that means less than the equivalent to one parasite cell (genome ~580 fg DNA. The efficiency was 0.99 and the correlation coefficient (R² was 0.97. When different copy numbers of the target amplicon were used as standards, the assay could detect at least 10 copies of the specific target. The primers used were designed to amplify a 106 bp DNA fragment (Tm 83ºC. The assay was highly specific for S. mansoni, and did not recognize DNA from closely related non-schistosome trematodes. The real time PCR allowed for accurate quantification of S. mansoni DNA and no time-consuming post-PCR detection of amplification products by gel electrophoresis was required. The assay is potentially able to quantify S. mansoni DNA (and indirectly parasite burden in a number of samples, such as snail tissue, serum and feces from patients, and cercaria infested water. Thus, these PCR protocols have potential to be used as tools for monitoring of schistosome transmission and quantitative diagnosis of human infection.

[REAL TIME POLYMERASE CHAIN REACTION IN TULAREMIA LABORATORY DIAGNOSTICS].

Science.gov (United States)

Kormilitsyna, M I; Mescheryakova, I S; Mikhailova, T V; Dobrovolsky, A A

2015-01-01

Enhancement of tularemia laboratory diagnostics by F. tularensis DNA determination in blood sera of patients using real time polymerase chain reaction (RT-PCR). 39 blood sera of patients obtained during transmissive epidemic outbreak of tularemia in Khanty-Mansiysk in 2013 were studied in agglutination reaction, passive hemagglutination, RT-PCR. Specific primers and fluorescent probes were used: ISFTu2F/R+ISFTu2P, Tu14GF/R+tul4-PR2. Advantages of using RT-PCR for early diagnostics of tularemia, when specific antibodies are not detected using traditional immunologic methods, were established. Use of a combination of primers and ISFTu2F/R+ISFTu2P probe allowed to detect F. tularensis DNA in 100% of sera, whereas Tul4G F/R+tul4-PR2 combination--92% of sera. The data were obtained when DNA was isolated from sera using "Proba Rapid" express method. Clinical-epidemiologic diagnosis oftularemia was confirmed by both immune-serologic and RT-PCR methods when sera were studied 3-4 weeks after the onset of the disease. RT-PCR with ISFTu2F/R primers and fluorescent probe ISFTu2P, having high sensitivity and specificity, allows to determine F. tularensis DNA in blood sera of patients at both the early stage and 3-4 weeks after the onset of the disease.

Molecular detection of the carriage rate of four intestinal protozoa with real-time polymerase chain reaction

DEFF Research Database (Denmark)

Efunshile, Michael A; Ngwu, Bethrand A F; Kurtzhals, Jørgen A L

2015-01-01

Diarrhea remains the second largest killer of children worldwide, and Nigeria ranks number two on the list of global deaths attributable to diarrhea. Meanwhile, prevalence studies on potentially diarrheagenic protozoa in asymptomatic carriers using molecular detection methods remain scarce in sub......-Saharan countries. To overcome sensitivity issues related to microscopic detection and identification of cysts in stool concentrates, real-time polymerase chain reaction (PCR) was used to analyze genomic DNAs extracted from stool samples from 199 healthy school children for Entamoeba histolytica, E. dispar, Giardia...

Babesia microti real-time polymerase chain reaction testing of Connecticut blood donors: potential implications for screening algorithms.

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Johnson, Stephanie T; Van Tassell, Eric R; Tonnetti, Laura; Cable, Ritchard G; Berardi, Victor P; Leiby, David A

2013-11-01

Babesia microti, an intraerythrocytic parasite, has been implicated in transfusion transmission. B. microti seroprevalence in Connecticut (CT) blood donors is approximately 1%; however, it is not known what percentage of donors is parasitemic and poses a risk for transmitting infection. Therefore, we determined the prevalence of demonstrable B. microti DNA in donors from a highly endemic area of CT and compared observed rates with concurrent immunofluorescence assay (IFA) testing results. Blood samples from consenting donors in southeastern CT were collected from mid-August through early October 2009 and tested by IFA for immunoglobulin G antibodies and real-time polymerase chain reaction (PCR) for B. microti DNA. IFA specificity was determined using blood donor samples collected in northwestern Vermont (VT), an area nonendemic for Babesia. Of 1002 CT donors, 25 (2.5%) were IFA positive and three (0.3%) were real-time PCR positive. Among the three real-time PCR-positive donors, two were also IFA positive, while one was IFA negative and may represent a window period infection. The two IFA- and real-time PCR-positive donors appeared to subsequently clear infection. The other real-time PCR-positive donor did not provide follow-up samples. Of 1015 VT donors tested by IFA, only one (0.1%) was positive, but may have acquired infection during travel to an endemic area. We prospectively identified several real-time PCR-positive blood donors, including an IFA-negative real-time PCR-positive donor, in an area highly endemic for B. microti. These results suggest the need to include nucleic acid testing in planned mitigation strategies for B. microti. © 2013 American Association of Blood Banks.

A Comprehensive Real-Time Traffic Map for Geographic Routing in VANETs

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Chi-Fu Huang

2017-01-01

Full Text Available Vehicular Ad Hoc Networks (VANETs have attracted a lot of attention during the last decade. VANETs can not only improve driving safety, but also convenience, and support most future Intelligent Transportation System (ITS. Due to the highly dynamic network topology of VANETs, many geographic routing protocols have been proposed and use real-time traffic information as an important metric to select a reliable forwarding path. However, most of the existing works do not describe how to gather real-time traffic. They either assume this information is already available, or can query an existing traffic center. Few studies have noticed this issue but the proposed solutions only consider a small region. In this paper, we propose a Comprehensive Real-Time Traffic Map (CRT Map to collect wide-ranging real-time traffic information with low overhead. In the design of a CRT Map, the concept of Crowdsensing is adopted. Vehicles cooperatively gather traffic information and share it with each other to construct an overview of the whole road network traffic. In addition, we design a CRT Map Based Routing (CBR, which takes into account the connectivity of consecutive roads in routing decisions. Simulation results show that the CBR can achieve a lower end-to-end delay and a higher packet delivery ratio.

Real time polymerase chain reaction in diagnosis of chronic myeloid leukemia

International Nuclear Information System (INIS)

Tashfeen, S.; Ahmed, S.; Bhatti, F.A.; Ali, N.

2014-01-01

Objective: To compare the sensitivity and specificity of Real Time Polymerase Chain Reaction (RT-PCR) with conventional cytogenetics in diagnosis of chronic myeloid leukemia. Study Design: A cross-sectional, analytical study. Place and Duration of Study: The Armed Forces Institute of Pathology (AFIP), Rawalpindi, from December 2010 to January 2012. Methodology: A total number of 40 patients were studied, in which all were diagnosed as CML on peripheral blood and bone marrow aspiration. The subjects were tested for the presence of Philadelphia (Ph) chromosome by cytogenetics and BCR-ABL fusion gene by RT-PCR. 2-3 ml of venous blood was collected, half in sodium heparin (anti-coagulant) for cytogenetics and half in EDTA for PCR. For cytogenetics, cells were cultured for 72 hours in RPMI 1640 medium and examined by arresting in metaphase using Colchicine to identify Philadelphia chromosome. For PCR, RNA extraction was done by Tri Reagent LS (MRC, USA) and cDNA was synthesized using reverse transcriptase and gene specific primer. RT- PCR was done on ABI-7500. The positive samples were identified when fluorescence exceeded threshold limit. Results of cytogenetics and RT PCR were compared. Results: Out of the 40 patients, PCR showed 37 (92.5%) were positive and 3 (7.5%) were negative for BCR-ABL fusion gene, whereas in cytogenetics 28 (70%) were positive for Ph chromosome and 12 (30%) were negative for Ph chromosome. Sensitivity and specificity of cytogenetics was 75.6% and 100% respectively. Conclusion: Real time PCR as compared to cytogenetics is less tedious, gives quick results, does not require multiple sampling due to culture failure and can be done on peripheral blood. (author)

On-site identification of meat species in processed foods by a rapid real-time polymerase chain reaction system.

Science.gov (United States)

Furutani, Shunsuke; Hagihara, Yoshihisa; Nagai, Hidenori

2017-09-01

Correct labeling of foods is critical for consumers who wish to avoid a specific meat species for religious or cultural reasons. Therefore, gene-based point-of-care food analysis by real-time Polymerase Chain Reaction (PCR) is expected to contribute to the quality control in the food industry. In this study, we perform rapid identification of meat species by our portable rapid real-time PCR system, following a very simple DNA extraction method. Applying these techniques, we correctly identified beef, pork, chicken, rabbit, horse, and mutton in processed foods in 20min. Our system was sensitive enough to detect the interfusion of about 0.1% chicken egg-derived DNA in a processed food sample. Our rapid real-time PCR system is expected to contribute to the quality control in food industries because it can be applied for the identification of meat species, and future applications can expand its functionality to the detection of genetically modified organisms or mutations. Copyright © 2017 Elsevier Ltd. All rights reserved.

Detection of medically important Candida species by absolute quantitation real-time polymerase chain reaction.

Science.gov (United States)

Than, Leslie Thian Lung; Chong, Pei Pei; Ng, Kee Peng; Seow, Heng Fong

2015-01-01

The number of invasive candidiasis cases has risen especially with an increase in the number of immunosuppressed and immunocom promised patients. The early detection of Candida species which is specific and sensitive is important in determining the correct administration of antifungal drugs to patients. This study aims to develop a method for the detection, identification and quantitation of medically important Candida species through quantitative polymerase chain reaction (qPCR). The isocitrate lyase (ICL) gene which is not found in mammals was chosen as the target gene of real-time PCR. Absolute quantitation of the gene copy number was achieved by constructing the plasmid containing the ICL gene which is used to generate standard curve. Twenty fungal species, two bacterial species and human DNA were tested to check the specificity of the detection method. All eight Candida species were successfully detected, identified and quantitated based on the ICL gene. A seven-log range of the gene copy number and a minimum detection limit of 10(3) copies were achieved. A one-tube absolute quantification real-time PCR that differentiates medically important Candida species via individual unique melting temperature was achieved. Analytical sensitivity and specificity were not compromised.

Real-time polymerase chain reaction as a tool for evaluation of magnetic poly(glycidyl methacrylate)-based microspheres in molecular diagnostics

Czech Academy of Sciences Publication Activity Database

Trachtová, S.; Španová, A.; Horák, Daniel; Kozáková, Hana; Rittich, B.

2016-01-01

Roč. 22, č. 5 (2016), s. 639-646 ISSN 1381-6128 R&D Projects: GA ČR GA15-07268S Institutional support: RVO:61389013 ; RVO:61388971 Keywords : magnetic microspheres * inhibitory effect * real-time polymerase chain Subject RIV: CD - Macromolecular Chemistry; CD - Macromolecular Chemistry (MBU-M) Impact factor: 2.611, year: 2016

Interlaboratory validation data on real-time polymerase chain reaction detection for unauthorized genetically modified papaya line PRSV-YK

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Kosuke Nakamura

2016-06-01

Real-time polymerase chain reaction (PCR detection method for unauthorized genetically modified (GM papaya (Carica papaya L. line PRSV-YK (PRSV-YK detection method was developed using whole genome sequence data (DDBJ Sequenced Read Archive under accession No. PRJDB3976. Interlaboratory validation datasets for PRSV-YK detection method were provided. Data indicating homogeneity of samples prepared for interlaboratory validation were included. Specificity and sensitivity test data for PRSV-YK detection method were also provided.

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

Science.gov (United States)

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

2017-08-15

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

Detection of viable Salmonella in ice cream by TaqMan real-time polymerase chain reaction assay combining propidium monoazide

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Yuexia Wang

2015-09-01

Full Text Available Real-time polymerase chain reaction (PCR allows rapid detection of Salmonella in frozen dairy products, but it might cause a false positive detection result because it might amplify DNA from dead target cells as well. In this study, Salmonella-free frozen ice cream was initially inoculated with heat-killed Salmonella Typhimurium cells and stored at −18°C. Bacterial DNA extracted from the sample was amplified using TaqMan probe-based real-time PCR targeting the invA gene. Our results indicated that DNA from the dead cells remained stable in frozen ice cream for at least 20 days, and could produce fluorescence signal for real-time PCR as well. To overcome this limitation, propidium monoazide (PMA was combined with real-time PCR. PMA treatment can effectively prevent PCR amplification from heat-killed Salmonella cells in frozen ice cream. The PMA real-time PCR assay can selectively detect viable Salmonella at as low as 103 CFU/mL. Combining 18 hours of pre-enrichment with the assay allows for the detection of viable Salmonella at 100 CFU/mL and avoiding the false-positive result of dead cells. The PMA real-time PCR assay provides an alternative specifically for detection of viable Salmonella in ice cream. However, when the PMA real-time PCR assay was evaluated in ice cream subjected to frozen storage, it obviously underestimated the contamination situation of viable Salmonella, which might lead to a false negative result. According to this result, the use of enrichment prior to PMA real-time PCR analysis remains as the more appropriate approach.

Detection of viable Salmonella in ice cream by TaqMan real-time polymerase chain reaction assay combining propidium monoazide.

Science.gov (United States)

Wang, Yuexia; Yang, Ming; Liu, Shuchun; Chen, Wanyi; Suo, Biao

2015-09-01

Real-time polymerase chain reaction (PCR) allows rapid detection of Salmonella in frozen dairy products, but it might cause a false positive detection result because it might amplify DNA from dead target cells as well. In this study, Salmonella-free frozen ice cream was initially inoculated with heat-killed Salmonella Typhimurium cells and stored at -18°C. Bacterial DNA extracted from the sample was amplified using TaqMan probe-based real-time PCR targeting the invA gene. Our results indicated that DNA from the dead cells remained stable in frozen ice cream for at least 20 days, and could produce fluorescence signal for real-time PCR as well. To overcome this limitation, propidium monoazide (PMA) was combined with real-time PCR. PMA treatment can effectively prevent PCR amplification from heat-killed Salmonella cells in frozen ice cream. The PMA real-time PCR assay can selectively detect viable Salmonella at as low as 10 3  CFU/mL. Combining 18 hours of pre-enrichment with the assay allows for the detection of viable Salmonella at 10 0  CFU/mL and avoiding the false-positive result of dead cells. The PMA real-time PCR assay provides an alternative specifically for detection of viable Salmonella in ice cream. However, when the PMA real-time PCR assay was evaluated in ice cream subjected to frozen storage, it obviously underestimated the contamination situation of viable Salmonella, which might lead to a false negative result. According to this result, the use of enrichment prior to PMA real-time PCR analysis remains as the more appropriate approach. Copyright © 2015. Published by Elsevier B.V.

Polymerase study: Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic real-time PCR

DEFF Research Database (Denmark)

Søndergaard, Mette Sofie Rousing; Löfström, Charlotta; Al-Habib, Zahra Fares Sayer

DNA extractions and intermediate or bad with the crude extractions, while TaKaRa ExTaq HS only performed well with the purest extractions of fecal samples and intermediate with semi-automated magnetic beads based extracted fecal samples. In conclusion, our data shows that exchanging the DNA polymerase......Diagnostic analyses of foodborne pathogens are increasingly based on molecular methods such as PCR, which can improve the sensitivity and reduce the analysis time. The core of PCR is the enzyme performing the reaction: the DNA polymerase. Changing the polymerase can influence the sensitivity...... commercially available polymerases and four master mixes in two validated PCR assays, for Campylobacter and Salmonella, respectively, to develop more sensitive, robust and cost effective assays. The polymerases were screened on purified DNA and the five best performing, for each PCR assay, were then applied...

Fast real-time polymerase chain reaction for quantitative detection of Lactobacillus delbrueckii bacteriophages in milk.

Science.gov (United States)

Martín, Maria Cruz; del Rio, Beatriz; Martínez, Noelia; Magadán, Alfonso H; Alvarez, Miguel A

2008-12-01

One of the main microbiological problems of the dairy industry is the susceptibility of starter bacteria to virus infections. Lactobacillus delbrueckii, a component of thermophilic starter cultures used in the manufacture of several fermented dairy products, including yogurt, is also sensitive to bacteriophage attacks. To avoid the problems associated with these viruses, quick and sensitive detection methods are necessary. In the present study, a fast real-time quantitative polymerase chain reaction assay for the direct detection and quantification of L. delbrueckii phages in milk was developed. A set of primers and a TaqMan MGB probe was designed, based on the lysin gene sequence of different L. delbrueckii phages. The results show the proposed method to be a rapid (total processing time 30 min), specific and highly sensitive technique for detecting L. delbrueckii phages in milk.

Trends and advances in food analysis by real-time polymerase chain reaction.

Science.gov (United States)

Salihah, Nur Thaqifah; Hossain, Mohammad Mosharraf; Lubis, Hamadah; Ahmed, Minhaz Uddin

2016-05-01

Analyses to ensure food safety and quality are more relevant now because of rapid changes in the quantity, diversity and mobility of food. Food-contamination must be determined to maintain health and up-hold laws, as well as for ethical and cultural concerns. Real-time polymerase chain reaction (RT-PCR), a rapid and inexpensive quantitative method to detect the presence of targeted DNA-segments in samples, helps in determining both accidental and intentional adulterations of foods by biological contaminants. This review presents recent developments in theory, techniques, and applications of RT-PCR in food analyses, RT-PCR addresses the limitations of traditional food analyses in terms of sensitivity, range of analytes, multiplexing ability, cost, time, and point-of-care applications. A range of targets, including species of plants or animals which are used as food ingredients, food-borne bacteria or viruses, genetically modified organisms, and allergens, even in highly processed foods can be identified by RT-PCR, even at very low concentrations. Microfluidic RT-PCR eliminates the separate sample-processing step to create opportunities for point-of-care analyses. We also cover the challenges related to using RT-PCR for food analyses, such as the need to further improve sample handling.

Detection of Brucella spp. in milk from seronegative cows by real-time polymerase chain reaction in the region of Batna, Algeria

Science.gov (United States)

Sabrina, Rabehi; Mossadak, Hamdi Taha; Bakir, Mamache; Asma, Meghezzi; Khaoula, Boushaba

2018-01-01

Aim: The aim of this study was to detect Brucella spp. DNA in milk samples collected from seronegative cows using the real-time polymerase chain reaction (PCR) assay for diagnosis of brucellosis in seronegative dairy cows to prevent transmission of disease to humans and to reduce economic losses in animal production. Materials and Methods: In this study, 65 milk samples were investigated for the detection of Brucella spp. The detection of the IS711 gene in all samples was done by real-time PCR assay by comparative cycle threshold method. Results: The results show that of the 65 DNA samples tested, 2 (3.08%) were positive for Brucella infection. The mean cyclic threshold values of IS711 real-time PCR test were 37.97 and 40.48, indicating a positive reaction. Conclusion: The results of the present study indicated that the real-time PCR appears to offer several advantages over serological tests. For this reason, the real-time PCR should be validated on representative numbers of Brucella-infected and free samples before being implemented in routine diagnosis in human and animal brucellosis for controlling this disease. PMID:29657430

Diagnosis of ventricular drainage-related bacterial meningitis by broad-range real-time polymerase chain reaction

DEFF Research Database (Denmark)

Deutch, Susanna; Dahlberg, Daniel; Hedegaard, Jesper

2007-01-01

OBJECTIVE: To compare a broad-range real-time polymerase chain reaction (PCR) diagnostic strategy with culture to evaluate additional effects on the etiological diagnosis and the quantification of the bacterial load during the course of ventricular drainage-related bacterial meningitis (VR......-BM). METHODS: We applied a PCR that targeted conserved regions of the 16S ribosomal ribonucleic acid gene to cerebrospinal fluid (CSF) samples from patients with external ventricular drainage or a ventriculoperitoneal shunt during the course of VR-BM. We compared the PCR results with CSF cultures. A total...... of 350 routine CSF samples were consecutively collected from 86 patients. The CSF deoxyribonucleic acid was automatically purified and subjected to PCR. Amplicons from the PCR samples that were positive for VR-BM were subsequently deoxyribonucleic acid sequenced for final identification. Clinical data...

Comparison of Real Time Polymerase Chain Reaction with Microscopy and Antigen Detection Assay for the Diagnosis of Malaria

International Nuclear Information System (INIS)

Khan, S. A.; Ahmed, S.; Khan, F. A.; Shamshad, G. U.; Joyia, Z.; Mushahid, N.; Saeed, S.

2013-01-01

Objective: To determine the sensitivity of a real time polymerase chain reaction (PCR) for malaria diagnosis and to compare its accuracy with microscopy and an antigen based rapid diagnostic test (OptiMal). Study Design: Cross-sectional analytical study. Place and Duration of Study: Military Hospital, Armed Forces Institute of Transfusion and Armed Forces Institute of Pathology, Rawalpindi, from July to December 2011. Methodology: Venous blood samples of 300 clinically suspected patients of malaria were tested for malaria parasite by microscopy and OptiMal; and malaria parasite index was calculated for the positive samples. Plasmodium genus specific real time PCR was performed on all specimens, targeting small subunit rRNA gene. Diagnostic accuracy of three tests was compared and cost analysis was done. Results: Out of 300 patients, malaria parasite was detected in 110, 106 and 123 patients by microscopy, OptiMAL and PCR respectively. Real time PCR was 100% sensitive while microscopy and OptiMal had sensitivity of 89.4% and 86.2% respectively. All methods were 100% specific. The cost per test was calculated to be 0.2, 2.75 and 3.30 US dollar by microscopy, OptiMal and PCR respectively, excluding the once capital cost on PCR equipment. Conclusion: Genus specific real time PCR for the diagnosis of malaria was successfully established as a highly sensitive and affordable technology that should be incorporated in the diagnostic algorithm in this country. (author)

Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction.

Science.gov (United States)

Abdeldaim, Guma M K; Strålin, Kristoffer; Kirsebom, Leif A; Olcén, Per; Blomberg, Jonas; Herrmann, Björn

2009-08-01

A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 10(4) DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

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

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

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I. Karthika Lakshmi

2018-04-01

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

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.

Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis

Science.gov (United States)

Chase, D.M.; Elliott, D.G.; Pascho, R.J.

2006-01-01

Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.

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.

Linear-after-the-exponential polymerase chain reaction and allied technologies. Real-time detection strategies for rapid, reliable diagnosis from single cells.

Science.gov (United States)

Pierce, Kenneth E; Wangh, Lawrence J

2007-01-01

Accurate detection of gene sequences in single cells is the ultimate challenge to polymerase chain reaction (PCR) sensitivity. Unfortunately, commonly used conventional and real-time PCR techniques are often too unreliable at that level to provide the accuracy needed for clinical diagnosis. Here we provide details of linear-after-the-exponential-PCR (LATE-PCR), a method similar to asymmetric PCR in the use of primers at different concentrations, but with novel design criteria to ensure high efficiency and specificity. Compared with conventional PCR, LATE-PCR increases the signal strength and allele discrimination capability of oligonucleotide probes such as molecular beacons and reduces variability among replicate samples. The analysis of real-time kinetics of LATE-PCR signals provides a means for improving the accuracy of single cell genetic diagnosis.

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...

Simultaneous detection of Acidovorax avenae subsp. citrulli and Didymella bryoniae in cucurbit seedlots using magnetic capture hybridization and real-time polymerase chain reaction.

Science.gov (United States)

Ha, Y; Fessehaie, A; Ling, K S; Wechter, W P; Keinath, A P; Walcott, R R

2009-06-01

To improve the simultaneous detection of two pathogens in cucurbit seed, a combination of magnetic capture hybridization (MCH) and multiplex real-time polymerase chain reaction (PCR) was developed. Single-stranded DNA hybridization capture probes targeting DNA of Acidovorax avenae subsp. citrulli, causal agent of bacterial fruit blotch, and Didymella bryoniae, causal agent of gummy stem blight, were covalently attached to magnetic particles and used to selectively concentrate template DNA from cucurbit seed samples. Sequestered template DNAs were subsequently amplified by multiplex real-time PCR using pathogen-specific TaqMan PCR assays. The MCH multiplex real-time PCR assay displayed a detection threshold of A. avenae subsp. citrulli at 10 CFU/ml and D. bryoniae at 10(5) conidia/ml in mixtures of pure cultures of the two pathogens, which was 10-fold more sensitive than the direct real-time PCR assays for the two pathogens separately. Although the direct real-time PCR assay displayed a detection threshold for A. avenae subsp. citrulli DNA of 100 fg/microl in 25% (1/4 samples) of the samples assayed, MCH real-time PCR demonstrated 100% detection frequency (4/4 samples) at the same DNA concentration. MCH did not improve detection sensitivity for D. bryoniae relative to direct real-time PCR using conidial suspensions or seed washes from D. bryoniae-infested cucurbit seed. However, MCH real-time PCR facilitated detection of both target pathogens in watermelon and melon seed samples (n = 5,000 seeds/sample) in which 0.02% of the seed were infested with A. avenae subsp. citrulli and 0.02% were infested with D. bryoniae.

Progesterone receptor isoform analysis by quantitative real-time polymerase chain reaction in formalin-fixed, paraffin-embedded canine mammary dysplasias and tumors

DEFF Research Database (Denmark)

Guil-Luna, S.; Stenvang, Jan; Brünner, Nils

2014-01-01

and its isoforms in formalin-fixed, paraffin-embedded tissue samples from canine mammary lesions (4 dysplasias, 10 benign tumors, and 46 carcinomas) using 1-step SYBR Green quantitative real-time polymerase chain reaction (RT-qPCR). Progesterone receptor was expressed in 75% of dysplasias, all benign...... in the expression of isoform A versus B. Analysis of progesterone receptor mRNA isoforms by RT-qPCR was successful in routinely formalin-fixed, paraffin-embedded tissue samples and enabled the distribution of isoforms A and B to be identified for the first time in dysplasias, benign tumors, and malignant tumors...

Comprehensive seismic monitoring of the Cascadia megathrust with real-time GPS

Science.gov (United States)

Melbourne, T. I.; Szeliga, W. M.; Santillan, V. M.; Scrivner, C. W.; Webb, F.

2013-12-01

We have developed a comprehensive real-time GPS-based seismic monitoring system for the Cascadia subduction zone based on 1- and 5-second point position estimates computed within the ITRF08 reference frame. A Kalman filter stream editor that uses a geometry-free combination of phase and range observables to speed convergence while also producing independent estimation of carrier phase biases and ionosphere delay pre-cleans raw satellite measurements. These are then analyzed with GIPSY-OASIS using satellite clock and orbit corrections streamed continuously from the International GNSS Service (IGS) and the German Aerospace Center (DLR). The resulting RMS position scatter is less than 3 cm, and typical latencies are under 2 seconds. Currently 31 coastal Washington, Oregon, and northern California stations from the combined PANGA and PBO networks are analyzed. We are now ramping up to include all of the remaining 400+ stations currently operating throughout the Cascadia subduction zone, all of which are high-rate and telemetered in real-time to CWU. These receivers span the M9 megathrust, M7 crustal faults beneath population centers, several active Cascades volcanoes, and a host of other hazard sources. To use the point position streams for seismic monitoring, we have developed an inter-process client communication package that captures, buffers and re-broadcasts real-time positions and covariances to a variety of seismic estimation routines running on distributed hardware. An aggregator ingests, re-streams and can rebroadcast up to 24 hours of point-positions and resultant seismic estimates derived from the point positions to application clients distributed across web. A suite of seismic monitoring applications has also been written, which includes position time series analysis, instantaneous displacement vectors, and peak ground displacement contouring and mapping. We have also implemented a continuous estimation of finite-fault slip along the Cascadia megathrust

Real time elastography endoscopic ultrasound (RTE-EUS), a comprehensive review

Energy Technology Data Exchange (ETDEWEB)

Dietrich, C.F., E-mail: christoph.dietrich@ckbm.de [Med. Klinik 2, Caritas-Krankenhaus Bad Mergentheim (Germany); Department of Gastroenterology, Research Center of Gastroenterology and Hepatology, University of Medicine and Pharmacy of Craiova (Romania); Klinik für Innere Medizin, Krankenhaus Märkisch Oderland (Germany); Săftoiu, A.; Jenssen, C. [Med. Klinik 2, Caritas-Krankenhaus Bad Mergentheim (Germany); Department of Gastroenterology, Research Center of Gastroenterology and Hepatology, University of Medicine and Pharmacy of Craiova (Romania); Klinik für Innere Medizin, Krankenhaus Märkisch Oderland (Germany)

2014-03-15

Real-time elastography (RTE) performed during endoscopic ultrasound (EUS) is a relatively new technique which allows the evaluation of tissue stiffness, with the intent of better characterising lesions during EUS examinations. The aim of this comprehensive review was to describe the technique of RTE-EUS, as well as the clinical applications, including the study of pancreatic lesions, but also hepatobiliary, gastrointestinal (GI) tract pathology (including anal canal), lymph nodes, adrenal glands, lung and mediastinum, as well as urogenital applications. One of the advantages of the RTE-EUS technique is especially the possibility to be used in various locations accessible from the GI tract. Future developments are also briefly discussed, as elastography is a tissue characterising technique that will certainly not replace biopsy, but will rather be an adjunct during EUS examinations, due to its ease of use and low cost.

Real time elastography endoscopic ultrasound (RTE-EUS), a comprehensive review

International Nuclear Information System (INIS)

Dietrich, C.F.; Săftoiu, A.; Jenssen, C.

2014-01-01

Real-time elastography (RTE) performed during endoscopic ultrasound (EUS) is a relatively new technique which allows the evaluation of tissue stiffness, with the intent of better characterising lesions during EUS examinations. The aim of this comprehensive review was to describe the technique of RTE-EUS, as well as the clinical applications, including the study of pancreatic lesions, but also hepatobiliary, gastrointestinal (GI) tract pathology (including anal canal), lymph nodes, adrenal glands, lung and mediastinum, as well as urogenital applications. One of the advantages of the RTE-EUS technique is especially the possibility to be used in various locations accessible from the GI tract. Future developments are also briefly discussed, as elastography is a tissue characterising technique that will certainly not replace biopsy, but will rather be an adjunct during EUS examinations, due to its ease of use and low cost

Near Real-Time Comprehension Classification with Artificial Neural Networks: Decoding e-Learner Non-Verbal Behavior

Science.gov (United States)

Holmes, Mike; Latham, Annabel; Crockett, Keeley; O'Shea, James D.

2018-01-01

Comprehension is an important cognitive state for learning. Human tutors recognize comprehension and non-comprehension states by interpreting learner non-verbal behavior (NVB). Experienced tutors adapt pedagogy, materials, and instruction to provide additional learning scaffold in the context of perceived learner comprehension. Near real-time…

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

Science.gov (United States)

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

2017-05-01

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

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

Directory of Open Access Journals (Sweden)

Miriam Ribas Zambenedetti

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

A comprehensive cloud-based real-time simulation framework for oblivious power routing in clusters of DC microgrids

DEFF Research Database (Denmark)

Amini, M. Hadi; Boroojeni, Kianoosh G.; Dragicevic, Tomislav

2017-01-01

of microgrids. The effectiveness of the proposed algorithm has been verified in MATLAB simulation. Furthermore, we propose a comprehensive simulation platform for further implementation of the proposed strategy on OPAL-RT real-time simulator system (RTDS). In our proposed platform, the communication path...

Real-time polymerase chain reaction with melting analysis of positive blood culture specimens in bloodstream infections: diagnostic value and turnaround time.

Science.gov (United States)

Angeletti, Silvia; Gherardi, Giovanni; De Florio, Lucia; Avola, Alessandra; Crea, Francesca; Riva, Elisabetta; Vitali, Massimiliano Andrea; Galluzzo, Sara; Dicuonzo, Giordano

2013-01-01

A Real-time polymerase chain reaction (PCR) with melting analysis was devised to target bacterial and fungal genes together with the most prevalent antimicrobial resistance genes in 250 positive blood culture broths. This method allowed the blood culture cultivated pathogens to be classified into clinically relevant groups such as Enterobacteriaceae, oxidase-positive bacilli, oxidase-positive coccobacilli, S. aureus and yeast. Enterococci and streptococci could be distinguished from CoNS only by the Gram stain. Gram-positive bacilli were discriminated from Gram-positive cocci by Gram stain. Furthermore, the most important antimicrobial resistant genes such as mecA, vanA, bla TEM , bla SHV and bla CTX-M could be identified. All results were obtained with a turnaround time of three hours from the moment of blood culture positivity compared to 24-72 hours for phenotypic methods. In conclusion, the proposed approach can allow the clinician to implement proper early management of sepsis patients.

Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds.

Science.gov (United States)

Rojas, María; González, Isabel; Pavón, Miguel Angel; Pegels, Nicolette; Lago, Adriana; Hernández, Pablo E; García, Teresa; Martín, Rosario

2010-06-01

Species-specific real-time polymerase chain reaction (PCR) assays using TaqMan probes have been developed for verifying the labeling of meat and commercial meat products from game birds, including quail, pheasant, partridge, guinea fowl, pigeon, Eurasian woodcock and song thrush. The method combines the use of species-specific primers and TaqMan probes that amplify small fragments (amplicons meat products from the target species demonstrated the suitability of the assay for the detection of the target DNAs.

Real-time PCR-based genotyping from whole blood using Taq DNA polymerase and a buffer supplemented with 1,2-propanediol and trehalose

Czech Academy of Sciences Publication Activity Database

Utekal, Pavol; Kocanda, Lukáš; Matoušek, P.; Wagner, P.; Bugajev, Viktor; Dráber, Petr

2015-01-01

Roč. 416, January (2015), s. 178-182 ISSN 0022-1759 R&D Projects: GA ČR(CZ) GBP302/12/G101; GA MPO FR-TI3/067; GA ČR(CZ) GA14-09807S; GA ČR(CZ) GA14-00703S Institutional support: RVO:68378050 Keywords : 1,2-Propanediol * real-time PCR * SYBR Green I * Taq DNA polymerase * trehalose * Unseparated blood Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.858, year: 2015

Use of a novel virus inactivation method for a multicenter avian influenza real-time reverse transcriptase-polymerase chain reaction proficiency study.

Science.gov (United States)

Spackman, Erica; Suarez, David L

2005-01-01

Proficiency assessments are important elements in quality control for diagnostic laboratories. Traditionally, proficiency testing for polymerase chain reaction (PCR)-based assays has involved the use of clinical samples, samples "spiked" with live agents or DNA plasmids. Because of government regulations and biosecurity concerns, distribution of live high-consequence pathogens of livestock and poultry, such as avian influenza, is not possible, and DNA plasmids are not technically suitable for evaluating RNA virus detection. Therefore, a proficiency testing panel using whole avian influenza in a diluent containing a phenolic disinfectant that inactivates the virus while preserving the RNA for at least 8 weeks at -70 C was developed and used in a multicenter proficiency assessment for a type A influenza real-time reverse transcriptase (RT)-PCR test. The test, which was highly standardized, except for variation in the real-time RT-PCR equipment used, was shown to be highly reproducible by proficiency testing in 12 laboratories in the United States, Canada, and Hong Kong. Variation in cycle threshold values among 35 data sets and 490 samples was minimal (CV = 5.19%), and sample identifications were highly accurate (96.7% correct identifications) regardless of real-time PCR instrumentation.

Designing a time-effective TaqMan probe-based real-time polymerase chain reaction protocol for the identification of Yersinia enterocolitica in raw pork meat

Directory of Open Access Journals (Sweden)

Milena Alicja Stachelska

2017-01-01

Full Text Available The aim of this study was to design a time-effective method comprising a short pre-enrichment step in a non-selective broth in combination with the TaqMan probe applied in the real-time polymerase chain reaction to detect Yersinia enterocolitica strains in raw pork meat. The method enabled to detect 1 colony forming unit per 25 mg of Yersinia enterocolitica in pork meat. The specificity and reliability of the method was not diminished by the company of microflora naturally present in meat. The method was found successful to detect pathogenic Yersinia enterocolitica strains in pork meat. It is advised to be used for assessing the microbial risk and for controlling the microbial quality of meat and meat products.

Validation of Geno-Sen's scrub typhus real time polymerase chain reaction kit by its comparison with a serological ELISA Test

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Velmurugan Anitharaj

2017-01-01

Full Text Available Background: In the recent past, scrub typhus (ST has been reported from different parts of India, based on Weil-Felix/enzyme-linked immunosorbent assay (ELISA/indirect immunofluorescence assay (IFA. Molecular tests are applied only by a few researchers. Aims: Evaluation of a new commercial real time polymerase chain reaction (PCR kit for molecular diagnosis of ST by comparing it with the commonly used IgM ELISA is our aim. Settings and Design: ST has been reported all over India including Puducherry and surrounding Tamil Nadu and identified as endemic for ST. This study was designed to correlate antibody detection by IgM ELISA and Orientia tsutsugamushi DNA in real time PCR. Materials and Methods: ST IgM ELISA (InBios Inc., USA was carried out for 170 consecutive patients who presented with the symptoms of acute ST during 11 months (November, 2015– September, 2016. All 77 of these patients with IgM ELISA positivity and 49 of 93 IgM ELISA negative patients were subjected to real time PCR (Geno-Sen's ST real time PCR, Himachal Pradesh, India. Statistical Analysis: Statistical analysis for clinical and laboratory results was performed using IBM SPSS Statistics 17 for Windows (SPSS Inc., Chicago, USA. Chi-square test with Yates correction (Fisher's test was employed for a small number of samples. Results and Conclusion: Among 77 suspected cases of acute ST with IgM ELISA positivity and 49 IgM negative patients, 42 and 7 were positive, respectively, for O. tsutsugamushi 56-kDa type-specific gene in real time PCR kit. Until ST IFA, the gold standard diagnostic test, is properly validated in India, diagnosis of acute ST will depend on both ELISA and quantitative PCR.

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.

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

Science.gov (United States)

Mo, Yiqun; Wan, Rong; Zhang, Qunwei

2012-01-01

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

Mathematical analysis of the real time array PCR (RTA PCR) process

NARCIS (Netherlands)

Dijksman, Johan Frederik; Pierik, A.

2012-01-01

Real time array PCR (RTA PCR) is a recently developed biochemical technique that measures amplification curves (like with quantitative real time Polymerase Chain Reaction (qRT PCR)) of a multitude of different templates in a sample. It combines two different methods in order to profit from the

On-chip real-time single-copy polymerase chain reaction in picoliter droplets

Energy Technology Data Exchange (ETDEWEB)

Beer, N R; Hindson, B; Wheeler, E; Hall, S B; Rose, K A; Kennedy, I; Colston, B

2007-04-20

The first lab-on-chip system for picoliter droplet generation and PCR amplification with real-time fluorescence detection has performed PCR in isolated droplets at volumes 10{sup 6} smaller than commercial real-time PCR systems. The system utilized a shearing T-junction in a silicon device to generate a stream of monodisperse picoliter 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 them to be thermal cycled through the PCR protocol without droplet motion. With this system a 10-pL droplet, encapsulating less than one copy of viral genomic DNA through Poisson statistics, showed real-time PCR amplification curves with a cycle threshold of {approx}18, twenty cycles earlier than commercial instruments. This combination of the established real-time PCR assay with digital microfluidics is ideal for isolating single-copy nucleic acids in a complex environment.

Generation and comprehensive analysis of an influenza virus polymerase cellular interaction network.

Science.gov (United States)

Tafforeau, Lionel; Chantier, Thibault; Pradezynski, Fabrine; Pellet, Johann; Mangeot, Philippe E; Vidalain, Pierre-Olivier; Andre, Patrice; Rabourdin-Combe, Chantal; Lotteau, Vincent

2011-12-01

The influenza virus transcribes and replicates its genome inside the nucleus of infected cells. Both activities are performed by the viral RNA-dependent RNA polymerase that is composed of the three subunits PA, PB1, and PB2, and recent studies have shown that it requires host cell factors to transcribe and replicate the viral genome. To identify these cellular partners, we generated a comprehensive physical interaction map between each polymerase subunit and the host cellular proteome. A total of 109 human interactors were identified by yeast two-hybrid screens, whereas 90 were retrieved by literature mining. We built the FluPol interactome network composed of the influenza virus polymerase (PA, PB1, and PB2) and the nucleoprotein NP and 234 human proteins that are connected through 279 viral-cellular protein interactions. Analysis of this interactome map revealed enriched cellular functions associated with the influenza virus polymerase, including host factors involved in RNA polymerase II-dependent transcription and mRNA processing. We confirmed that eight influenza virus polymerase-interacting proteins are required for virus replication and transcriptional activity of the viral polymerase. These are involved in cellular transcription (C14orf166, COPS5, MNAT1, NMI, and POLR2A), translation (EIF3S6IP), nuclear transport (NUP54), and DNA repair (FANCG). Conversely, we identified PRKRA, which acts as an inhibitor of the viral polymerase transcriptional activity and thus is required for the cellular antiviral response.

The diagnosis of microorganism involved in infective endocarditis (IE by polymerase chain reaction (PCR and real-time PCR: A systematic review

Directory of Open Access Journals (Sweden)

Reza Faraji

2018-02-01

Full Text Available Broad-range bacterial rDNA polymerase chain reaction (PCR followed by sequencing may be identified as the etiology of infective endocarditis (IE from surgically removed valve tissue; therefore, we reviewed the value of molecular testing in identifying organisms' DNA in the studies conducted until 2016. We searched Google Scholar, Scopus, ScienceDirect, Cochrane, PubMed, and Medline electronic databases without any time limitations up to December 2016 for English studies reporting microorganisms involved in infective endocarditis microbiology using PCR and real-time PCR. Most studies were prospective. Eleven out of 12 studies used valve tissue samples and blood cultures while only 1 study used whole blood. Also, 10 studies used the molecular method of PCR while 2 studies used real-time PCR. Most studies used 16S rDNA gene as the target gene. The bacteria were identified as the most common microorganisms involved in infective endocarditis. Streptococcus spp. and Staphylococcus spp. were, by far, the most predominant bacteria detected. In all studies, PCR and real-time PCR identified more pathogens than blood and tissue cultures; moreover, the sensitivity and specificity of PCR and real-time PCR were more than cultures in most of the studies. The highest sensitivity and specificity were 96% and 100%, respectively. The gram positive bacteria were the most frequent cause of infective endocarditis. The molecular methods enjoy a greater sensitivity compared to the conventional blood culture methods; yet, they are applicable only to the valve tissue of the patients undergoing cardiac valve surgery.

The diagnosis of microorganism involved in infective endocarditis (IE) by polymerase chain reaction (PCR) and real-time PCR: A systematic review.

Science.gov (United States)

Faraji, Reza; Behjati-Ardakani, Mostafa; Moshtaghioun, Seyed Mohammad; Kalantar, Seyed Mehdi; Namayandeh, Seyedeh Mahdieh; Soltani, Mohammadhossien; Emami, Mahmood; Zandi, Hengameh; Firoozabadi, Ali Dehghani; Kazeminasab, Mahmood; Ahmadi, Nastaran; Sarebanhassanabadi, Mohammadtaghi

2018-02-01

Broad-range bacterial rDNA polymerase chain reaction (PCR) followed by sequencing may be identified as the etiology of infective endocarditis (IE) from surgically removed valve tissue; therefore, we reviewed the value of molecular testing in identifying organisms' DNA in the studies conducted until 2016. We searched Google Scholar, Scopus, ScienceDirect, Cochrane, PubMed, and Medline electronic databases without any time limitations up to December 2016 for English studies reporting microorganisms involved in infective endocarditis microbiology using PCR and real-time PCR. Most studies were prospective. Eleven out of 12 studies used valve tissue samples and blood cultures while only 1 study used whole blood. Also, 10 studies used the molecular method of PCR while 2 studies used real-time PCR. Most studies used 16S rDNA gene as the target gene. The bacteria were identified as the most common microorganisms involved in infective endocarditis. Streptococcus spp. and Staphylococcus spp. were, by far, the most predominant bacteria detected. In all studies, PCR and real-time PCR identified more pathogens than blood and tissue cultures; moreover, the sensitivity and specificity of PCR and real-time PCR were more than cultures in most of the studies. The highest sensitivity and specificity were 96% and 100%, respectively. The gram positive bacteria were the most frequent cause of infective endocarditis. The molecular methods enjoy a greater sensitivity compared to the conventional blood culture methods; yet, they are applicable only to the valve tissue of the patients undergoing cardiac valve surgery. Copyright © 2017. Published by Elsevier Taiwan.

Clinical Usefulness of Real-Time Polymerase Chain Reaction for the Diagnosis of Vibrio vulnificus Infection Using Skin and Soft Tissues.

Science.gov (United States)

Lee, Jun-Young; Kim, Seok Won; Kim, Dong-Min; Yun, Na Ra; Kim, Choon-Mee; Lee, Sang-Hong

2017-08-01

Vibrio vulnificus is a halophilic gram-negative bacillus isolated in seawater, fish, and shellfish. Infection by V. vulnificus is the most severe food-borne infection reported in the United States of America. Here, we aimed to examine the clinical usefulness of polymerase chain reaction (PCR) using tissue specimens other than blood samples as a diagnostic tool for V. vulnificus infection. A retrospective study was conducted with patients who underwent real-time PCR of toxR in both blood and skin tissues, including serum, bullae, swab, and operation room specimens, between 2006 and 2009. The median V. vulnificus DNA load of 14 patients in real-time PCR analysis of serum at the time of admission was 638.5 copies/mL blood, which was within the interquartile range (IQR: 37-3,225). In contrast, the median value by real-time PCR using the first tissue specimen at the time of admission was 16,650 copies/mL tissue fluid (IQR: 4,419-832,500). This difference was statistically significant ( P = 0.022). DNA copy numbers in tissues were less affected by short-term antibiotic administration than that in blood samples, and antibiotic administration increased the DNA copy number in some patients. We found, for the first time, that DNA copy numbers in tissues of patients infected by V. vulnificus were higher than those in blood samples. Additionally, skin lesions were more useful than blood samples as specimens for PCR analysis in patients administered antibiotics for V. vulnificus infection before admission.

Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis.

Science.gov (United States)

Peeters, M; Huang, C L; Vonk, L A; Lu, Z F; Bank, R A; Helder, M N; Doulabi, B Zandieh

2016-11-01

Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised.Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016

A sensitive detection method for MPLW515L or MPLW515K mutation in chronic myeloproliferative disorders with locked nucleic acid-modified probes and real-time polymerase chain reaction.

Science.gov (United States)

Pancrazzi, Alessandro; Guglielmelli, Paola; Ponziani, Vanessa; Bergamaschi, Gaetano; Bosi, Alberto; Barosi, Giovanni; Vannucchi, Alessandro M

2008-09-01

Acquired mutations in the juxtamembrane region of MPL (W515K or W515L), the receptor for thrombopoietin, have been described in patients with primary myelofibrosis or essential thrombocythemia, which are chronic myeloproliferative disorders. We have developed a real-time polymerase chain reaction assay for the detection and quantification of MPL mutations that is based on locked nucleic acid fluorescent probes. Mutational analysis was performed using DNA from granulocytes. Reference curves were obtained using cloned fragments of MPL containing either the wild-type or mutated sequence; the predicted sensitivity level was at least 0.1% mutant allele in a wild-type background. None of the 60 control subjects presented with a MPLW515L/K mutation. Of 217 patients with myelofibrosis, 19 (8.7%) harbored the MPLW515 mutation, 10 (52.6%) with the W515L allele. In one case, both the W515L and W515K alleles were detected by real-time polymerase chain reaction. By comparing results obtained with conventional sequencing, no erroneous genotype attribution using real-time polymerase chain reaction was found, whereas one patient considered wild type according to sequence analysis actually harbored a low W515L allele burden. This is a simple, sensitive, and cost-effective procedure for large-scale screening of the MPLW515L/K mutation in patients suspected to have a myeloproliferative disorder. It can also provide a quantitative estimate of mutant allele burden that might be useful for both patient prognosis and monitoring response to therapy.

Real-time PCR in virology.

Science.gov (United States)

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

2002-03-15

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

Real-time polymerase chain reaction assay for the rapid detection and characterization of chloroquine-resistant Plasmodium falciparum malaria in returned travelers.

Science.gov (United States)

Farcas, Gabriella A; Soeller, Rainer; Zhong, Kathleen; Zahirieh, Alireza; Kain, Kevin C

2006-03-01

Imported drug-resistant malaria is a growing problem in industrialized countries. Rapid and accurate diagnosis is essential to prevent malaria-associated mortality in returned travelers. However, outside of a limited number of specialized centers, the microscopic diagnosis of malaria is slow, unreliable, and provides little information about drug resistance. Molecular diagnostics have the potential to overcome these limitations. We developed and evaluated a rapid, real-time polymerase chain reaction (PCR) assay to detect Plasmodium falciparum malaria and chloroquine (CQ)-resistance determinants in returned travelers who are febrile. A real-time PCR assay based on detection of the K76T mutation in PfCRT (K76T) of P. falciparum was developed on a LightCycler platform (Roche). The performance characteristics of the real-time assay were compared with those of the nested PCR-restriction fragment-length polymorphism (RFLP) and the sequence analyses of samples obtained from 200 febrile returned travelers, who included 125 infected with P. falciparum (48 of whom were infected CQ-susceptible [K76] and 77 of whom were CQ-resistant [T76] P. falciparum), 22 infected with Plasmodium vivax, 10 infected with Plasmodium ovale, 3 infected with Plasmodium malariae malaria, and 40 infected with other febrile syndromes. All patient samples were coded, and all analyses were performed blindly. The real-time PCR assay detected multiple pfcrt haplotypes associated with CQ resistance in geographically diverse malaria isolates acquired by travelers. Compared with nested-PCR RFLP (the reference standard), the real-time assay was 100% sensitive and 96.2% specific for detection of the P. falciparum K76T mutation. This assay is rapid, sensitive, and specific for the detection and characterization of CQ-resistant P. falciparum malaria in returned travelers. This assay is automated, standardized, and suitable for routine use in clinical diagnostic laboratories.

Quantification of Xylella fastidiosa from Citrus Trees by Real-Time Polymerase Chain Reaction Assay.

Science.gov (United States)

Oliveira, Antonio C; Vallim, Marcelo A; Semighini, Camile P; Araújo, Welington L; Goldman, Gustavo H; Machado, Marcos A

2002-10-01

ABSTRACT Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease of sweet orange cultivars in Brazil. Polymerase chain reaction (PCR)-based assays constitute the principal diagnostic method for detection of these bacteria. In this work, we established a real-time quantitative PCR (QPCR) assay to quantify X. fastidiosa in naturally and artificially infected citrus. The X. fastidiosa cell number detected in the leaves increased according to the age of the leaf, and bacteria were not detected in the upper midrib section in young leaves, indicating temporal and spatial distribution patterns of bacteria, respectively. In addition, the X. fastidiosa cell number quantified in leaves of 'Pera' orange and 'Murcott' tangor reflected the susceptible and resistant status of these citrus cultivars. None of the 12 endophytic citrus bacteria or the four strains of X. fastidiosa nonpathogenic to citrus that were tested showed an increase in the fluorescence signal during QPCR. In contrast, all 10 CVC-causing strains exhibited an increase in fluorescence signal, thus indicating the specificity of this QPCR assay. Our QPCR provides a powerful tool for studies of different aspects of the Xylella-citrus interactions, and can be incorporated into breeding programs in order to select CVC-resistant plants more quickly.

Real-time polymerase chain reaction assay for endogenous reference gene for specific detection and quantification of common wheat-derived DNA (Triticum aestivum L.).

Science.gov (United States)

Vautrin, Sonia; Zhang, David

2007-01-01

A species-specific endogenous reference gene system was developed for polymerase chain reaction (PCR)-based analysis in common wheat (Triticum aestivum L.) by targeting the ALMT1 gene, an aluminium-activated malate transporter. The primers and probe were elaborated for real-time PCR-based qualitative and quantitative assay. The size of amplified product is 95 base pairs. The specificity was assessed on 17 monocot and dicot plant species. The established real-time PCR assay amplified only T. aestivum-derived DNA; no amplification occurred on other phylogenetically related species, including durum wheat (T. durum). The robustness of the system was tested on the DNA of 15 common wheat cultivars using 20 000 genomic copies per PCR the mean cycle threshold (Ct) values of 24.02 +/- 0.251 were obtained. The absolute limits of detection and quantification of the real-time PCR assay were estimated to 2 and 20 haploid genome copies of common wheat, respectively. The linearity was experimentally validated on 2-fold serial dilutions of DNA from 650 to 20 000 haploid genome copies. All these results show that the real-time PCR assay developed on the ALMT1 gene is suitable to be used as an endogenous reference gene for PCR-based specific detection and quantification of T. aestivum-derived DNA in various applications, in particular for the detection and quantification of genetically modified materials in common wheat.

Development and validation of a SYBR Green I-based real-time polymerase chain reaction method for detection of haptoglobin gene deletion in clinical materials.

Science.gov (United States)

Soejima, Mikiko; Tsuchiya, Yuji; Egashira, Kouichi; Kawano, Hiroyuki; Sagawa, Kimitaka; Koda, Yoshiro

2010-06-01

Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin (Hp) antibodies. Being homozygous for the Hp gene deletion (HP(del)) is the only known cause of congenital anhaptoglobinemia, and clinical diagnosis of HP(del) before transfusion is important to prevent anaphylactic shock. We recently developed a 5'-nuclease (TaqMan) real-time polymerase chain reaction (PCR) method. A SYBR Green I-based duplex real-time PCR assay using two forward primers and a common reverse primer followed by melting curve analysis was developed to determine HP(del) zygosity in a single tube. In addition, to obviate initial DNA extraction, we examined serially diluted blood samples as PCR templates. Allelic discrimination of HP(del) yielded optimal results at blood sample dilutions of 1:64 to 1:1024. The results from 2231 blood samples were fully concordant with those obtained by the TaqMan-based real-time PCR method. The detection rate of the HP(del) allele by the SYBR Green I-based method is comparable with that using the TaqMan-based method. This method is readily applicable due to its low initial cost and analyzability using economical real-time PCR machines and is suitable for high-throughput analysis as an alternative method for allelic discrimination of HP(del).

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

DEFF Research Database (Denmark)

Hoffmann, Bernd; Blome, Sandra; Bonilauri, Paolo

2011-01-01

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

Genetic traits of avascular necrosis of the femoral head analyzed by array comparative genomic hybridization and real-time polymerase chain reaction.

Science.gov (United States)

Hwang, Jung-Taek; Baik, Seung-Ho; Choi, Jin-Soo; Lee, Kweon-Haeng; Rhee, Seung-Koo

2011-01-03

In an attempt to observe the genetic traits of avascular necrosis of the femoral head, we analyzed the genomic alterations in blood samples of 18 patients with avascular necrosis of the femoral head (9 idiopathic and 9 alcoholic cases) using the array comparative genomic hybridization method and real-time polymerase chain reaction. Several candidate genes were identified that may induce avascular necrosis of the femoral head, and we investigated their role in the pathomechanism of osteonecrosis of bone. The frequency of each candidate gene over all the categories of avascular necrosis of the femoral head was also calculated by real-time polymerase chain reaction. The highest frequency specific genes in each category were FLJ40296, CYP27C1, and CTDP1. FLJ40296 and CYP27C1 had the highest frequency (55.6%) in the idiopathic category. FLJ40296 had a high frequency (44.4%) in the alcoholic category, but CYP27C1 had a relatively low frequency (33.3%) in the alcoholic category. However, CTDP1 showed a significantly high frequency (55.6%) in the alcoholic category and a low frequency (22.2%) in the idiopathic category. Although we statistically analyzed the frequency of each gene with Fisher's exact test, we could not prove statistical significance due to the small number of samples. Further studies are needed with larger sample numbers. If the causal genes of avascular necrosis of the femoral head are found, they may be used for early detection, prognosis prediction, and genomic treatment of avascular necrosis of the femoral head in the future. Copyright 2011, SLACK Incorporated.

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.

Quantification of Staphylococcus aureus and Staphylococcus epidermidis on the hands of health-care workers using a real-time polymerase chain reaction method

DEFF Research Database (Denmark)

Horn, P; Schouenborg, P Øland; Brandslund, I

2007-01-01

OBJECTIVE: The objective of this study was to test a polymerase chain reaction (PCR) assay intended as a tool for monitoring hand hygiene in hospital wards. METHODS: The hands of 20 health-care workers were sampled for 10 days using real-time PCR for quantification of Staphylococcus aureus and S....... epidermidis. Reference intervals (CI) and biological variation were evaluated using index of individuality (II) and critical difference (CD). RESULTS: 45% of the participants were positive for S. aureus on all 10 days. Intra-individual biological variation (CVI) was 129% for S. aureus and 62% for S...

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.

Evaluating viral interference between Influenza virus and Newcastle disease virus using real-time reverse transcription–polymerase chain reaction in chicken eggs

Directory of Open Access Journals (Sweden)

Ge Shengqiang

2012-07-01

Full Text Available Abstract Background Simultaneous and sequential allantoic cavity inoculations of Specific-pathogen-free (SPF chicken eggs with Influenza virus (AIV and Newcastle disease virus (NDV demonstrated that the interaction of AIV and NDV during co-infection was variable. Our research revisited the replication interference potential of AIV and NDV using real-time reverse transcription–polymerase chain reaction (real-time RT-PCR for AIV and NDV to specifically detect the viral genomes in mixed infections. Results Data from this survey showed that when different doses of NDV (Lasota or F48E8 and AIV (F98 or H5N1 were simultaneously inoculated into embryonating chicken eggs (ECE, interference with the growth of NDV occurred, while interference with the growth of AIV did not occur. When equal amount of the two viruses were sequentially employed, the degree of interference was dependent upon the time of superinfection and the virulence of virus. Conclusion AIV have a negative impact on NDV growth if they are inoculated simultaneously or sequentially and that the degree of interference depended upon the quantity and relative virulence of the virus strains used; however, interference with AIV was not observed. Only if NDV were inoculated at an earlier time will NDV able to interfere with the growth of AIV.

Real-time PCR in virology

OpenAIRE

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

2002-01-01

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

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

Directory of Open Access Journals (Sweden)

Laurent Dacheux

2016-07-01

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

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

Science.gov (United States)

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

2016-07-01

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

Cost analysis of real-time polymerase chain reaction microbiological diagnosis in patients with septic shock.

Science.gov (United States)

Alvarez, J; Mar, J; Varela-Ledo, E; Garea, M; Matinez-Lamas, L; Rodriguez, J; Regueiro, B

2012-11-01

Antibiotic treatment for septic shock is generally prescribed on an empirical basis using broad-spectrum antibiotics. Molecular diagnostic techniques can detect the presence of microbial DNA in blood within a few hours and facilitate early, targeted treatment. The aim of this study was to evaluate the economic impact of a real-time polymerase chain reaction technique, LightCycler SeptiFast (LSC), in patients with sepsis. A cost-minimisation study was carried out in patients admitted with a diagnosis of severe sepsis or septic shock to the intensive care unit of a university hospital. The stay in the intensive care unit, hospital admission, 28-day and six-month mortality, and the economic cost of the clinical process were also evaluated. The study involved 48 patients in the LSC group and 54 patients in the control group. The total cost was €42,198 in the control group versus €32,228 in the LCS group with statistically significant differences (P average net saving of €9970 per patient. The mortality rate was similar in both groups. The main finding of this study was the significant economic saving afforded by the use of the LCS technique, due to the shortening of intensive care unit stay and the use of fewer antibiotics.

A centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria.

Science.gov (United States)

Choi, Goro; Jung, Jae Hwan; Park, Byung Hyun; Oh, Seung Jun; Seo, Ji Hyun; Choi, Jong Seob; Kim, Do Hyun; Seo, Tae Seok

2016-06-21

In this study, we developed a centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria contaminated milk samples. The microdevice was designed to contain identical triplicate functional units and each unit has four reaction chambers, thereby making it possible to perform twelve direct-RPA reactions simultaneously. The integrated microdevice consisted of two layers: RPA reagents were injected in the top layer, while spiked milk samples with food poisoning bacteria were loaded into sample reservoirs in the bottom layer. For multiplex bacterial detection, the target gene-specific primers and probes were dried in each reaction chamber. The introduced samples and reagents could be equally aliquoted and dispensed into each reaction chamber by centrifugal force, and then the multiplex direct-RPA reaction was executed. The target genes of bacteria spiked in milk could be amplified at 39 °C without a DNA extraction step by using the direct-RPA cocktails, which were a combination of a direct PCR buffer and RPA enzymes. As the target gene amplification proceeded, the increased fluorescence signals coming from the reaction chambers were recorded in real-time at an interval of 2 min. The entire process, including the sample distribution, the direct-RPA reaction, and the real-time analysis, was accomplished with a custom-made portable genetic analyzer and a miniaturized optical detector. Monoplex, duplex, and triplex food poisoning bacteria (Salmonella enterica, Escherichia coli O157:H7, and Vibrio parahaemolyticus) detection was successfully performed with a detection sensitivity of 4 cells per 3.2 μL of milk samples within 30 min. By implementing the direct-PRA on the miniaturized centrifugal microsystem, the on-site food poisoning bacteria analysis would be feasible with high speed, sensitivity, and multiplicity.

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

DEFF Research Database (Denmark)

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

2015-01-01

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

Generation and Comprehensive Analysis of an Influenza Virus Polymerase Cellular Interaction Network▿†§

Science.gov (United States)

Tafforeau, Lionel; Chantier, Thibault; Pradezynski, Fabrine; Pellet, Johann; Mangeot, Philippe E.; Vidalain, Pierre-Olivier; Andre, Patrice; Rabourdin-Combe, Chantal; Lotteau, Vincent

2011-01-01

The influenza virus transcribes and replicates its genome inside the nucleus of infected cells. Both activities are performed by the viral RNA-dependent RNA polymerase that is composed of the three subunits PA, PB1, and PB2, and recent studies have shown that it requires host cell factors to transcribe and replicate the viral genome. To identify these cellular partners, we generated a comprehensive physical interaction map between each polymerase subunit and the host cellular proteome. A total of 109 human interactors were identified by yeast two-hybrid screens, whereas 90 were retrieved by literature mining. We built the FluPol interactome network composed of the influenza virus polymerase (PA, PB1, and PB2) and the nucleoprotein NP and 234 human proteins that are connected through 279 viral-cellular protein interactions. Analysis of this interactome map revealed enriched cellular functions associated with the influenza virus polymerase, including host factors involved in RNA polymerase II-dependent transcription and mRNA processing. We confirmed that eight influenza virus polymerase-interacting proteins are required for virus replication and transcriptional activity of the viral polymerase. These are involved in cellular transcription (C14orf166, COPS5, MNAT1, NMI, and POLR2A), translation (EIF3S6IP), nuclear transport (NUP54), and DNA repair (FANCG). Conversely, we identified PRKRA, which acts as an inhibitor of the viral polymerase transcriptional activity and thus is required for the cellular antiviral response. PMID:21994455

VERSE - Virtual Equivalent Real-time Simulation

Science.gov (United States)

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

2005-01-01

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

Standardization of a two-step real-time polymerase chain reaction based method for species-specific detection of medically important Aspergillus species.

Science.gov (United States)

Das, P; Pandey, P; Harishankar, A; Chandy, M; Bhattacharya, S; Chakrabarti, A

2017-01-01

Standardization of Aspergillus polymerase chain reaction (PCR) poses two technical challenges (a) standardization of DNA extraction, (b) optimization of PCR against various medically important Aspergillus species. Many cases of aspergillosis go undiagnosed because of relative insensitivity of conventional diagnostic methods such as microscopy, culture or antigen detection. The present study is an attempt to standardize real-time PCR assay for rapid sensitive and specific detection of Aspergillus DNA in EDTA whole blood. Three nucleic acid extraction protocols were compared and a two-step real-time PCR assay was developed and validated following the recommendations of the European Aspergillus PCR Initiative in our setup. In the first PCR step (pan-Aspergillus PCR), the target was 28S rDNA gene, whereas in the second step, species specific PCR the targets were beta-tubulin (for Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus), gene and calmodulin gene (for Aspergillus niger). Species specific identification of four medically important Aspergillus species, namely, A. fumigatus, A. flavus, A. niger and A. terreus were achieved by this PCR. Specificity of the PCR was tested against 34 different DNA source including bacteria, virus, yeast, other Aspergillus sp., other fungal species and for human DNA and had no false-positive reactions. The analytical sensitivity of the PCR was found to be 102 CFU/ml. The present protocol of two-step real-time PCR assays for genus- and species-specific identification for commonly isolated species in whole blood for diagnosis of invasive Aspergillus infections offers a rapid, sensitive and specific assay option and requires clinical validation at multiple centers.

Real-time polymerase chain reaction-based approach for quantification of the pat gene in the T25 Zea mays event.

Science.gov (United States)

Weighardt, Florian; Barbati, Cristina; Paoletti, Claudia; Querci, Maddalena; Kay, Simon; De Beuckeleer, Marc; Van den Eede, Guy

2004-01-01

In Europe, a growing interest for reliable techniques for the quantification of genetically modified component(s) of food matrixes is arising from the need to comply with the European legislative framework on novel food products. Real-time polymerase chain reaction (PCR) is currently the most powerful technique for the quantification of specific nucleic acid sequences. Several real-time PCR methodologies based on different molecular principles have been developed for this purpose. The most frequently used approach in the field of genetically modified organism (GMO) quantification in food or feed samples is based on the 5'-3'-exonuclease activity of Taq DNA polymerase on specific degradation probes (TaqMan principle). A novel approach was developed for the establishment of a TaqMan quantification system assessing GMO contents around the 1% threshold stipulated under European Union (EU) legislation for the labeling of food products. The Zea mays T25 elite event was chosen as a model for the development of the novel GMO quantification approach. The most innovative aspect of the system is represented by the use of sequences cloned in plasmids as reference standards. In the field of GMO quantification, plasmids are an easy to use, cheap, and reliable alternative to Certified Reference Materials (CRMs), which are only available for a few of the GMOs authorized in Europe, have a relatively high production cost, and require further processing to be suitable for analysis. Strengths and weaknesses of the use of novel plasmid-based standards are addressed in detail. In addition, the quantification system was designed to avoid the use of a reference gene (e.g., a single copy, species-specific gene) as normalizer, i.e., to perform a GMO quantification based on an absolute instead of a relative measurement. In fact, experimental evidences show that the use of reference genes adds variability to the measurement system because a second independent real-time PCR-based measurement

ArControl: An Arduino-Based Comprehensive Behavioral Platform with Real-Time Performance.

Science.gov (United States)

Chen, Xinfeng; Li, Haohong

2017-01-01

Studying animal behavior in the lab requires reliable delivering stimulations and monitoring responses. We constructed a comprehensive behavioral platform (ArControl: Arduino Control Platform) that was an affordable, easy-to-use, high-performance solution combined software and hardware components. The hardware component was consisted of an Arduino UNO board and a simple drive circuit. As for software, the ArControl provided a stand-alone and intuitive GUI (graphical user interface) application that did not require users to master scripts. The experiment data were automatically recorded with the built in DAQ (data acquisition) function. The ArControl also allowed the behavioral schedule to be entirely stored in and operated on the Arduino chip. This made the ArControl a genuine, real-time system with high temporal resolution (<1 ms). We tested the ArControl, based on strict performance measurements and two mice behavioral experiments. The results showed that the ArControl was an adaptive and reliable system suitable for behavioral research.

Use of sodC versus ctrA for real-time polymerase chain reaction-based detection of Neisseria meningitidis in sterile body fluids

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Fábio Takenori Higa

2013-04-01

Full Text Available We evaluated the use of a newly described sodC-based real-time-polymerase chain reaction (RT-PCR assay for detecting Neisseria meningitidis in normally sterile sites, such as cerebrospinal fluid and serum. The sodC-based RT-PCR assay has an advantage over ctrA for detecting nongroupable N. meningitidis isolates, which are commonly present in asymptomatic pharyngeal carriage. However, in our study, sodC-based RT-PCR was 7.5% less sensitive than ctrA. Given the public health impact of possible false-negative results due to the use of the sodC target gene alone, sodC-based RT-PCR for the diagnosis of meningococcal meningitis should be used with caution.

Development of Real-Time Quantitative Polymerase Chain Reaction Assays to Track Treatment Response in Retinoid Resistant Acute Promyelocytic Leukemia

Energy Technology Data Exchange (ETDEWEB)

Jovanovic, Jelena V. [Cancer Genetics Laboratory, Department of Medical and Molecular Genetics, King’s College London School of Medicine, London (United Kingdom); Rennie, Kristian [GSTS Pathology, Guy’s Hospital, London (United Kingdom); Culligan, Dominic [Department of Haematology, Aberdeen Royal Infirmary, Aberdeen (United Kingdom); Peniket, Andrew [Department of Haematology, John Radcliffe Hospital, Oxford (United Kingdom); Lennard, Anne [Department of Haematology, Royal Victoria Infirmary, Newcastle (United Kingdom); Harrison, Justin [Department of Haematology, Hemel Hempstead Hospital, Hemel Hempstead (United Kingdom); Vyas, Paresh [Medical Research Council Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford (United Kingdom); Grimwade, David, E-mail: david.grimwade@genetics.kcl.ac.uk [Cancer Genetics Laboratory, Department of Medical and Molecular Genetics, King’s College London School of Medicine, London (United Kingdom)

2011-10-25

Molecular detection of minimal residual disease (MRD) has become established to assess remission status and guide therapy in patients with ProMyelocytic Leukemia–RARA+ acute promyelocytic leukemia (APL). However, there are few data on tracking disease response in patients with rarer retinoid resistant subtypes of APL, characterized by PLZF–RARA and STAT5b–RARA. Despite their rarity (<1% of APL) we identified 6 cases (PLZF–RARA, n = 5; STAT5b–RARA, n = 1), established the respective breakpoint junction regions and designed reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) assays to detect leukemic transcripts. The relative level of fusion gene expression in diagnostic samples was comparable to that observed in t(15;17) – associated APL, affording assay sensitivities of ∼1 in 10{sup 4}−10{sup 5}. Serial samples were available from two PLZF–RARA APL patients. One showed persistent polymerase chain reaction positivity, predicting subsequent relapse, and remains in CR2, ∼11 years post-autograft. The other, achieved molecular remission (CRm) with combination chemotherapy, remaining in CR1 at 6 years. The STAT5b–RARA patient failed to achieve CRm following frontline combination chemotherapy and ultimately proceeded to allogeneic transplant on the basis of a steadily rising fusion transcript level. These data highlight the potential of RT-qPCR detection of MRD to facilitate development of more individualized approaches to the management of rarer molecularly defined subsets of acute leukemia.

Development of Real-Time Quantitative Polymerase Chain Reaction Assays to Track Treatment Response in Retinoid Resistant Acute Promyelocytic Leukemia

International Nuclear Information System (INIS)

Jovanovic, Jelena V.; Rennie, Kristian; Culligan, Dominic; Peniket, Andrew; Lennard, Anne; Harrison, Justin; Vyas, Paresh; Grimwade, David

2011-01-01

Molecular detection of minimal residual disease (MRD) has become established to assess remission status and guide therapy in patients with ProMyelocytic Leukemia–RARA+ acute promyelocytic leukemia (APL). However, there are few data on tracking disease response in patients with rarer retinoid resistant subtypes of APL, characterized by PLZF–RARA and STAT5b–RARA. Despite their rarity (<1% of APL) we identified 6 cases (PLZF–RARA, n = 5; STAT5b–RARA, n = 1), established the respective breakpoint junction regions and designed reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) assays to detect leukemic transcripts. The relative level of fusion gene expression in diagnostic samples was comparable to that observed in t(15;17) – associated APL, affording assay sensitivities of ∼1 in 10 4 −10 5 . Serial samples were available from two PLZF–RARA APL patients. One showed persistent polymerase chain reaction positivity, predicting subsequent relapse, and remains in CR2, ∼11 years post-autograft. The other, achieved molecular remission (CRm) with combination chemotherapy, remaining in CR1 at 6 years. The STAT5b–RARA patient failed to achieve CRm following frontline combination chemotherapy and ultimately proceeded to allogeneic transplant on the basis of a steadily rising fusion transcript level. These data highlight the potential of RT-qPCR detection of MRD to facilitate development of more individualized approaches to the management of rarer molecularly defined subsets of acute leukemia.

Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots

Science.gov (United States)

Sang, Fuming; Yang, Yang; Yuan, Lin; Ren, Jicun; Zhang, Zhizhou

2015-09-01

Hot start (HS) PCR is an excellent alternative for high-throughput real time PCR due to its ability to prevent nonspecific amplification at low temperature. Development of a cost-effective and simple HS PCR technique to guarantee high-throughput PCR specificity and consistency still remains a great challenge. In this study, we systematically investigated the HS characteristics of QDs triggered in real time PCR with EvaGreen and SYBR Green I dyes by the analysis of amplification curves, standard curves and melting curves. Two different kinds of DNA polymerases, Pfu and Taq, were employed. Here we showed that high specificity and efficiency of real time PCR were obtained in a plasmid DNA and an error-prone two-round PCR assay using QD-based HS PCR, even after an hour preincubation at 50 °C before real time PCR. Moreover, the results obtained by QD-based HS PCR were comparable to a commercial Taq antibody DNA polymerase. However, no obvious HS effect of QDs was found in real time PCR using Taq DNA polymerase. The findings of this study demonstrated that a cost-effective high-throughput real time PCR based on QD triggered HS PCR could be established with high consistency, sensitivity and accuracy.Hot start (HS) PCR is an excellent alternative for high-throughput real time PCR due to its ability to prevent nonspecific amplification at low temperature. Development of a cost-effective and simple HS PCR technique to guarantee high-throughput PCR specificity and consistency still remains a great challenge. In this study, we systematically investigated the HS characteristics of QDs triggered in real time PCR with EvaGreen and SYBR Green I dyes by the analysis of amplification curves, standard curves and melting curves. Two different kinds of DNA polymerases, Pfu and Taq, were employed. Here we showed that high specificity and efficiency of real time PCR were obtained in a plasmid DNA and an error-prone two-round PCR assay using QD-based HS PCR, even after an hour

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

A Comprehensive Optimization Strategy for Real-time Spatial Feature Sharing and Visual Analytics in Cyberinfrastructure

Science.gov (United States)

Li, W.; Shao, H.

2017-12-01

For geospatial cyberinfrastructure enabled web services, the ability of rapidly transmitting and sharing spatial data over the Internet plays a critical role to meet the demands of real-time change detection, response and decision-making. Especially for the vector datasets which serve as irreplaceable and concrete material in data-driven geospatial applications, their rich geometry and property information facilitates the development of interactive, efficient and intelligent data analysis and visualization applications. However, the big-data issues of vector datasets have hindered their wide adoption in web services. In this research, we propose a comprehensive optimization strategy to enhance the performance of vector data transmitting and processing. This strategy combines: 1) pre- and on-the-fly generalization, which automatically determines proper simplification level through the introduction of appropriate distance tolerance (ADT) to meet various visualization requirements, and at the same time speed up simplification efficiency; 2) a progressive attribute transmission method to reduce data size and therefore the service response time; 3) compressed data transmission and dynamic adoption of a compression method to maximize the service efficiency under different computing and network environments. A cyberinfrastructure web portal was developed for implementing the proposed technologies. After applying our optimization strategies, substantial performance enhancement is achieved. We expect this work to widen the use of web service providing vector data to support real-time spatial feature sharing, visual analytics and decision-making.

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

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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.

Comparison between culture and a multiplex quantitative real-time polymerase chain reaction assay detecting Ureaplasma urealyticum and U. parvum.

Science.gov (United States)

Frølund, Maria; Björnelius, Eva; Lidbrink, Peter; Ahrens, Peter; Jensen, Jørgen Skov

2014-01-01

A novel multiplex quantitative real-time polymerase chain reaction (qPCR) for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR.

Comparison between culture and a multiplex quantitative real-time polymerase chain reaction assay detecting Ureaplasma urealyticum and U. parvum.

Directory of Open Access Journals (Sweden)

Maria Frølund

Full Text Available A novel multiplex quantitative real-time polymerase chain reaction (qPCR for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR.

A quantitative real time polymerase chain reaction approach for estimating processed animal proteins in feed: preliminary data

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Maria Cesarina Abete

2013-04-01

Full Text Available Lifting of the ban on the use of processed animal proteins (PAPs from non-ruminants in non-ruminant feed is in the wind, avoiding intraspecies recycling. Discrimination of species will be performed through polymerase chain reaction (PCR, which is at a moment a merely qualitative method. Nevertheless, quantification of PAPs in feed is needed. The aim of this study was to approach the quantitative determination of PAPs in feed through Real Time (RT-PCR technique; three different protocols picked up from the literature were tested. Three different kind of matrices were examined: pure animal meals (bovine, chicken and pork; one feed sample certified by the European reference laboratory on animal proteins (EURL AP in feed spiked with 0.1% bovine meal; and genomic DNAs from bovine, chicken and pork muscles. The limit of detection (LOD of the three protocols was set up. All the results obtained from the three protocols considered failed in the quantification process, most likely due to the uncertain copy numbers of the analytical targets chosen. This preliminary study will allow us to address further investigations, with the purpose of developing a RT-PCR quantitative method.

Evaluation of the efficacy of real-time polymerase chain reaction for the routine early detection of Pseudomonas aeruginosa in cystic fibrosis sputum and throat swab specimens.

LENUS (Irish Health Repository)

Logan, Catriona

2012-02-01

A longitudinal study of 2099 sputa and throat swabs received from 183 pediatric cystic fibrosis patients over a 29-month period was used to evaluate the efficacy of real-time polymerase chain reaction (PCR) for the early detection of Pseudomonas aeruginosa as compared to microbiologic culture. Real-time PCR resulted in an increased number of specimens identified as P. aeruginosa positive. The sensitivity of culture was 82% (373\\/453) and of PCR was 93% (420\\/453) when considering both positive culture and PCR results as true positives. Of the 80 specimens identified as PCR positive\\/culture negative for P. aeruginosa, the subsequent patient sample in 32.5% (26\\/80) of specimens concerned was identified as P. aeruginosa culture positive, suggesting that PCR has the potential to detect P. aeruginosa earlier than the microbiologic culture. Real-time PCR analysis found no evidence of the Liverpool and Manchester epidemic P. aeruginosa strains in the cohort examined. The findings of this study highlight the importance of specimen collection protocols to ensure that adequate samples are received at the laboratory for testing, thereby minimizing the potential for reporting of false-negative P. aeruginosa culture results.

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

Science.gov (United States)

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

2011-12-01

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

Relationship Between Ebola Virus Real-Time Quantitative Polymerase Chain Reaction-Based Threshold Cycle Value and Virus Isolation From Human Plasma.

Science.gov (United States)

Spengler, Jessica R; McElroy, Anita K; Harmon, Jessica R; Ströher, Ute; Nichol, Stuart T; Spiropoulou, Christina F

2015-10-01

We performed a longitudinal analysis of plasma samples obtained from 4 patients with Ebola virus (EBOV) disease (EVD) to determine the relationship between the real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)-based threshold cycle (Ct) value and the presence of infectious EBOV. EBOV was not isolated from plasma samples with a Ct value of >35.5 or >12 days after onset of symptoms. EBOV was not isolated from plasma samples in which anti-EBOV nucleoprotein immunoglobulin G was detected. These data demonstrate the utility of interpreting qRT-PCR results in the context of the course of EBOV infection and associated serological responses for patient-management decisions. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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.

Real-Time Polymerase Chain Reaction Detection of Trichomonas vaginalis from vaginal swabs: Validation of a Diagnostic Method and Preliminary Epidemiological Application

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Carlo Mengoli

2009-06-01

Full Text Available Background Trichomonas vaginalis is the most common nonviral sexually trasmitted diseases (STDs agent. For females, the diagnostic gold standard is the culture of vaginal swab, which is labour-exacting.The direct microscopic examination of vaginal secretions is the most used approach, but its sensitivity depends on the skill of the observer. Objectives We evaluated an original real-time TaqMan-based Polymerase Chain Reaction (PCR technique.The scope of the study was to confirm the effectiveness of the molecular approach in a clinical context and to explore its relevance to an epidemiological investigation. Study Design a ß-tubulin gene was chosen as target sequence.The assay was designed to exploit the quantitative potential of the TaqMan procedure.The population sample was 583 adult females presenting at the Service from January 2005 to December 2005.Three vaginal swabs were collected from each patient, one for wet mount microscopy, one for broth culture, and one for the molecular assay. Results The prevalence was 3.3% (culture, 3.1% (microscopy, 3.8% (PCR.An excess risk was detected in the immigrant population (risk ratio by PCR = 28. Conclusions The molecular approach was the most accurate way to detect the protozoon.The real-time PCR is convenient in a busy laboratory, provided the necessary equipment is available, and it is suitable for epidemiological investigation.

A sensitive detection method for MPLW515L or MPLW515K mutation in chronic myeloproliferative disorders with locked nucleic acid-modified probes and real-time polymerase chain reaction.

OpenAIRE

Alessandro, Pancrazzi; Paola, Guglielmelli; Vanessa, Ponziani; Gaetano, Bergamaschi; Alberto, Bosi; Giovanni, Barosi; Alessandro M, Vannucchi

2008-01-01

Acquired mutations in the juxtamembrane region of MPL (W515K or W515L), the receptor for thrombopoietin, have been described in patients with primary myelofibrosis or essential thrombocythemia, which are chronic myeloproliferative disorders. We have developed a real-time polymerase chain reaction assay for the detection and quantification of MPL mutations that is based on locked nucleic acid fluorescent probes. Mutational analysis was performed using DNA from granulocytes. Reference curves we...

Development of a diagnostic real-time polymerase chain reaction assay for the detection of invasive Haemophilus influenzae in clinical samples.

LENUS (Irish Health Repository)

Meyler, Kenneth L

2012-12-01

Since the introduction of the Haemophilus influenzae serotype b vaccine, invasive H. influenzae disease has become dominated by nontypeable (NT) strains. Several widely used molecular diagnostic methods have been shown to lack sensitivity or specificity in the detection of some of these strains. Novel real-time assays targeting the fucK, licA, and ompP2 genes were developed and evaluated. The fucK assay detected all strains of H. influenzae tested (n = 116) and had an analytical sensitivity of 10 genome copies\\/polymerase chain reaction (PCR). This assay detected both serotype b and NT H. influenzae in 12 previously positive specimens (culture and\\/or bexA PCR) and also detected H. influenzae in a further 5 of 883 culture-negative blood and cerebrospinal fluid (CSF) samples. The fucK assay has excellent potential as a diagnostic test for detection of typeable and nontypeable strains of invasive H. influenzae in clinical samples of blood and CSF.

Development of a diagnostic real-time polymerase chain reaction assay for the detection of invasive Haemophilus influenzae in clinical samples.

Science.gov (United States)

Meyler, Kenneth L; Meehan, Mary; Bennett, Desiree; Cunney, Robert; Cafferkey, Mary

2012-12-01

Since the introduction of the Haemophilus influenzae serotype b vaccine, invasive H. influenzae disease has become dominated by nontypeable (NT) strains. Several widely used molecular diagnostic methods have been shown to lack sensitivity or specificity in the detection of some of these strains. Novel real-time assays targeting the fucK, licA, and ompP2 genes were developed and evaluated. The fucK assay detected all strains of H. influenzae tested (n = 116) and had an analytical sensitivity of 10 genome copies/polymerase chain reaction (PCR). This assay detected both serotype b and NT H. influenzae in 12 previously positive specimens (culture and/or bexA PCR) and also detected H. influenzae in a further 5 of 883 culture-negative blood and cerebrospinal fluid (CSF) samples. The fucK assay has excellent potential as a diagnostic test for detection of typeable and nontypeable strains of invasive H. influenzae in clinical samples of blood and CSF. Copyright © 2012 Elsevier Inc. All rights reserved.

Dynamic Reconfiguration in Real-Time Systems Energy, Performance, and Thermal Perspectives

CERN Document Server

Wang, Weixun; Ranka, Sanjay

2013-01-01

Given the widespread use of real-time multitasking systems, there are tremendous optimization opportunities if reconfigurable computing can be effectively incorporated while maintaining performance and other design constraints of typical applications. The focus of this book is to describe the dynamic reconfiguration techniques that can be safely used in real-time systems. This book provides comprehensive approaches by considering synergistic effects of computation, communication as well as storage together to significantly improve overall performance, power, energy and temperature.  Provides a comprehensive introduction to optimization and dynamic reconfiguration techniques in real-time embedded systems; Covers state-of-the-art techniques and ongoing research in reconfigurable architectures; Focuses on algorithms tuned for dynamic reconfiguration techniques in real-time systems;  Provides reference for anyone designing low-power systems, energy-/temperature-constrained devices, and power-performance efficie...

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

Science.gov (United States)

Johnston, Stephen; Gallaher, Zachary; Czaja, Krzysztof

2012-05-15

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

A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

Directory of Open Access Journals (Sweden)

Meng Shuang

2010-06-01

Full Text Available Abstract Background The hepatitis C virus (HCV genome is extremely heterogeneous. Several HCV infections can not be detected using currently available commercial assays, probably because of mismatches between the template and primers/probes. By aligning the HCV sequences, we developed a duplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR assay using 2 sets of primers/probes and a specific armored RNA as internal control. The 2 detection probes were labelled with the same fluorophore, namely, 6-carboxyfluorescein (FAM, at the 5' end; these probes could mutually combine, improving the power of the test. Results The limit of detection of the duplex primer/probe assay was 38.99 IU/ml. The sensitivity of the assay improved significantly, while the specificity was not affected. All HCV genotypes in the HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques could be detected. In the testing of 109 serum samples, the performance of the duplex real-time RT-PCR assay was identical to that of the COBAS AmpliPrep (CAP/COBAS TaqMan (CTM assay and superior to 2 commercial HCV assay kits. Conclusions The duplex real-time RT-PCR assay is an efficient and effective viral assay. It is comparable with the CAP/CTM assay with regard to the power of the test and is appropriate for blood-donor screening and laboratory diagnosis of HCV infection.

Clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary Epstein-Barr virus infection in children.

Science.gov (United States)

Pitetti, Raymond D; Laus, Stella; Wadowsky, Robert M

2003-08-01

Epstein-Barr virus (EBV) infectious mononucleosis is often diagnosed based on characteristic clinical features and either a positive heterophil antibody test or serology, both of which can be unreliable in young children. Real time quantitative PCR assays that measure EBV DNA load in serum or plasma are highly sensitive in young children, but serum and plasma contain inhibitors of PCR which must be removed by DNA extraction techniques. A real time TaqMan PCR assay was designed and evaluated for simultaneously measuring EBV DNA load and validating the removal of PCR inhibitors from serum samples. A serum sample was available from patients classified serologically as primary EBV infection (n = 28), EBV-seronegative (n = 25) and EBV-seropositive (n = 26). Patients were classified as having EBV infectious mononucleosis if they had specified clinical findings and > or =10% atypical lymphocytes in peripheral blood or had a positive Monospot test result. DNA was purified by a spin column method and tested in PCR reactions with primers for EBV DNA polymerase gene and internal control targets. Amplification of the two PCR products was measured in real time with separate TaqMan DNA probes labeled with various fluorescent reporters. The mean age of study patients was 9 years, 4 months. Twenty-one (75%) of the patients in the primary EBV infection group, one (4%) of the seronegatives and none of the seropositives had detectable EBV DNA. Within the primary infection group, those with detectable virus were more likely than those without detectable virus to have evidence of lymphadenopathy (14 of 16 vs.1 of 5; P = 0.011), higher mean atypical (11.7 vs.0.9%; P = 0.002) and absolute atypical (1.5 vs.0.1 x 109/l; P = 0.004) lymphocyte count, higher mean absolute lymphocyte count (4.7 vs.2.3 x 109/l; P = 0.026) and higher mean aspartate aminotransferase value (119.8 vs.37.3 IU/l; P = 0.036). Ten patients, all in the primary infection group, had EBV infectious mononucleosis, and all

Newborn screening for congenital cytomegalovirus using real-time polymerase chain reaction in umbilical cord blood.

Science.gov (United States)

Barkai, Galia; Barzilai, Asher; Mendelson, Ella; Tepperberg-Oikawa, Michal; Roth, Daphne Ari-Even; Kuint, Jacob

2013-06-01

Congenital cytomegalovirus (C-CMV) infection affects 0.4-2% of newborn infants in Israel, most of whom are asymptomatic. Of these, 10-20% will subsequently develop hearing impairment and may have benetitted from early detection by neonatal screeing. To retrospectively anaIyze the results of a screening program for C-CMV performed at the Sheba Medical Center, Tel, Hashomer, during a 1 year period, using real-time polymerase chain reaction (rt-PCR) from umbilical cord blood. CMV DNA was detected by rt-PCR performed on infants' cord blood. C-CMV was confirmed by urine culture (Shell-vial). All confirmed cases were further investigated for C-CMV manifestations by head ultrasound, complete blood count, liver enzyme measurement, ophthalmology examination and hearing investigation. During the period 1 June 2009 to 31 May 2010, 11,022 infants were born at the Sheba Medical Center, of whom 8105 (74%) were screened. Twenty-three (0.28%) were positive for CMV and 22 of them (96%) were confirmed by urine culture. Two additional infants, who had not been screened, were detected after clinical suspicion. All 24 infants were further Investigated, and 3 (12.5%) had central nervous system involvement (including hearing impairment) and were offered intravenous ganciclovir for 6 weeks. Eighteen infants (82%) would not otherwise have been diagnosed. The relatively low incidence of C-CMV detected in our screening program probably reflects the low sensitivity of cord blood screening. Nevertheless, this screening program reliably detected a non-negligible number of infants who could benefit from early detection. Other screening methods using saliva should be investigated further.

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…

Quantification of algal endosymbionts (Symbiodinium) in coral tissue using real-time PCR

NARCIS (Netherlands)

Mieog, J. C.; Van Oppen, M. J. H.; Berkelmans, R.; Stam, W. T.; Olsen, J. L.

Understanding the flexibility of the endosymbioses between scleractinian corals and single-cell algae of the genus Symbiodinium will provide valuable insights into the future of coral reefs. Here, a real-time polymerase chain reaction (PCR) assay is presented to accurately determine the cell

Molecular Detection of the Carriage Rate of Four Intestinal Protozoa with Real-Time Polymerase Chain Reaction: Possible Overdiagnosis of Entamoeba histolytica in Nigeria.

Science.gov (United States)

Efunshile, Michael A; Ngwu, Bethrand A F; Kurtzhals, Jørgen A L; Sahar, Sumrin; König, Brigitte; Stensvold, Christen R

2015-08-01

Diarrhea remains the second largest killer of children worldwide, and Nigeria ranks number two on the list of global deaths attributable to diarrhea. Meanwhile, prevalence studies on potentially diarrheagenic protozoa in asymptomatic carriers using molecular detection methods remain scarce in sub-Saharan countries. To overcome sensitivity issues related to microscopic detection and identification of cysts in stool concentrates, real-time polymerase chain reaction (PCR) was used to analyze genomic DNAs extracted from stool samples from 199 healthy school children for Entamoeba histolytica, E. dispar, Giardia intestinalis, and Cryptosporidium. Questionnaires were administered for epidemiological data collection. E. histolytica was not detected in any of the samples, whereas Giardia (37.2%), E. dispar (18.6%), and Cryptosporidium (1%) were found. Most of the children sourced their drinking water from community wells (91%), while the majority disposed of feces in the bush (81.9%). Our study is the first to use real-time PCR to evaluate the epidemiology of E. histolytica, Giardia, and Cryptosporidium in Nigeria where previous studies using traditional diagnostic techniques have suggested higher and lower carriage rates of E. histolytica and Giardia, respectively. It is also the first study to accurately identify the prevalence of common potentially diarrheagenic protozoa in asymptomatic carriers in sub-Saharan Africa. © The American Society of Tropical Medicine and Hygiene.

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.

Improvement of a real-time RT-PCR assay for the detection of enterovirus RNA

Directory of Open Access Journals (Sweden)

Bruynseels Peggy

2009-07-01

Full Text Available Abstract We describe an improvement of an earlier reported real-time RT-PCR assay for the detection of enterovirus RNA, based on the 5' exonuclease digestion of a dual-labeled fluorogenic probe by Taq DNA polymerase. A different extraction method, real-time RT-PCR instrument and primer set were evaluated. Our data show that the optimized assay yields a higher sensitivity and reproducibility and resulted in a significant reduced hands-on time per sample.

Real Time Polymerase Chain Reaction : Perangkat Diagnostic Alternatif untuk Melacak Virus Nipah (REAL TIME POLYMERASE CHAIN REACTION : AN ALTERNATIVE DIAGNOSTIC TOOL TO DETECT NIPAH VIRUS

Directory of Open Access Journals (Sweden)

Indrawati Sendow

2014-05-01

Full Text Available Nipah is a dangerous zoonotic disease with a high social, economical and psychological impact. Fruitbat Pteropus sp. is one of the nipah virus  reservoir host. As the virus is categorized as a dangerous zoonoticdisease that cause fatal in human, all works related to live virus should be conducted in a laboratory withBSL4 facilities. The detection of nipah virus using real time PCR to replace virus isolastion can thereforebe conducted in a laboratory without BSL4 facilities. The results was further  confirmed at referencelaboratory at   Australian Animal Health Laboratory ( AAHL Geelong, Australia, indicated that nipahvirus can be detected in saliva of fruit bat P. vampyrus in Medan North Sumatera.

Real-time PCR Machine System Modeling and a Systematic Approach for the Robust Design of a Real-time PCR-on-a-Chip System

Directory of Open Access Journals (Sweden)

Da-Sheng Lee

2010-01-01

Full Text Available Chip-based DNA quantification systems are widespread, and used in many point-of-care applications. However, instruments for such applications may not be maintained or calibrated regularly. Since machine reliability is a key issue for normal operation, this study presents a system model of the real-time Polymerase Chain Reaction (PCR machine to analyze the instrument design through numerical experiments. Based on model analysis, a systematic approach was developed to lower the variation of DNA quantification and achieve a robust design for a real-time PCR-on-a-chip system. Accelerated lift testing was adopted to evaluate the reliability of the chip prototype. According to the life test plan, this proposed real-time PCR-on-a-chip system was simulated to work continuously for over three years with similar reproducibility in DNA quantification. This not only shows the robustness of the lab-on-a-chip system, but also verifies the effectiveness of our systematic method for achieving a robust design.

Real-time PCR array as a universal platform for the detection of genetically modified crops and its application in identifying unapproved genetically modified crops in Japan.

Science.gov (United States)

Mano, Junichi; Shigemitsu, Natsuki; Futo, Satoshi; Akiyama, Hiroshi; Teshima, Reiko; Hino, Akihiro; Furui, Satoshi; Kitta, Kazumi

2009-01-14

We developed a novel type of real-time polymerase chain reaction (PCR) array with TaqMan chemistry as a platform for the comprehensive and semiquantitative detection of genetically modified (GM) crops. Thirty primer-probe sets for the specific detection of GM lines, recombinant DNA (r-DNA) segments, endogenous reference genes, and donor organisms were synthesized, and a 96-well PCR plate was prepared with a different primer-probe in each well as the real-time PCR array. The specificity and sensitivity of the array were evaluated. A comparative analysis with the data and publicly available information on GM crops approved in Japan allowed us to assume the possibility of unapproved GM crop contamination. Furthermore, we designed a Microsoft Excel spreadsheet application, Unapproved GMO Checker version 2.01, which helps process all the data of real-time PCR arrays for the easy assumption of unapproved GM crop contamination. The spreadsheet is available free of charge at http://cse.naro.affrc.go.jp/jmano/index.html .

TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease.

Science.gov (United States)

Bohuski, Elizabeth; Lorch, Jeffrey M; Griffin, Kathryn M; Blehert, David S

2015-04-15

Fungal skin infections associated with Ophidiomyces ophiodiicola, a member of the Chrysosporium anamorph of Nannizziopsis vriesii (CANV) complex, have been linked to an increasing number of cases of snake fungal disease (SFD) in captive snakes around the world and in wild snake populations in eastern North America. The emergence of SFD in both captive and wild situations has led to an increased need for tools to better diagnose and study the disease. We developed two TaqMan real-time polymerase chain reaction (PCR) assays to rapidly detect O. ophiodiicola in clinical samples. One assay targets the internal transcribed spacer region (ITS) of the fungal genome while the other targets the more variable intergenic spacer region (IGS). The PCR assays were qualified using skin samples collected from 50 snakes for which O. ophiodiicola had been previously detected by culture, 20 snakes with gross skin lesions suggestive of SFD but which were culture-negative for O. ophiodiicola, and 16 snakes with no clinical signs of infection. Both assays performed equivalently and proved to be more sensitive than traditional culture methods, detecting O. ophiodiicola in 98% of the culture-positive samples and in 40% of the culture-negative snakes that had clinical signs of SFD. In addition, the assays did not cross-react with a panel of 28 fungal species that are closely related to O. ophiodiicola or that commonly occur on the skin of snakes. The assays did, however, indicate that some asymptomatic snakes (~6%) may harbor low levels of the fungus, and that PCR should be paired with histology when a definitive diagnosis is required. These assays represent the first published methods to detect O. ophiodiicola by real-time PCR. The ITS assay has great utility for assisting with SFD diagnoses whereas the IGS assay offers a valuable tool for research-based applications.

DNA polymerase hybrids derived from the family-B enzymes of Pyrococcus furiosus and Thermococcus kodakarensis: improving performance in the polymerase chain reaction.

Science.gov (United States)

Elshawadfy, Ashraf M; Keith, Brian J; Ee Ooi, H'Ng; Kinsman, Thomas; Heslop, Pauline; Connolly, Bernard A

2014-01-01

The polymerase chain reaction (PCR) is widely applied across the biosciences, with archaeal Family-B DNA polymerases being preferred, due to their high thermostability and fidelity. The enzyme from Pyrococcus furiosus (Pfu-Pol) is more frequently used than the similar protein from Thermococcus kodakarensis (Tkod-Pol), despite the latter having better PCR performance. Here the two polymerases have been comprehensively compared, confirming that Tkod-Pol: (1) extends primer-templates more rapidly; (2) has higher processivity; (3) demonstrates superior performance in normal and real time PCR. However, Tkod-Pol is less thermostable than Pfu-Pol and both enzymes have equal fidelities. To understand the favorable properties of Tkod-Pol, hybrid proteins have been prepared. Single, double and triple mutations were used to site arginines, present at the "forked-point" (the junction of the exonuclease and polymerase channels) of Tkod-Pol, at the corresponding locations in Pfu-Pol, slightly improving PCR performance. The Pfu-Pol thumb domain, responsible for double-stranded DNA binding, has been entirely replaced with that from Tkod-Pol, again giving better PCR properties. Combining the "forked-point" and thumb swap mutations resulted in a marked increase in PCR capability, maintenance of high fidelity and retention of the superior thermostability associated with Pfu-Pol. However, even the arginine/thumb swap mutant falls short of Tkod-Pol in PCR, suggesting further improvement within the Pfu-Pol framework is attainable. The significance of this work is the observation that improvements in PCR performance are easily attainable by blending elements from closely related archaeal polymerases, an approach that may, in future, be extended by using more polymerases from these organisms.

Real-Time Polymerase Chain Reaction-Based Detection of Bordetella pertussis in Mexican Infants and Their Contacts: A 3-Year Multicenter Study.

Science.gov (United States)

Aquino-Andrade, Alejandra; Martínez-Leyva, Gabriel; Mérida-Vieyra, Jocelin; Saltigeral, Patricia; Lara, Antonino; Domínguez, Wendy; García de la Puente, Silvestre; De Colsa, Agustín

2017-09-01

To evaluate the usefulness of real-time polymerase chain reaction (RT-PCR) as a diagnostic method for the detection of Bordetella pertussis in hospitalized patients aged pertussis detection and symptoms in household contacts of patients diagnosed with pertussis were studied. A total of 286 patients were included; of these, 67.1% had B pertussis and 4.5% had Bordetella spp. Complications occurred in 20% of patients, and the mortality rate was 6.7%. Of 434 contacts studied, 111 were mothers of study infants, representing the most frequently B pertussis-infected group and the main symptomatic contact. The use of RT-PCR permits improved detection and diagnosis of pertussis and a better understanding of the epidemiology of sources of infection. The complications and mortality rate of pertussis continue to be high. Household contacts are confirmed as a frequent source of infection of B pertussis in young children. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Quantitative Tetraplex Real-Time Polymerase Chain Reaction Assay with TaqMan Probes Discriminates Cattle, Buffalo, and Porcine Materials in Food Chain.

Science.gov (United States)

Hossain, M A Motalib; Ali, Md Eaqub; Sultana, Sharmin; Asing; Bonny, Sharmin Quazi; Kader, Md Abdul; Rahman, M Aminur

2017-05-17

Cattle, buffalo, and porcine materials are widely adulterated, and their quantification might safeguard health, religious, economic, and social sanctity. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays have been documented but they are just suitable for identification, cannot quantify adulterations. We described here a quantitative tetraplex real-time PCR assay with TaqMan Probes to quantify contributions from cattle, buffalo, and porcine materials simultaneously. Amplicon-sizes were very short (106-, 90-, and 146-bp for cattle, buffalo, and porcine) because longer targets could be broken down, bringing serious ambiguity in molecular diagnostics. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 27 frankfurters and 27 meatballs reflected 84-115% target recovery at 0.1-10% adulterations. Finally, a test of 36 commercial products revealed 71% beef frankfurters, 100% meatballs, and 85% burgers contained buffalo adulteration, but no porcine was found in beef products.

A two-step lyssavirus real-time polymerase chain reaction using degenerate primers with superior sensitivity to the fluorescent antigen test.

Science.gov (United States)

Suin, Vanessa; Nazé, Florence; Francart, Aurélie; Lamoral, Sophie; De Craeye, Stéphane; Kalai, Michael; Van Gucht, Steven

2014-01-01

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤ 1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.

Comparison of real-time and quantitative polymerase chain reaction assays in detection of cytomegalovirus DNA in clinical specimens

International Nuclear Information System (INIS)

Gokahmetoglu, S.; Deniz, E.

2007-01-01

To compare the real-time (RT) and qualitative (Q) polymerase chain reaction (PCR) assays for detection of Cytomegalovirus (CMV) DNA. The study took place in the Department of Microbiology, Erciyes University, Kayseri and in Iontek Laboratory, Istanbul, Turkey, from August to December 2006. One hundred and seven clinical specimens from 67 patients were included in the study. Cytomegalovirus DNA was investigated using RT-PCR kit (Fluorion Iontek, Turkey) and Q-PCR kit (Fluorion Iontek, Turkey). Deoxyribonucleic acid sequencing was applied to the samples that yielded discrepant results in both assays. Mac Nema's Chi Square test was used for statistical analysis. Of the specimens, 27 were found positive with both assays: 9 with only RT-PCR, and 11 with only Q-PCR assay. Both assays were found negative in 60 of the specimens. There was a good agreement between the 2 assays in 87(81.3%) of the specimens. There was no statistical significant difference between the assays (p>0.05). Two of the 11 samples that RT-PCR negative Q-PCR positive, and 3 of 9 samples that RT-PCR positive Q-PCR negative were found to be CMV DNA positive by DNA sequencing. A good level of concordance between RT-PCR and Q-PCR assays for CMV DNA detection has been found. (author)

A novel rapid genotyping technique for Collie eye anomaly: SYBR Green-based real-time polymerase chain reaction method applicable to blood and saliva specimens on Flinders Technology Associates filter paper.

Science.gov (United States)

Chang, Hye-Sook; Mizukami, Keijiro; Yabuki, Akira; Hossain, Mohammad A; Rahman, Mohammad M; Uddin, Mohammad M; Arai, Toshiro; Yamato, Osamu

2010-09-01

Collie eye anomaly (CEA) is a canine inherited ocular disease that shows a wide variety of manifestations and severity of clinical lesions. Recently, a CEA-associated mutation was reported, and a DNA test that uses conventional polymerase chain reaction (PCR) has now become available. The objective of the current study was to develop a novel rapid genotyping technique by using SYBR Green-based real-time PCR for future large-scale surveys as a key part in the strategy to eradicate CEA by selective breeding. First, a SYBR Green-based real-time PCR assay for genotyping of CEA was developed and evaluated by using purified DNA samples from normal, carrier, and affected Border Collies in which genotypes had previously been determined by conventional PCR. This real-time PCR assay demonstrated appropriate amplifications in all genotypes, and the results were consistent with those of conventional PCR. Second, the availability of Flinders Technology Associates filter paper (FTA card) as DNA templates for the real-time PCR assay was evaluated by using blood and saliva specimens to determine suitability for CEA screening. DNA-containing solution prepared from a disc of blood- or saliva-spotted FTA cards was available directly as templates for the real-time PCR assay when the volume of solution was 2.5% of the PCR mixture. In conclusion, SYBR Green-based real-time PCR combined with FTA cards is a rapid genotyping technique for CEA that can markedly shorten the overall time required for genotyping as well as simplify the sample preparation. Therefore, this newly developed technique suits large-scale screening in breeding populations of Collie-related breeds.

Real-time PCR Machine System Modeling and a Systematic Approach for the Robust Design of a Real-time PCR-on-a-Chip System

OpenAIRE

Lee, Da-Sheng

2010-01-01

Chip-based DNA quantification systems are widespread, and used in many point-of-care applications. However, instruments for such applications may not be maintained or calibrated regularly. Since machine reliability is a key issue for normal operation, this study presents a system model of the real-time Polymerase Chain Reaction (PCR) machine to analyze the instrument design through numerical experiments. Based on model analysis, a systematic approach was developed to lower the variation of DN...

GNSS global real-time augmentation positioning: Real-time precise satellite clock estimation, prototype system construction and performance analysis

Science.gov (United States)

Chen, Liang; Zhao, Qile; Hu, Zhigang; Jiang, Xinyuan; Geng, Changjiang; Ge, Maorong; Shi, Chuang

2018-01-01

Lots of ambiguities in un-differenced (UD) model lead to lower calculation efficiency, which isn't appropriate for the high-frequency real-time GNSS clock estimation, like 1 Hz. Mixed differenced model fusing UD pseudo-range and epoch-differenced (ED) phase observations has been introduced into real-time clock estimation. In this contribution, we extend the mixed differenced model for realizing multi-GNSS real-time clock high-frequency updating and a rigorous comparison and analysis on same conditions are performed to achieve the best real-time clock estimation performance taking the efficiency, accuracy, consistency and reliability into consideration. Based on the multi-GNSS real-time data streams provided by multi-GNSS Experiment (MGEX) and Wuhan University, GPS + BeiDou + Galileo global real-time augmentation positioning prototype system is designed and constructed, including real-time precise orbit determination, real-time precise clock estimation, real-time Precise Point Positioning (RT-PPP) and real-time Standard Point Positioning (RT-SPP). The statistical analysis of the 6 h-predicted real-time orbits shows that the root mean square (RMS) in radial direction is about 1-5 cm for GPS, Beidou MEO and Galileo satellites and about 10 cm for Beidou GEO and IGSO satellites. Using the mixed differenced estimation model, the prototype system can realize high-efficient real-time satellite absolute clock estimation with no constant clock-bias and can be used for high-frequency augmentation message updating (such as 1 Hz). The real-time augmentation message signal-in-space ranging error (SISRE), a comprehensive accuracy of orbit and clock and effecting the users' actual positioning performance, is introduced to evaluate and analyze the performance of GPS + BeiDou + Galileo global real-time augmentation positioning system. The statistical analysis of real-time augmentation message SISRE is about 4-7 cm for GPS, whlile 10 cm for Beidou IGSO/MEO, Galileo and about 30 cm

Potential testing of reprocessing procedures by real-time polymerase chain reaction: A multicenter study of colonoscopy devices.

Science.gov (United States)

Valeriani, Federica; Agodi, Antonella; Casini, Beatrice; Cristina, Maria Luisa; D'Errico, Marcello Mario; Gianfranceschi, Gianluca; Liguori, Giorgio; Liguori, Renato; Mucci, Nicolina; Mura, Ida; Pasquarella, Cesira; Piana, Andrea; Sotgiu, Giovanni; Privitera, Gaetano; Protano, Carmela; Quattrocchi, Annalisa; Ripabelli, Giancarlo; Rossini, Angelo; Spagnolo, Anna Maria; Tamburro, Manuela; Tardivo, Stefano; Veronesi, Licia; Vitali, Matteo; Romano Spica, Vincenzo

2018-02-01

Reprocessing of endoscopes is key to preventing cross-infection after colonoscopy. Culture-based methods are recommended for monitoring, but alternative and rapid approaches are needed to improve surveillance and reduce turnover times. A molecular strategy based on detection of residual traces from gut microbiota was developed and tested using a multicenter survey. A simplified sampling and DNA extraction protocol using nylon-tipped flocked swabs was optimized. A multiplex real-time polymerase chain reaction (PCR) test was developed that targeted 6 bacteria genes that were amplified in 3 mixes. The method was validated by interlaboratory tests involving 5 reference laboratories. Colonoscopy devices (n = 111) were sampled in 10 Italian hospitals. Culture-based microbiology and metagenomic tests were performed to verify PCR data. The sampling method was easily applied in all 10 endoscopy units and the optimized DNA extraction and amplification protocol was successfully performed by all of the involved laboratories. This PCR-based method allowed identification of both contaminated (n = 59) and fully reprocessed endoscopes (n = 52) with high sensibility (98%) and specificity (98%), within 3-4 hours, in contrast to the 24-72 hours needed for a classic microbiology test. Results were confirmed by next-generation sequencing and classic microbiology. A novel approach for monitoring reprocessing of colonoscopy devices was developed and successfully applied in a multicenter survey. The general principle of tracing biological fluids through microflora DNA amplification was successfully applied and may represent a promising approach for hospital hygiene. Copyright © 2018 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Use of palivizumab and infection control measures to control an outbreak of respiratory syncytial virus in a neonatal intensive care unit confirmed by real-time polymerase chain reaction.

LENUS (Irish Health Repository)

O'Connell, K

2011-04-01

Respiratory syncytial virus (RSV) is a potentially life-threatening infection in premature infants. We report an outbreak involving four infants in the neonatal intensive care unit (NICU) of our hospital that occurred in February 2010. RSV A infection was confirmed by real-time polymerase chain reaction. Palivizumab was administered to all infants in the NICU. There were no additional symptomatic cases and repeat RSV surveillance confirmed that there was no further cross-transmission within the unit. The outbreak highlighted the infection control challenge of very high bed occupancy in the unit and the usefulness of molecular methods in facilitating detection and management.

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

Science.gov (United States)

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

2015-10-01

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

Quantification of bovine leukemia virus proviral DNA using a low-cost real-time polymerase chain reaction.

Science.gov (United States)

Petersen, M I; Alvarez, I; Trono, K G; Jaworski, J P

2018-04-11

The detection of bovine leukemia virus (BLV) proviral DNA is an important tool to address whether an animal is infected with BLV. Compared with serological assays, real-time PCR accounts for greater sensitivity and can serve as a confirmatory test for the clarification of inconclusive or discordant serological test results. However, the high cost related to real-time PCR assays has limited their systematic inclusion in BLV surveillance and eradication programs. The aim of the present study was to validate a low-cost quantitative real-time PCR. Interestingly, by using SYBR Green detection dye, we were able to reduce the cost of a single reaction by a factor of 5 compared with most common assays based on the use of fluorogenic probes (i.e., TaqMan technology). This approach allowed a highly sensitive and specific detection and quantification of BLV proviral DNA from purified peripheral blood leukocytes and a milk matrix. Due to its simplicity and low cost, our in-house BLV SYBR quantitative real-time PCR might be used either as a screening or as a confirmatory test in BLV control programs. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Real-time control environment for the RFX experiment

International Nuclear Information System (INIS)

Barana, O.; Cavinato, M.; Luchetta, A.; Manduchi, G.; Taliercio, C.

2005-01-01

A comprehensive set of control schemes can be presently implemented on RFX due to the enhanced load assembly and renewed power supply system. The schemes include: plasma equilibrium control and resistive wall mode stabilization, aiming at controlling actively the discharge when the passive action of the shell vanishes; the rotation of the localised helical deformation to minimize the enhanced plasma-wall interaction; the MHD mode control and the 'intelligent shell', aiming at achieving a better comprehension of the underlying physics. To the purpose, an integrated, distributed, digital system has been developed consisting of a set of computing nodes. Each node can act either as pre-processing or control station, the former acquiring raw data and computing intermediate control parameters, the latter executing control algorithms and driving the power amplifiers. An overview of the system architecture is presented in the paper with reference to the software real-time environment providing both basic functions, such as data read-out and real-time communication, and useful tools to program control algorithms, to perform simulations and to commission the system. To simulate the control schemes, the real-time environment is extended to include a so called 'simulation mode', in which the real-time nodes exchange their input/output signals with one station running a suitable model of the experiment, for instance the two dimensional FEM code MAXFEA in the case of the equilibrium control. In this way the control system can be tested offline and the time needed for the commissioning of algorithms reduced

Real time quantitative amplification detection on a microarray: towards high multiplex quantitative PCR.

NARCIS (Netherlands)

Pierik, A.; Moamfa, M; van Zelst, M.; Clout, D.; Stapert, H.; Dijksman, Johan Frederik; Broer, D.; Wimberger-Friedl, R.

2012-01-01

Quantitative real-time polymerase chain reaction (qrtPCR) is widely used as a research and diagnostic tool. Notwithstanding its many powerful features, the method is limited in the degree of multiplexing to about 6 due to spectral overlap of the available fluorophores. A new method is presented that

Real time quantitative amplification detection on a microarray : towards high multiplex quantitative PCR

NARCIS (Netherlands)

Pierik, Anke; Boamfa, M.; Zelst, van M.; Clout, D.; Stapert, H.R.; Dijksman, J.F.; Broer, D.J.; Wimberger-Friedl, R.

2012-01-01

Quantitative real-time polymerase chain reaction (qrtPCR) is widely used as a research and diagnostic tool. Notwithstanding its many powerful features, the method is limited in the degree of multiplexing to about 6 due to spectral overlap of the available fluorophores. A new method is presented that

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...

Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

Directory of Open Access Journals (Sweden)

Roberta Flávia Ribeiro Rolando

2012-06-01

Full Text Available This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR. A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

Quantitative detection and typing of hepatitis D virus in human serum by real-time polymerase chain reaction and melting curve analysis.

Science.gov (United States)

Hofmann, Joerg; Frenzel, Katrin; Minh, Bui Q; von Haeseler, Arndt; Edelmann, Anke; Ross, Stefan R; Berg, Thomas; Krüger, Detlev H; Meisel, Helga

2010-06-01

Hepatitis D virus (HDV) infection is an important etiologic agent of fulminant hepatitis and may aggravate the clinical course of chronic hepatitis B infection resulting in cirrhosis and liver failure. This report describes the establishment of a real-time reverse transcriptase polymerase chain reaction method that allows the quantitative detection of HDV-1 and HDV-3 with a sensitivity in a linear range of 2 x 10(3) to 10(8) copies/mL. Additionally, the new assay provides the opportunity to distinguish HDV-1 from HDV-3 by a subsequent melting curve analysis, an important option because these HDV types are highly associated with severe clinical outcome. The results of the melting curve analysis of 42 HDV sequences obtained in this study and the phylogenetic analysis based on 139 full-length sequences from GenBank were consistent and showed that all sequences described here cluster within the HDV-1 clade. Therefore, this assay is useful for monitoring of antiviral treatment and molecular epidemiologic studies of HDV distribution. Copyright 2010 Elsevier Inc. All rights reserved.

Determination of real-time polymerase chain reaction uncertainty of measurement using replicate analysis and a graphical user interface with Fieller's theorem.

Science.gov (United States)

Stuart, James Ian; Delport, Johan; Lannigan, Robert; Zahariadis, George

2014-07-01

Disease monitoring of viruses using real-time polymerase chain reaction (PCR) requires knowledge of the precision of the test to determine what constitutes a significant change. Calculation of quantitative PCR confidence limits requires bivariate statistical methods. To develop a simple-to-use graphical user interface to determine the uncertainty of measurement (UOM) of BK virus, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) real-time PCR assays. Thirty positive clinical samples for each of the three viral assays were repeated once. A graphical user interface was developed using a spreadsheet (Excel, Microsoft Corporation, USA) to enable data entry and calculation of the UOM (according to Fieller's theorem) and PCR efficiency. The confidence limits for the BK virus, CMV and EBV tests were ∼0.5 log, 0.5 log to 1.0 log, and 0.5 log to 1.0 log, respectively. The efficiencies of these assays, in the same order were 105%, 119% and 90%. The confidence limits remained stable over the linear range of all three tests. A >5 fold (0.7 log) and a >3-fold (0.5 log) change in viral load were significant for CMV and EBV when the results were ≤1000 copies/mL and >1000 copies/mL, respectively. A >3-fold (0.5 log) change in viral load was significant for BK virus over its entire linear range. PCR efficiency was ideal for BK virus and EBV but not CMV. Standardized international reference materials and shared reporting of UOM among laboratories are required for the development of treatment guidelines for BK virus, CMV and EBV in the context of changes in viral load.

A Two-Step Lyssavirus Real-Time Polymerase Chain Reaction Using Degenerate Primers with Superior Sensitivity to the Fluorescent Antigen Test

Directory of Open Access Journals (Sweden)

Vanessa Suin

2014-01-01

Full Text Available A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR, based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.

Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis

DEFF Research Database (Denmark)

Andersen, Ulla Vestergaard; Haakansson, I. T.; Roust, Tina

2013-01-01

extent developmental stages can affect the variation of diagnostic test results. This study investigated the influence of developmental stages of strongyle eggs on the variation real-time polymerase chain reaction (PCR) results. Mixed species strongyle eggs were obtained from the faeces of a naturally...

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.

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.

Investigating Comprehension in Real World Tasks: Understanding Jury Instructions.

Science.gov (United States)

Charrow, Veda R.; Charrow, Robert

This paper discusses the results of part of an ongoing project studying an aspect of real world language usage, the comprehension of standard jury instructions. Problems in the comprehension of these instructions include the memory load that they impose, the fact that most instructions are read only once, and the fact that instructions are written…

[The implementation of polymerase chain reaction technique: the real time to reveal and differentiate the viruses of human papilloma of high carcinogenic risk].

Science.gov (United States)

Andosova, L D; Kontorshchikova, K N; Blatova, O L; Kudel'kina, S Iu; Kuznetsova, I A; Belov, A V; Baĭkova, R A

2011-07-01

The polymerase chain reaction technique was applied in "real time" format to evaluate the occurrence rate and infection ratio of various genotypes of human papilloma of high carcinogenic risk in virus-positive women and contact persons. The examination sampling consisted of 738 women aged of 17-50 years. The examination results permitted to establish high percentage of infection of 546 patients (74%) by carcinogenic papilloma viruses. The analysis of detection rate of various genotypes of human papilloma of high carcinogenic risk established that the 56th and 16th types of high carcinogenic risk are revealed more often than others--in 33% and 15.4% correspondingly. In males, first place in occurrence rate is for those types of virus of human papilloma: the 56th n = 10 (33.3%), 16th n = 3 (10%), 45th n = 3 (10%), 51th n = 3 (10%). The rest of genotypes are detected in 3-7% cases.

Reverse transcriptase-quantitative polymerase chain reaction (RT ...

African Journals Online (AJOL)

zino

2014-02-05

Feb 5, 2014 ... ecological studies - A review ... The objective of this review is to assess the importance of RT-qPCR in soil related ... phenol extraction step with heat inactivation of the added .... Real time polymerase chain reaction (PCR).

Real-time polymerase chain reaction and culture in the diagnosis of invasive group B streptococcal disease in infants: a retrospective study.

Science.gov (United States)

Meehan, M; Cafferkey, M; Corcoran, S; Foran, A; Hapnes, N; LeBlanc, D; McGuinness, C; Nusgen, U; O'Sullivan, N; Cunney, R; Drew, R

2015-12-01

Group B streptococcus (GBS) is a leading cause of invasive disease in infants. Accurate and rapid diagnosis is crucial to reduce morbidity and mortality. Real-time polymerase chain reaction (PCR) targeting the dltR gene was utilised for the direct detection of GBS DNA in blood and cerebrospinal fluid (CSF) from infants at an Irish maternity hospital. A retrospective review of laboratory and patient records during the period 2011-2013 was performed in order to evaluate PCR and culture for the diagnosis of invasive GBS disease. A total of 3570 blood and 189 CSF samples from 3510 infants had corresponding culture and PCR results. Culture and PCR exhibited concordance in 3526 GBS-negative samples and 13 (25%) GBS-positive samples (n = 53). Six (11%) and 34 (64%) GBS-positive samples were positive only in culture or PCR, respectively. Culture and PCR identified more GBS-positive infants (n = 47) than PCR (n = 43) or culture (n = 16) alone. Using culture as the reference standard, the sensitivity, specificity, and positive and negative predictive values for PCR on blood samples were 71.4%, 99.2%, 25% and 99.9%, and for CSF samples, they were 60%, 97.8%, 42.9% and 98.9%, respectively. The sensitivity and positive predictive values were improved (blood: 84.6% and 55%; CSF: 77.8% and 100%, respectively) when maternal risk factors and other laboratory test results were considered. The findings in this study recommend the use of direct GBS real-time PCR for the diagnosis of GBS infection in infants with a clinical suspicion of invasive disease and as a complement to culture, but should be interpreted in the light of other laboratory and clinical findings.

A plastome primer set for comprehensive quantitative real time RT-PCR analysis of Zea mays: a starter primer set for other Poaceae species

Directory of Open Access Journals (Sweden)

Dunn Sade N

2008-06-01

Full Text Available Abstract Background Quantitative Real Time RT-PCR (q2(RTPCR is a maturing technique which gives researchers the ability to quantify and compare very small amounts of nucleic acids. Primer design and optimization is an essential yet time consuming aspect of using q2(RTPCR. In this paper we describe the design and empirical optimization of primers to amplify and quantify plastid RNAs from Zea mays that are robust enough to use with other closely related species. Results Primers were designed and successfully optimized for 57 of the 104 reported genes in the maize plastome plus two nuclear genes. All 59 primer pairs produced single amplicons after end-point reverse transcriptase polymerase chain reactions (RT-PCR as visualized on agarose gels and subsequently verified by q2(RTPCR. Primer pairs were divided into several categories based on the optimization requirements or the uniqueness of the target gene. An in silico test suggested the majority of the primer sets should work with other members of the Poaceae family. An in vitro test of the primer set on two unsequenced species (Panicum virgatum and Miscanthus sinensis supported this assumption by successfully producing single amplicons for each primer pair. Conclusion Due to the highly conserved chloroplast genome in plant families it is possible to utilize primer pairs designed against one genomic sequence to detect the presence and abundance of plastid genes or transcripts from genomes that have yet to be sequenced. Analysis of steady state transcription of vital system genes is a necessary requirement to comprehensively elucidate gene expression in any organism. The primer pairs reported in this paper were designed for q2(RTPCR of maize chloroplast genes but should be useful for other members of the Poaceae family. Both in silico and in vitro data are presented to support this assumption.

Determination of real-time polymerase chain reaction uncertainty of measurement using replicate analysis and a graphical user interface with Fieller’s theorem

Science.gov (United States)

Stuart, James Ian; Delport, Johan; Lannigan, Robert; Zahariadis, George

2014-01-01

BACKGROUND: Disease monitoring of viruses using real-time polymerase chain reaction (PCR) requires knowledge of the precision of the test to determine what constitutes a significant change. Calculation of quantitative PCR confidence limits requires bivariate statistical methods. OBJECTIVE: To develop a simple-to-use graphical user interface to determine the uncertainty of measurement (UOM) of BK virus, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) real-time PCR assays. METHODS: Thirty positive clinical samples for each of the three viral assays were repeated once. A graphical user interface was developed using a spreadsheet (Excel, Microsoft Corporation, USA) to enable data entry and calculation of the UOM (according to Fieller’s theorem) and PCR efficiency. RESULTS: The confidence limits for the BK virus, CMV and EBV tests were ∼0.5 log, 0.5 log to 1.0 log, and 0.5 log to 1.0 log, respectively. The efficiencies of these assays, in the same order were 105%, 119% and 90%. The confidence limits remained stable over the linear range of all three tests. DISCUSSION: A >5 fold (0.7 log) and a >3-fold (0.5 log) change in viral load were significant for CMV and EBV when the results were ≤1000 copies/mL and >1000 copies/mL, respectively. A >3-fold (0.5 log) change in viral load was significant for BK virus over its entire linear range. PCR efficiency was ideal for BK virus and EBV but not CMV. Standardized international reference materials and shared reporting of UOM among laboratories are required for the development of treatment guidelines for BK virus, CMV and EBV in the context of changes in viral load. PMID:25285125

Comparison of real-time reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis.

Science.gov (United States)

Doenges, Stephanie J; Weber, Karin; Dorsch, Roswitha; Fux, Robert; Hartmann, Katrin

2017-04-01

Objectives Diagnosis of feline infectious peritonitis (FIP) remains challenging, especially in cats without effusions. The objective of this study was to evaluate the sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) detecting feline coronavirus (FCoV) RNA in peripheral blood mononuclear cells (PBMCs) and serum in comparison with the same real-time RT-PCR in cell-free body cavity effusion. Methods This prospective case-control study included 92 cats. Forty-three cats had a definitive diagnosis of FIP, established either by histopathological examination (n = 28) or by positive immunofluorescence staining of FCoV antigen in macrophages of effusions (n = 11), or by both methods (n = 4). Forty-nine control cats had other diseases but similar clinical signs. Real-time RT-PCR was performed on PBMCs of 37 cats (21 cats with FIP, 16 controls), on serum of 51 cats (26 cats with FIP, 25 controls) and on cell-free body cavity effusion of 69 cats (36 cats with FIP, 33 controls). Sensitivity, specificity, positive and negative predictive value, including 95% confidence intervals (CI), were calculated. Results Real-time RT-PCR of PBMCs, serum and cell-free body cavity effusion showed a specificity of 100% (95% CI 79.4-100% in PBMCs, 86.3-100% in serum, 89.4-100% in cell-free body cavity effusion) and a sensitivity of 28.6% (95% CI 11.3-52.2%) in PBMCs, 15.4% (95% CI 4.4-34.9%) in serum and 88.9% (95% CI 73.9-96.9%) in cell-free body cavity effusion to diagnose FIP. Conclusions and relevance Although it is known that RT-PCR can often provide false-positive results in healthy cats, this real-time RT-PCR was shown to be a specific tool for the diagnosis of FIP when applied in a clinical setting. Sensitivity in cell-free body cavity effusion was high but low in PBMCs and serum. PBMC samples showed a higher sensitivity than serum samples, and are therefore a better choice if no effusion is present.

Development of real-time quantitative polymerase chain reaction assays to track treatment response in retinoid resistant acute promyelocytic leukemia

Directory of Open Access Journals (Sweden)

Jelena V Jovanovic

2011-10-01

Full Text Available Molecular detection of minimal residual disease (MRD has become established to assess remission status and guide therapy in patients with PML-RARA+ acute promyelocytic leukemia (APL. However, there are few data on tracking disease response in patients with rarer retinoid resistant subtypes of APL, characterized by PLZF-RARA and STAT5b-RARA. Despite their relative rarity (<1% of APL we identified 6 cases (PLZF-RARA, n=5; STAT5b-RARA, n=1, established the respective breakpoint junction regions and designed real-time quantitative polymerase chain reaction (RQ-PCR assays to detect leukemic transcripts. The relative level of fusion gene expression in diagnostic samples was comparable to that observed in t(15;17-associated APL, affording assay sensitivities of ~1 in 104-105. Serial samples were available from 2 PLZF-RARA APL patients. One showed persistent PCR positivity, predicting subsequent relapse, and remains in CR2, ~11 years post-autograft. The other, achieved molecular remission (CRm with combination chemotherapy, remaining in CR1 at 6 years. The STAT5b-RARA patient failed to achieve CRm following frontline combination chemotherapy and ultimately proceeded to allogeneic transplant on the basis of a steadily rising fusion transcript level. These data highlight the potential of RQ-PCR detection of MRD to facilitate development of more individualized approaches to the management of rarer molecularly-defined subsets of acute leukemia.

Detection and quantification of periodontal pathogens in smokers and never-smokers with chronic periodontitis by real-time polymerase chain reaction.

Science.gov (United States)

Guglielmetti, Mariana R; Rosa, Ecinele F; Lourenção, Daniele S; Inoue, Gislene; Gomes, Elaine F; De Micheli, Giorgio; Mendes, Fausto Medeiros; Hirata, Rosário D C; Hirata, Mario H; Pannuti, Claudio M

2014-10-01

The purpose of the present investigation is to compare the presence and number of periodontal pathogens in the subgingival microbiota of smokers versus never-smokers with chronic periodontitis and matched probing depths (PDs) using real-time polymerase chain reaction (RT-PCR). Forty current smokers and 40 never-smokers, matched for age, sex, and mean PD of sampling site, were included in this investigation. A full-mouth periodontal examination was performed, and a pooled subgingival plaque sample was collected from the deepest site in each quadrant of each participant. To confirm smoking status, expired carbon monoxide (CO) concentrations were measured with a CO monitor. The presence and quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola were determined using RT-PCR. Smokers had greater overall mean PD (P = 0.001) and attachment loss (P = 0.006) and fewer bleeding on probing sites (P = 0.001). An association was observed between smoking status and the presence of A. actinomycetemcomitans (P <0.001). The counts of A. actinomycetemcomitans (P <0.001), P. gingivalis (P = 0.042), and T. forsythia (P <0.001) were significantly higher in smokers. Smokers showed significantly greater amounts of P. gingivalis, A. actinomycetemcomitans, and T. forsythia than never-smokers. There was a significant association between smoking and the presence of A. actinomycetemcomitans.

Probabilistic real-time contingency ranking method

International Nuclear Information System (INIS)

Mijuskovic, N.A.; Stojnic, D.

2000-01-01

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

Detection of Toxoplasma gondii and Epstein-Barr virus in HIV patients with clinical symptoms of suspected central nervous system infection using duplex real-time polymerase chain reaction

Science.gov (United States)

Rahmawati, E.; Ibrahim, F.; Imran, D.; Sudarmono, P.

2017-08-01

Focal brain lesion is a neurological complication in HIV, which is marked as a space occupying lesion (SOL) and needs rapid and effective treatment. This lesion is mainly caused by encephalitis toxoplasma and primary central nervous system lymphoma related to the Epstein-Barr virus (EBV) infection, which is difficult to distinguish using CT scan or magnetic resonance imaging (MRI). The gold standard of diagnosing focal brain lesion has been brain biopsy, but this examination is an invasive procedure that causes complications. The objective of this study is to obtain the rapid laboratory diagnosis of Toxoplasma gondii (T. gondii) and EBV infection. In this experimental study, blood and cerebrospinal fluid were obtained from HIV patients who were admitted to the Neurology Department of Cipto Mangunkusumo Hospital. The samples were examined using duplex real-time polymerase chain reaction (PCR) to detect T. gondii and EBV. The first step was the optimization of duplex real-time PCR, including the annealing temperature, primer and probe concentration, elution volume, and template volume. Minimal DNA detection was used to measure minimal T. gondii and EBV. Cross reactions were determined for technical specificity using the bacteria and viruses Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Mycobacterium tuberculosis H37Rv, Candida spp, cytomegalovirus, herpes zoster virus, and varicella zoster virus. Duplex real-time PCR was applied optimally to patients. In the optimization of duplex real-time PCR, the annealing temperature of T. gondii and EBV were 58 °C, the concentration of primer forward and reverse for T. gondii and EBV were 0.2 μM, the concentration of probe for T. gondii and EBV were 0.4μM and 0.2 μM, respectively. Minimal DNA detection of T. gondii and EBV were 5.68 copy/ml and 1.31 copy/ml, respectively. There was no cross reaction between another bacteria and virus that were used as the primer and probe for T. gondii and EBV. The

Recombinase Polymerase Amplification Compared to Real-Time Polymerase Chain Reaction Test for the Detection of Fasciola hepatica in Human Stool

Science.gov (United States)

Cabada, Miguel M.; Malaga, Jose L.; Castellanos-Gonzalez, Alejandro; Bagwell, Kelli A.; Naeger, Patrick A.; Rogers, Hayley K.; Maharsi, Safa; Mbaka, Maryann; White, A. Clinton

2017-01-01

Fasciola hepatica is the most widely distributed trematode infection in the world. Control efforts may be hindered by the lack of diagnostic capacity especially in remote endemic areas. Polymerase chain reaction (PCR)–based methods offer high sensitivity and specificity but require expensive technology. However, the recombinase polymerase amplification (RPA) is an efficient isothermal method that eliminates the need for a thermal cycler and has a high deployment potential to resource-limited settings. We report on the characterization of RPA and PCR tests to detect Fasciola infection in clinical stool samples with low egg burdens. The sensitivity of the RPA and PCR were 87% and 66%, respectively. Both tests were 100% specific showing no cross-reactivity with trematode, cestode, or nematode parasites. In addition, RPA and PCR were able to detect 47% and 26% of infections not detected by microscopy, respectively. The RPA adapted to a lateral flow platform was more sensitive than gel-based detection of the reaction products. In conclusion, the Fasciola RPA is a highly sensitive and specific test to diagnose chronic infection using stool samples. The Fasciola RPA lateral flow has the potential for deployment to endemic areas after further characterization. PMID:27821691

REAL-TIME PCR DETECTION OF LISTERIA MONOCYTOGENES IN FOOD SAMPLES OF ANIMAL ORIGIN

Directory of Open Access Journals (Sweden)

Jaroslav Pochop

2013-02-01

Full Text Available The aim of this study was to follow the contamination of food with Listeria monocytogenes by using Step One real time polymerase chain reaction (PCR. We used the PrepSEQ Rapid Spin Sample Preparation Kit for isolation of DNA and SensiFAST SYBR Hi-ROX Kit for the real-time PCR performance. In 24 samples of food of animal origin without incubation were detected strains of Listeria monocytogenes in 15 samples (swabs. Nine samples were negative. Our results indicated that the real-time PCR assay developed in this study could sensitively detect Listeria monocytogenes in food of animal origin without incubation. This could prevent infection caused by Listeria monocytogenes, and also could benefit food manufacturing companies by extending their product’s shelf-life as well as saving the cost of warehousing their food products while awaiting pathogen testing results. The rapid real-time PCR-based method performed very well compared to the conventional method. It is a fast, simple, specific and sensitive way to detect nucleic acids, which could be used in clinical diagnostic tests in the future.

Listeria monocytogenes Identification in Food of Animal Origin Used with Real Time PCR

Directory of Open Access Journals (Sweden)

Jaroslav Pochop

2013-10-01

Full Text Available The aim of this study was to follow the contamination of food with Listeria monocytogenes by using Step One real time polymerase chain reaction (RT PCR. We used the PrepSEQ Rapid Spin Sample Preparation Kit for isolation of DNA and SensiFAST SYBR Hi-ROX Kit for the real-time PCR performance. In 20 samples of food of animal origin with incubation were detected strains of Listeria monocytogenes in 9 samples (swabs. Eleven samples were negative. Our results indicated that the real-time PCR assay developed in this study could sensitively detect Listeria monocytogenes in food of animal origin without incubation. This could prevent infection caused by Listeria monocytogenes, and also could benefit food manufacturing companies by extending their product’s shelf-life as well as saving the cost of warehousing their food products while awaiting pathogen testing results. The rapid real-time PCR-based method performed very well compared to the conventional method. It is a fast, simple, specific and sensitive way to detect nucleic acids, which could be used in clinical diagnostic tests in the future.

Development of real-time PCR for detection and quantitation of Streptococcus parauberis.

Science.gov (United States)

Nguyen, T L; Lim, Y J; Kim, D-H; Austin, B

2016-01-01

Streptococcus parauberis is an increasing threat to aquaculture of olive flounder, Paralichthys olivaceus Temminck & Schlegel, in South Korea. We developed a real-time polymerase chain reaction (PCR) method using the TaqMan probe assay to detect and quantify S. parauberis by targeting the gyrB gene sequences, which are effective for molecular analysis of the genus Streptococcus. Our real-time PCR assay is capable of detecting 10 fg of genomic DNA per reaction. The intra- and interassay coefficient of variation (CV) values ranged from 0.42-1.95%, demonstrating that the assay has good reproducibility. There was not any cross-reactivity to Streptococcus iniae or to other streptococcal/lactococcal fish pathogens, such as S. agalactiae and Lactococcus garvieae, indicating that the assay is highly specific to S. parauberis. The results of the real-time PCR assay corresponded well to those of conventional culture assays for S. parauberis from inoculated tissue homogenates (r = 0.957; P < 0.05). Hence, this sensitive and specific real-time PCR is a valuable tool for diagnostic quantitation of S. parauberis in clinical samples. © 2014 John Wiley & Sons Ltd.

Development of a highly sensitive one-tube nested real-time PCR for detecting Mycobacterium tuberculosis.

Science.gov (United States)

Choi, Yeonim; Jeon, Bo-Young; Shim, Tae Sun; Jin, Hyunwoo; Cho, Sang-Nae; Lee, Hyeyoung

2014-12-01

Rapid, accurate detection of Mycobacterium tuberculosis is crucial in the diagnosis of tuberculosis (TB), but conventional diagnostic methods have limited sensitivity and specificity or are time consuming. A new highly sensitive nucleic acid amplification test, combined nested and real-time polymerase chain reaction (PCR) in a single tube (one-tube nested real-time PCR), was developed for detecting M. tuberculosis, which takes advantage of two PCR techniques, i.e., nested PCR and real-time PCR. One-tube nested real-time PCR was designed to have two sequential reactions with two sets of primers and dual probes for the insertion sequence (IS) 6110 sequence of M. tuberculosis in a single closed tube. The minimum limits of detection of IS6110 real-time PCR and IS6110 one-tube nested real-time PCR were 100 fg/μL and 1 fg/μL of M. tuberculosis DNA, respectively. AdvanSure TB/non-tuberculous mycobacteria (NTM) real-time PCR, IS6110 real-time PCR, and two-tube nested real-time PCR showed 100% sensitivity and 100% specificity for clinical M. tuberculosis isolates and NTM isolates. In comparison, the sensitivities of AdvanSure TB/NTM real-time PCR, single IS6110 real-time PCR, and one-tube nested real-time PCR were 91% (152/167), 94.6% (158/167), and 100% (167/167) for sputum specimens, respectively. In conclusion, IS6110 one-tube nested real-time PCR is useful for detecting M. tuberculosis due to its high sensitivity and simple manipulation. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

Zornhagen, K W; Kristensen, A T; Hansen, A E; Oxboel, J; Kjaer, A

2015-12-01

Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a sensitive technique for quantifying gene expression. Stably expressed reference genes are necessary for normalization of RT-qPCR data. Only a few articles have been published on reference genes in canine tumours. The objective of this study was to demonstrate how to identify suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using RT-qPCR. Primer pairs for 17 potential reference genes were designed and tested in archival tumour biopsies from six dogs. The geNorm algorithm was used to analyse the most suitable reference genes. Eight potential reference genes were excluded from this final analysis because of their dissociation curves. β-Glucuronidase (GUSB) and proteasome subunit, beta type, 6 (PSMB6) were most stably expressed with an M value of 0.154 and a CV of 0.053 describing their average stability. We suggest that choice of reference genes should be based on specific testing in every new experimental set-up. © 2014 John Wiley & Sons Ltd.

Real Time Revisited

Science.gov (United States)

Allen, Phillip G.

1985-12-01

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

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System.

Science.gov (United States)

DeShields, Joseph B; Bomberger, Rachel A; Woodhall, James W; Wheeler, David L; Moroz, Natalia; Johnson, Dennis A; Tanaka, Kiwamu

2018-02-23

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Fluorescence resonance energy transfer-based real-time polymerase chain reaction method without DNA extraction for the genotyping of F5, F2, F12, MTHFR, and HFE.

Science.gov (United States)

Martinez-Serra, Jordi; Robles, Juan; Nicolàs, Antoni; Gutierrez, Antonio; Ros, Teresa; Amat, Juan Carlos; Alemany, Regina; Vögler, Oliver; Abelló, Aina; Noguera, Aina; Besalduch, Joan

2014-01-01

Blood samples are extensively used for the molecular diagnosis of many hematological diseases. The daily practice in a clinical laboratory of molecular diagnosis in hematology involves using a variety of techniques, based on the amplification of nucleic acids. Current methods for polymerase chain reaction (PCR) use purified genomic DNA, mostly isolated from total peripheral blood cells or white blood cells (WBC). In this paper we describe a real-time fluorescence resonance energy transfer-based method for genotyping directly from blood cells. Our strategy is based on an initial isolation of the WBCs, allowing the removal of PCR inhibitors, such as the heme group, present in the erythrocytes. Once the erythrocytes have been lysed, in the LightCycler(®) 2.0 Instrument, we perform a real-time PCR followed by a melting curve analysis for different genes (Factors 2, 5, 12, MTHFR, and HFE). After testing 34 samples comparing the real-time crossing point (CP) values between WBC (5×10(6) WBC/mL) and purified DNA (20 ng/μL), the results for F5 Leiden were as follows: CP mean value for WBC was 29.26±0.566 versus purified DNA 24.79±0.56. Thus, when PCR was performed from WBC (5×10(6) WBC/mL) instead of DNA (20 ng/μL), we observed a delay of about 4 cycles. These small differences in CP values were similar for all genes tested and did not significantly affect the subsequent analysis by melting curves. In both cases the fluorescence values were high enough, allowing a robust genotyping of all these genes without a previous DNA purification/extraction.

INVESTIGATION OF RANGES AND FREQUENCY OF MUTATIONS IN THE embB GENE IN MYCOBACTERIUMTUBERCULOSIS ASSOCIATED WITH RESISTANCE TO ETHAMBUTOL USING REAL-TIME POLYMERASE CHAINREACTION

Directory of Open Access Journals (Sweden)

Yu. S. Аlyapkinа

2017-01-01

Full Text Available Based on real-time allele-specific polymerase chain reaction, the ranges of potential mutations in codons of 306 and 405 of the embBgene in Mycobacterium tuberculosis associated with resistance to ethambutol were investigated. 5 different mutations were detected in codon 306 and 3 mutations were found in codon 406 of the embB gene. The detected mutations were confirmed by sequencing and mass spectrometry. By analyzing the frequency of detected mutations of , the set of reagents was developed for rapid testing of susceptibility tuberculous mycobacteria to ethambutol by multi-competitive allele-specific real-time PCR. Out of 107 tested specimens of clinical isolates, mutations of the embB gene of M. tuberculosis were detected in 49 (45.8% specimens, and no mutations were found in 58 (52.2% specimens. 39 (36.4% specimens had mutations in codon 306 of the embB gene, and 9 (8.4% specimens had a mutation in codon 406, and 1 (0.9% specimen had mutations in both codons 306 and 406. The high level of agreement in the results of molecular genetic and bacteriological tests (84% proved the significance of mutations in codons 306 and 406 of the embB gene in M. tuberculosis and the need for their identification in order to detect ethambutol resistant strains of M. tuberculosis. When using molecular genetic tests, the sensitivity level made 75.8%, while the specificity of standard culture-based methods makes 95.6%.

Encefalitis herpética confirmada por reacción en cadena de la polimerasa en tiempo real: reporte de caso Herpetic encephalitis confirmed by real time polymerase chain reaction: case report

Directory of Open Access Journals (Sweden)

Beatriz H. Aristizábal

2006-04-01

, migraine and alteration in the conscience level. Due to the commitment of the temporary lobe, the clinical manifestations can also include hallucinations, aphasia and changes of personality. The sequels in the treated patients are significant. Objective: to show the importance of early molecular diagnosis in patients with suspected herpetic encephalitis infection. Methods: the diagnosis of the herpetic encephalitis has changed in the last years thanks to the coming of the real time polymerase chain reaction for herpes simplex virus in cerebrospinal fluid, a fast strategy with high sensitivity and specificity that has allowed to replace the suspect diagnoses made by tomography axial computerized or electroencephalogram, or the low yields of the viral isolation in the cerebrospinal fluid. Results: A clinical case report of a patient attended in our hospital with image and neuropsychological studies compatible for herpetic encephalitis, and confirmed diagnosis by real time polymerase chain reaction is described. Conclusions: the results of laboratory and the early diagnosis are critical for the precocious treatment and the evolution of the patient.

Real-Time and Post-Processed Georeferencing for Hyperpspectral Drone Remote Sensing

Science.gov (United States)

Oliveira, R. A.; Khoramshahi, E.; Suomalainen, J.; Hakala, T.; Viljanen, N.; Honkavaara, E.

2018-05-01

The use of drones and photogrammetric technologies are increasing rapidly in different applications. Currently, drone processing workflow is in most cases based on sequential image acquisition and post-processing, but there are great interests towards real-time solutions. Fast and reliable real-time drone data processing can benefit, for instance, environmental monitoring tasks in precision agriculture and in forest. Recent developments in miniaturized and low-cost inertial measurement systems and GNSS sensors, and Real-time kinematic (RTK) position data are offering new perspectives for the comprehensive remote sensing applications. The combination of these sensors and light-weight and low-cost multi- or hyperspectral frame sensors in drones provides the opportunity of creating near real-time or real-time remote sensing data of target object. We have developed a system with direct georeferencing onboard drone to be used combined with hyperspectral frame cameras in real-time remote sensing applications. The objective of this study is to evaluate the real-time georeferencing comparing with post-processing solutions. Experimental data sets were captured in agricultural and forested test sites using the system. The accuracy of onboard georeferencing data were better than 0.5 m. The results showed that the real-time remote sensing is promising and feasible in both test sites.

Quantitative assessment of Azumiobodo hoyamushi distribution in the tunic of soft tunic syndrome-affected ascidian Halocynthia roretzi using real-time polymerase chain reaction.

Science.gov (United States)

Shin, Yun-Kyung; Nam, Ki-Woong; Park, Kwan Ha; Yoon, Jong-Man; Park, Kyung-Il

2014-11-26

The kinetoplastid parasite, Azumiobodo hoyamushi, is the causative agent of soft tunic syndrome (STS) in ascidians and leads to their mass mortality in Korean waters. This study was conducted to quantify A. hoyamushi density during the development of STS in the tunics of ascidians (Halocynthia roretzi) using real-time polymerase chain reaction (qPCR). The infection intensity of A. hoyamushi, as measured by qPCR, varied depending on the part of the tunic analyzed, as well as the stage of STS development. The highest infection intensity was recorded in the tunics of the siphons. The infection intensity of A. hoyamushi in the siphons was only 2.9 cell/tunic (area, 0.25 cm(2)) or 106.0 cell/gram tunic (GT) in the early phase of STS, but this value increased dramatically to 16,066 cells/tunic (0.25 cm(2)) or 617,004 cell/GT at the time of death. The number of A. hoyamushi parasites increased gradually and their distribution spread from the siphons to the other parts of the tunics. qPCR enabled the quantitation of A. hoyamushi and the results revealed that parasite density increased as STS progressed. In addition, our results suggested that the siphons might function as the portal of entry for A. hoyamushi during infection.

Real-time sensor failure detection by dynamic modelling of a PWR plant

International Nuclear Information System (INIS)

Turkcan, E.; Ciftcioglu, O.

1992-06-01

Signal validation and sensor failure detection is an important problem in real-time nuclear power plant (NPP) surveillance. Although conventional sensor redundancy, in a way, is a solution, identification of faulty sensor is necessary for further preventive actions to be taken. A comprehensive solution for the system so that any sensory reading is verified by its model based estimated counterpart, in real-time. Such a realization is accomplished by means of dynamic system's states estimation methodology using Kalman filter modelling technique. The method is investigated by means of real-time data of the steam generator of Borssele nuclear power plant and the method has proved to be satisfactory for real-time sensor failure detection as well as model validation verification. (author). 5 refs.; 6 figs.; 1 tab

Integration of Real-Time Data Into Building Automation Systems

Energy Technology Data Exchange (ETDEWEB)

Mark J. Stunder; Perry Sebastian; Brenda A. Chube; Michael D. Koontz

2003-04-16

The project goal was to investigate the possibility of using predictive real-time information from the Internet as an input to building management system algorithms. The objectives were to identify the types of information most valuable to commercial and residential building owners, managers, and system designers. To comprehensively investigate and document currently available electronic real-time information suitable for use in building management systems. Verify the reliability of the information and recommend accreditation methods for data and providers. Assess methodologies to automatically retrieve and utilize the information. Characterize equipment required to implement automated integration. Demonstrate the feasibility and benefits of using the information in building management systems. Identify evolutionary control strategies.

Development of a Real-time PCR test for porcine group A rotavirus diagnosis

Directory of Open Access Journals (Sweden)

Elizabeth C.M. Marconi

2015-01-01

Full Text Available Group A Rotavirus (RVA is one of the most common causes of diarrhea in humans and several animal species. A SYBR-Green Real-Time polymerase chain reaction (PCR was developed to diagnose RVA from porcine fecal samples, targeting amplification of a 137-bp fragment of nonstructural protein 5 (NSP5 gene using mRNA of bovine NADH-desidrogenase-5 as exogenous internal control. Sixty-five samples were tested (25 tested positive for conventional PCR and genetic sequencing. The overall agreement (kappa was 0.843, indicating 'very good' concordance between tests, presenting 100% of relative sensitivity (25+ Real Time PCR/25+ Conventional PCR and 87.5% of relative sensitivity (35- Real Time PCR/40- Conventional PCR. The results also demonstrated high intra- and inter-assay reproducibility (coefficient of variation ≤1.42%; thus, this method proved to be a fast and sensitive approach for the diagnosis of RVA in pigs.

A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR

NARCIS (Netherlands)

J.P.P. Meijerink (Jules); C. Mandigers; L. van de Locht; E. Tonnissen; F. Goodsaid; J. Raemaekers (John)

2001-01-01

textabstractQuantification of residual disease by real-time polymerase chain reaction (PCR) will become a pivotal tool in the development of patient-directed therapy. In recent years, various protocols to quantify minimal residual disease in leukemia or lymphoma patients have been

Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms

NARCIS (Netherlands)

Gasparic, M.B.; Cankar, K.; Zel, J.; Gruden, K.

2008-01-01

Background: The real-time polymerase chain reaction is currently the method of choice for quantifying nucleic acids in different DNA based quantification applications. It is widely used also for detecting and quantifying genetically modified components in food and feed, predominantly employing

Self-organization comprehensive real-time state evaluation model for oil pump unit on the basis of operating condition classification and recognition

Science.gov (United States)

Liang, Wei; Yu, Xuchao; Zhang, Laibin; Lu, Wenqing

2018-05-01

In oil transmission station, the operating condition (OC) of an oil pump unit sometimes switches accordingly, which will lead to changes in operating parameters. If not taking the switching of OCs into consideration while performing a state evaluation on the pump unit, the accuracy of evaluation would be largely influenced. Hence, in this paper, a self-organization Comprehensive Real-Time State Evaluation Model (self-organization CRTSEM) is proposed based on OC classification and recognition. However, the underlying model CRTSEM is built through incorporating the advantages of Gaussian Mixture Model (GMM) and Fuzzy Comprehensive Evaluation Model (FCEM) first. That is to say, independent state models are established for every state characteristic parameter according to their distribution types (i.e. the Gaussian distribution and logistic regression distribution). Meanwhile, Analytic Hierarchy Process (AHP) is utilized to calculate the weights of state characteristic parameters. Then, the OC classification is determined by the types of oil delivery tasks, and CRTSEMs of different standard OCs are built to constitute the CRTSEM matrix. On the other side, the OC recognition is realized by a self-organization model that is established on the basis of Back Propagation (BP) model. After the self-organization CRTSEM is derived through integration, real-time monitoring data can be inputted for OC recognition. At the end, the current state of the pump unit can be evaluated by using the right CRTSEM. The case study manifests that the proposed self-organization CRTSEM can provide reasonable and accurate state evaluation results for the pump unit. Besides, the assumption that the switching of OCs will influence the results of state evaluation is also verified.

Quantitative Real-time Polymerase Chain Reaction for Enteropathogenic Escherichia coli: A Tool for Investigation of Asymptomatic Versus Symptomatic Infections

Science.gov (United States)

Barletta, Francesca; Mercado, Erik; Ruiz, Joaquim; Ecker, Lucie; Lopez, Giovanni; Mispireta, Monica; Gil, Ana I.; Lanata, Claudio F.; Cleary, Thomas G.

2011-01-01

Background. Enteropathogenic Escherichia coli (EPEC) strains are pediatric pathogens commonly isolated from both healthy and sick children with diarrhea in areas of endemicity. The aim of this study was to compare the bacterial load of EPEC isolated from stool samples from children with and without diarrhea to determine whether bacterial load might be a useful tool for further study of this phenomenon. Methods. EPEC was detected by polymerase chain reaction (PCR) of colonies isolated on MacConkey plates from 53 diarrheal and 90 healthy children aged <2 years. DNA was isolated from stool samples by cetyltrimethylammonium bromide extraction. To standardize quantification by quantitative real-time PCR (qRT-PCR), the correlation between fluorescence threshold cycle and copy number of the intimin gene of EPEC E2348/69 was determined. Results. The detection limit of qRT-PCR was 5 bacteria/mg stool. The geometric mean load in diarrhea was 299 bacteria/mg (95% confidence interval [CI], 77–1164 bacteria/mg), compared with 29 bacteria/mg (95% CI, 10–87 bacteria/mg) in control subjects (P = .016). Bacterial load was significantly higher in children with diarrhea than in control subjects among children <12 months of age (178 vs 5 bacteria/mg; P = .006) and among children with EPEC as the sole pathogen (463 vs 24 bacteria/mg; P = .006). Conclusions. EPEC load measured by qRT-PCR is higher in diarrheal than in healthy children. qRT-PCR may be useful to study the relationship between disease and colonization in settings of endemicity. PMID:22028433

Securing Real-Time Sessions in an IMS-Based Architecture

Science.gov (United States)

Cennamo, Paolo; Fresa, Antonio; Longo, Maurizio; Postiglione, Fabio; Robustelli, Anton Luca; Toro, Francesco

The emerging all-IP mobile network infrastructures based on 3rd Generation IP Multimedia Subsystem philosophy are characterised by radio access technology independence and ubiquitous connectivity for mobile users. Currently, great focus is being devoted to security issues since most of the security threats presently affecting the public Internet domain, and the upcoming ones as well, are going to be suffered by mobile users in the years to come. While a great deal of research activity, together with standardisation efforts and experimentations, is carried out on mechanisms for signalling protection, very few integrated frameworks for real-time multimedia data protection have been proposed in a context of IP Multimedia Subsystem, and even fewer experimental results based on testbeds are available. In this paper, after a general overview of the security issues arising in an advanced IP Multimedia Subsystem scenario, a comprehensive infrastructure for real-time multimedia data protection, based on the adoption of the Secure Real-Time Protocol, is proposed; then, the development of a testbed incorporating such functionalities, including mechanisms for key management and cryptographic context transfer, and allowing the setup of Secure Real-Time Protocol sessions is presented; finally, experimental results are provided together with quantitative assessments and comparisons of system performances for audio sessions with and without the adoption of the Secure Real-Time Protocol framework.

Quantitative real-time polymerase chain reaction for Streptococcus mutans and Streptococcus sobrinus in dental plaque samples and its association with early childhood caries.

Science.gov (United States)

Choi, Eun-Jung; Lee, Sung-Hoon; Kim, Young-Jae

2009-03-01

Streptococcus mutans and Streptococcus sobrinus are closely associated with the development of early childhood caries (ECC). Recently, quantitative real-time polymerase chain reaction (qRT-PCR) has been used for rapid and accurate quantification of these bacterial species. This study aims to detect quantitatively the levels of S. mutans and S. sobrinus in plaque samples by qRT-PCR, and to assess their association with the prevalence of ECC in Korean preschool children. One hundred and five children (71 months old or younger) were examined and classified into three groups (caries-free, ECC, severe ECC). Dental plaque samples were collected and qRT-PCR was conducted using oligonucleotide primers specific for glucosyltransferase gene (S. mutans-gtfB, S. sobrinus-gtfU) and universal primer. Pearson's correlation test was conducted to evaluate the relationship between the dmfs (decayed, missing, or filled surfaces primary teeth) scores and the microbiological findings. There was a significant difference between the levels of S. mutans and S. sobrinus in the plaque samples of the three groups (P plaque samples. The children with higher ratio of S. sobrinus to S. mutans in their dental plaque showed higher incidence of ECC.

A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR.

NARCIS (Netherlands)

Meijerink, J.P.P.; Mandigers, C.M.P.W.; Locht, A.T.F. van de; Tonnissen, E.L.R.T.M.; Goodsaid, F.; Raemaekers, J.M.M.

2001-01-01

Quantification of residual disease by real-time polymerase chain reaction (PCR) will become a pivotal tool in the development of patient-directed therapy. In recent years, various protocols to quantify minimal residual disease in leukemia or lymphoma patients have been developed. These assays assume

Fluorescence resonance energy transfer-based real-time polymerase chain reaction method without DNA extraction for the genotyping of F5, F2, F12, MTHFR, and HFE

Directory of Open Access Journals (Sweden)

Martinez-Serra J

2014-06-01

Full Text Available Jordi Martinez-Serra,1 Juan Robles,2 Antoni Nicolàs,3 Antonio Gutierrez,1 Teresa Ros,1 Juan Carlos Amat,1 Regina Alemany,4 Oliver Vögler,4 Aina Abelló,2 Aina Noguera,2 Joan Besalduch1 1Department of Hematology, 2Department of Clinical Analysis, Hospital Universitary Son Espases, Palma de Mallorca, Spain; 3ECOGEN, Barcelona, 4Department of Cell Biology, University of the Balearic Islands, Palma de Mallorca, Spain Abstract: Blood samples are extensively used for the molecular diagnosis of many hematological diseases. The daily practice in a clinical laboratory of molecular diagnosis in hematology involves using a variety of techniques, based on the amplification of nucleic acids. Current methods for polymerase chain reaction (PCR use purified genomic DNA, mostly isolated from total peripheral blood cells or white blood cells (WBC. In this paper we describe a real-time fluorescence resonance energy transfer-based method for genotyping directly from blood cells. Our strategy is based on an initial isolation of the WBCs, allowing the removal of PCR inhibitors, such as the heme group, present in the erythrocytes. Once the erythrocytes have been lysed, in the LightCycler® 2.0 Instrument, we perform a real-time PCR followed by a melting curve analysis for different genes (Factors 2, 5, 12, MTHFR, and HFE. After testing 34 samples comparing the real-time crossing point (CP values between WBC (5×106 WBC/mL and purified DNA (20 ng/µL, the results for F5 Leiden were as follows: CP mean value for WBC was 29.26±0.566 versus purified DNA 24.79±0.56. Thus, when PCR was performed from WBC (5×106 WBC/mL instead of DNA (20 ng/µL, we observed a delay of about 4 cycles. These small differences in CP values were similar for all genes tested and did not significantly affect the subsequent analysis by melting curves. In both cases the fluorescence values were high enough, allowing a robust genotyping of all these genes without a previous DNA purification

Evaluation of dual target-specific real-time PCR for the detection of Kingella kingae in a Danish paediatric population

DEFF Research Database (Denmark)

de Knegt, Victoria Elizabeth; Kristiansen, Gitte Qvist; Schønning, Kristian

2017-01-01

BACKGROUND: We aimed to evaluate the relevance of dual target real-time polymerase chain (PCR) assays targeting the rtxA and cpn60 genes of the paediatric pathogen Kingella kingae. We also studied for the first time the clinical and epidemiological features of K. kingae infections in a Danish pop......-value: peak in autumn. CONCLUSION: Dual target-specific real-time PCR markedly improved the detection of K. kingae in clinical specimens when compared to culture methods....

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

Science.gov (United States)

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

2012-04-01

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

Real-time detection of musical onsets with linear prediction and sinusoidal modeling

Science.gov (United States)

Glover, John; Lazzarini, Victor; Timoney, Joseph

2011-12-01

Real-time musical note onset detection plays a vital role in many audio analysis processes, such as score following, beat detection and various sound synthesis by analysis methods. This article provides a review of some of the most commonly used techniques for real-time onset detection. We suggest ways to improve these techniques by incorporating linear prediction as well as presenting a novel algorithm for real-time onset detection using sinusoidal modelling. We provide comprehensive results for both the detection accuracy and the computational performance of all of the described techniques, evaluated using Modal, our new open source library for musical onset detection, which comes with a free database of samples with hand-labelled note onsets.

Comparative analysis of fluorescence in situ hybridizationand real time polymerase chain reaction in diagnosis of chronic myeloid leukemia

International Nuclear Information System (INIS)

Ali, J.; Khan, S.A.; Rauf, S.E.; Ayyub, M.; Ali, N.

2017-01-01

To compare the sensitivity and specificity of fluorescence in situ hybridization (FISH) with real time polymerase chain reaction (RT-PCR) in the diagnosis of Chronic Myeloid Leukemia (CML). Study Design: A cross-sectional, analytical study. Place and Duration of Study: Haematology Department, Armed Forces Institute of Pathology, Rawalpindi, from January 2012 to February 2014. Methodology:A total number of 87 patients of CML were studied. The diagnosis was made on the basis of clinical history, peripheral blood and bone marrow aspiration. These patients were tested for the presence of BCR-ABL1 fusion gene by RT-PCR and FISH. About 5 ml of venous blood was collected, half was taken in heparin for FISH and half in ethylenediamine tetra-acetic acid (EDTA) for CBC and PCR. For FISH, cells were cultured for 24 hours in RPMI 1640 medium and evaluated using BX51 fluorescence microscope for dual fusion signal of yellow colour. Samples having 20 or more interphases positive for dual fusion signals were taken as positive. For PCR, RNA extraction was done by Tri-Reagent LS (MRC, USA) and cDNA was synthesized using reverse transcriptase and gene specific primer. RT-PCR was done on ABI-7500. The positive samples were identified when fluorescence exceeded threshold limit. Results of RT-PCR and FISH were compared. Results: Out of the 87 patients, 85 (97.7%) were PCR positive and 2 (2.3%) were PCR negative, whereas in FISH 83 (95.4%) were positive and 4 (4.5%) were negative. Sensitivity and specificity of FISH was 97.6% and 100%, respectively. Conclusion: FISH is a reliable supplementary method to PCR for detection of BCR-ABL1 fusion gene in the diagnosis of CML. (author)

Miltenberger blood group typing by real-time polymerase chain reaction (qPCR) melting curve analysis in Thai population.

Science.gov (United States)

Vongsakulyanon, A; Kitpoka, P; Kunakorn, M; Srikhirin, T

2015-12-01

To develop reliable and convenient methods for Miltenberger (Mi(a) ) blood group typing. To apply real-time polymerase chain reaction (qPCR) melting curve analysis to Mi(a) blood group typing. The Mi(a) blood group is the collective set of glycophorin hybrids in the MNS blood group system. Mi(a+) blood is common among East Asians and is also found in the Thai population. Incompatible Mi(a) blood transfusions pose the risk of life-threatening haemolysis; therefore, Mi(a) blood group typing is necessary in ethnicities where the Mi(a) blood group is prevalent. One hundred and forty-three blood samples from Thai blood donors were used in the study. The samples included 50 Mi(a+) samples and 93 Mi(a-) samples, which were defined by serology. The samples were typed by Mi(a) typing qPCR, and 50 Mi(a+) samples were sequenced to identify the Mi(a) subtypes. Mi(a) subtyping qPCR was performed to define GP.Mur. Both Mi(a) typing and Mi(a) subtyping were tested on a conventional PCR platform. The results of Mi(a) typing qPCR were all concordant with serology. Sequencing of the 50 Mi(a+) samples revealed 47 GP.Mur samples and 3 GP.Hop or Bun samples. Mi(a) subtyping qPCR was the supplementary test used to further define GP.Mur from other Mi(a) subtypes. Both Mi(a) typing and Mi(a) subtyping performed well using a conventional PCR platform. Mi(a) typing qPCR correctly identified Mi(a) blood groups in a Thai population with the feasibility of Mi(a) subtype discrimination, and Mi(a) subtyping qPCR was able to further define GP.Mur from other Mi(a) subtypes. © 2015 British Blood Transfusion Society.

Wheat-specific gene, ribosomal protein l21, used as the endogenous reference gene for qualitative and real-time quantitative polymerase chain reaction detection of transgenes.

Science.gov (United States)

Liu, Yi-Ke; Li, He-Ping; Huang, Tao; Cheng, Wei; Gao, Chun-Sheng; Zuo, Dong-Yun; Zhao, Zheng-Xi; Liao, Yu-Cai

2014-10-29

Wheat-specific ribosomal protein L21 (RPL21) is an endogenous reference gene suitable for genetically modified (GM) wheat identification. This taxon-specific RPL21 sequence displayed high homogeneity in different wheat varieties. Southern blots revealed 1 or 3 copies, and sequence analyses showed one amplicon in common wheat. Combined analyses with sequences from common wheat (AABBDD) and three diploid ancestral species, Triticum urartu (AA), Aegilops speltoides (BB), and Aegilops tauschii (DD), demonstrated the presence of this amplicon in the AA genome. Using conventional qualitative polymerase chain reaction (PCR), the limit of detection was 2 copies of wheat haploid genome per reaction. In the quantitative real-time PCR assay, limits of detection and quantification were about 2 and 8 haploid genome copies, respectively, the latter of which is 2.5-4-fold lower than other reported wheat endogenous reference genes. Construct-specific PCR assays were developed using RPL21 as an endogenous reference gene, and as little as 0.5% of GM wheat contents containing Arabidopsis NPR1 were properly quantified.

Development of a non invasion real-time PCR assay for the quantitation of chicken parvovirus in fecal swabs

Science.gov (United States)

The present study describes the development of a real time Taqman polymerase chain reaction (PCR) assay using a fluorescent labeled probe for the detection and quantitation of chicken parvovirus (ChPV) in feces. The primers and probes were designed based on the nucleotide sequence of the non struct...

Adhesion of mutans streptococci to self-ligating ceramic brackets: in vivo quantitative analysis with real-time polymerase chain reaction.

Science.gov (United States)

Jung, Woo-Sun; Yang, Il-Hyung; Lim, Won Hee; Baek, Seung-Hak; Kim, Tae-Woo; Ahn, Sug-Joon

2015-12-01

To analyze in vivo mutans streptococci (MS) adhesion to self-ligating ceramic brackets [Clarity-SL (CSL) and Clippy-C (CC)] and the relationships between bacterial adhesion and oral hygiene indices. Four central incisor brackets from the maxilla and mandible were collected from 40 patients (20 patients per each bracket type) at debonding immediately after plaque and gingival indices were measured. Adhesions of Streptococcus mutans, S. sobrinus, and total bacteria were quantitatively determined using real-time polymerase chain reaction after genomic DNA was extracted. Factorial analysis of variance was used to analyze bacterial adhesion to the brackets with respect to the bracket type and jaw position. Correlation coefficients were calculated to determine the relationships of bacterial adhesion to oral hygiene indices. Adhesion of total bacteria and S. mutans to CSL was higher than that to CC (P brackets was higher than that to the maxillary ones (P brackets were higher than that in the mandibular ones (P brackets and jaw positions. Interestingly, no significant relationships were found between bacterial adhesions and oral hygiene indices. Complex bracket configurations may significantly influence bacterial adhesion to orthodontic brackets. Further in vivo study using bracket raw materials will help to define the relationships between bacteria adhesion and enamel demineralization. Because oral hygiene indices were not significantly correlated with adhesions of MS to self-ligating ceramic brackets, careful examinations around the brackets should be needed to prevent enamel demineralization, regardless of oral hygiene status. © The Author 2015. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

Science.gov (United States)

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

2018-04-01

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

Viewing Human DNA Polymerase β Faithfully and Unfaithfully Bypass an Oxidative Lesion by Time-Dependent Crystallography

Science.gov (United States)

Vyas, Rajan; Reed, Andrew J.; Tokarsky, E. John; Suo, Zucai

2015-01-01

One common oxidative DNA lesion, 8-oxo-7,8-dihydro-2′-deoxyguanine (8-oxoG), is highly mutagenic in vivo due to its anti-conformation forming a Watson–Crick base pair with correct deoxycytidine 5′-triphosphate (dCTP) and its syn-conformation forming a Hoogsteen base pair with incorrect deoxyadenosine 5′-triphosphate (dATP). Here, we utilized time-resolved X-ray crystallography to follow 8-oxoG bypass by human DNA polymerase β (hPolβ). In the 12 solved structures, both Watson–Crick (anti-8-oxoG:anti-dCTP) and Hoogsteen (syn-8-oxoG:anti-dATP) base pairing were clearly visible and were maintained throughout the chemical reaction. Additionally, a third Mg2+ appeared during the process of phosphodiester bond formation and was located between the reacting α- and β-phosphates of the dNTP, suggesting its role in stabilizing reaction intermediates. After phosphodiester bond formation, hPolβ reopened its conformation, pyrophosphate was released, and the newly incorporated primer 3′-terminal nucleotide stacked, rather than base paired, with 8-oxoG. These structures provide the first real-time pictures, to our knowledge, of how a polymerase correctly and incorrectly bypasses a DNA lesion. PMID:25825995

TEST KIT FOR THE DETECTION AND GENOTYPING OF HIGHLY PATHOGENIC INFLUENZA VIRUS A H5N1 BY REAL-TIME POLYMERASE CHAIN REACTION

Directory of Open Access Journals (Sweden)

S. V. Stepaniuk

2014-06-01

Full Text Available Results of the annual monitoring of epizooties indicate that highly pathogenic HPAI/H5N1 avian influenza widely circulated in Eurasian region. Over a period of 2010–2013 years more than 165 cases of outbreaks in 14 countries were found out. Ukraine became one of the first countries in Europe where in Autonomous Republic of Crimea in October 2005 outbreak of avian epizootic with HPAI/H5N1 was documented and until February 2008 more than 236,000 poultry were killed. Since then the question of monitoring of infected both migrating birds and poultry in places of cross contact in Ukraine remains of high priority. The test system is developed for identification and genotyping A H5N1 on three genes (M, H5 and N1 HPAI/H5N1 in real-time mode for polymerase chain reaction. Test kit capacity to detect HPAI/h5n1avian influenza virus and differentiate it from the other viral infection agents of birds and animals were studied by testing of HPAI/H5N1 virus isolated during mass infection outbreak in Crimea in 2005 and cultural specimens of other viral pathogens. It was established that the «DIA Real Avian Influenza» test kit was capable to detect RNA influenza A virus of high pathogenic H5N1 strains having high sensitivity (100% while RNA of the Crimean HPAI/H5N1 isolate studying and specificity (100% while RNA viruses of Newcastle birds disease, fowl powershift, syndrome of drop in egg production and horse influenza studying.

Development of multiplex real-time PCR assay for the detection of Brucella spp., Leptospira spp. and Campylobacter foetus

Directory of Open Access Journals (Sweden)

Abdelfattah M. Selim

2014-12-01

Full Text Available Abortion among dairy cattle is one of the major causes of economic losses in the livestock industry. This study describes a 1-step multiplex real-time polymerase chain reaction (PCR to detect Brucella spp., Leptospira spp. and Campylobacter foetus, these are significant bacteria commonly implicated in bovine abortion. ß-actin was added to the same PCR reaction as an internal control to detect any extraction failure or PCR inhibition. The detection limit of multiplex real-time PCR using purified DNA from cultured organisms was set to 5 fg for Leptospira spp. and C. foetus and to 50 fg for Brucella spp. The multiplex real-time PCR did not produce any non-specific amplification when tested with different strains of the 3 pathogens. This multiplex real-time PCR provides a valuable tool for diagnosis, simultaneous and rapid detection for the 3 pathogens causing abortion in bovine.

Comparison of Parasite Burden Using Real-Time Polymerase Chain Reaction Assay and Limiting Dilution Assay in Leishma-nia major Infected Mouse

Directory of Open Access Journals (Sweden)

Somayeh GHOTLOO

2015-12-01

Full Text Available Background:Limiting dilution assay is considered as the gold standard method for quantifying the number of parasites in the animal model of Leishmania infection. Nowadays, real-time PCR is being increasingly applied to quantify infectious agents. In the present study, a real-time PCR assay was developed to estimate para­site burdens in lymph nodes of Leishmania major infected BALB/C mice. Enumera­tion of parasites was also performed by limiting dilution assay and compared with the results of real-time PCR based quantification.Methods:The SYBR Green based real- time PCR assay was performed to amplify a 75 bp fragment of superoxide dismutase B1 gene in the lymph nodes of L. major infected BALB/C mice 8 weeks post infection. Mice were infected subcutaneously at the base of their tail with 2 × 105L. major promastigotes in the stationary phase of growth. To compare parasite burdens obtained by real-time PCR assay with those of limiting dilution assay, twelve 8-fold serial dilutions of the lymph node homoge­nates were prepared in the Schneider medium and incubated at 26°C.After 7 days, wells containing motile parasites were identified by direct observation under an inverted light microscope and the total number of parasites was estimated using the ELIDA software.Results:Spearman's correlation coefficient of the parasite burdens between real-time PCR and limiting dilution assay was 0.72 (Pvalue = 0.008.Conclusion:Real-time PCR assay is an appropriate replacement to existing limit­ing dilution assay in quantifying parasite burden in the experimental model of Leishma­nia infection.

Performance of 2 commercial real-time polymerase chain reaction assays for the detection of Aspergillus and Pneumocystis DNA in bronchoalveolar lavage fluid samples from critical care patients.

Science.gov (United States)

Orsi, Carlotta Francesca; Gennari, William; Venturelli, Claudia; La Regina, Annunziata; Pecorari, Monica; Righi, Elena; Machetti, Marco; Blasi, Elisabetta

2012-06-01

This article investigates the performance of 2 commercial real-time polymerase chain reaction (PCR) assays, MycAssay™ Aspergillus (Myc(Asp)Assay) and MycAssay™ Pneumocystis (Myc(PCP)Assay), on the ABI 7300 platform for the detection of Aspergillus (Asp) or Pneumocystis jirovecii (Pj) DNA in bronchoalveolar lavage (BAL) samples from 20 patients. Operationally, patients enrolled were clustered into 3 groups: invasive aspergillosis group (IA, 7 patients), Pj pneumonia group (PCP, 8 patients), and negative control group (5 patients). All the IA patients were Myc(Asp)Assay positive, whereas 12 non-IA patients returned negative PCR results. Furthermore, 7 of 8 PCP patients were Myc(PCP)Assay positive, while 9 non-PCP patients were PCR negative. In conclusion, these data provide an early indication of the effectiveness of both the Myc(Asp)Assay and Myc(PCP)Assay on the ABI 7300 platform for the detection of either Asp or Pj DNA in BAL from patients with deep fungal infections. Copyright © 2012 Elsevier Inc. All rights reserved.

Development of a Real-time PCR test for porcine group A rotavirus diagnosis

OpenAIRE

Marconi, Elizabeth C.M.; Bernardes, Nara T.C.G.; Beserra, Laila A.R.; Silva, Fernanda D.F.; Gregori, Fabio

2015-01-01

Group A Rotavirus (RVA) is one of the most common causes of diarrhea in humans and several animal species. A SYBR-Green Real-Time polymerase chain reaction (PCR) was developed to diagnose RVA from porcine fecal samples, targeting amplification of a 137-bp fragment of nonstructural protein 5 (NSP5) gene using mRNA of bovine NADH-desidrogenase-5 as exogenous internal control. Sixty-five samples were tested (25 tested positive for conventional PCR and genetic sequencing). The overall agreement (...

Multipurpose assessment for the quantification of Vibrio spp. and total bacteria in fish and seawater using multiplex real-time polymerase chain reaction

Science.gov (United States)

Kim, Ji Yeun; Lee, Jung-Lim

2014-01-01

Background This study describes the first multiplex real-time polymerase chain reaction assay developed, as a multipurpose assessment, for the simultaneous quantification of total bacteria and three Vibrio spp. (V. parahaemolyticus, V. vulnificus and V. anguillarum) in fish and seawater. The consumption of raw finfish as sushi or sashimi has been increasing the chance of Vibrio outbreaks in consumers. Freshness and quality of fishery products also depend on the total bacterial populations present. Results The detection sensitivity of the specific targets for the multiplex assay was 1 CFU mL−1 in pure culture and seawater, and 10 CFU g−1 in fish. While total bacterial counts by the multiplex assay were similar to those obtained by cultural methods, the levels of Vibrio detected by the multiplex assay were generally higher than by cultural methods of the same populations. Among the natural samples without Vibrio spp. inoculation, eight out of 10 seawater and three out of 20 fish samples were determined to contain Vibrio spp. Conclusion Our data demonstrate that this multiplex assay could be useful for the rapid detection and quantification of Vibrio spp. and total bacteria as a multipurpose tool for surveillance of fish and water quality as well as diagnostic method. © 2014 The Authors. Journal of the Science of Food and Agriculture published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:24752974

Dependable Real-Time Systems

Science.gov (United States)

1991-09-30

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

LabVIEW Real-Time

CERN Multimedia

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

2003-01-01

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

Concepts of real time and semi-real time material control

International Nuclear Information System (INIS)

Lovett, J.E.

1975-01-01

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

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....

Real time expert systems

International Nuclear Information System (INIS)

Asami, Tohru; Hashimoto, Kazuo; Yamamoto, Seiichi

1992-01-01

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

Single tube multiplex real-time PCR for the rapid detection of herpesvirus infections of the central nervous system.

Science.gov (United States)

Sankuntaw, Nipaporn; Sukprasert, Saovaluk; Engchanil, Chulapan; Kaewkes, Wanlop; Chantratita, Wasun; Pairoj, Vantanit; Lulitanond, Viraphong

2011-01-01

Human herpesvirus infection of immunocompromised hosts may lead to central nervous system (CNS) infection and diseases. In this study, a single tube multiplex real-time PCR was developed for the detection of five herpesviruses (HSV-1, HSV-2, VZV, EBV and CMV) in clinical cerebrospinal fluid (CSF) specimens. Two primer pairs specific for the herpesvirus polymerase gene and five hybridization probe pairs for the specific identification of the herpesvirus types were used in a LightCycler multiplex real-time PCR. A singleplex real-time PCR was first optimized and then applied to the multiplex real-time PCR. The singleplex and multiplex real-time PCRs showed no cross-reactivity. The sensitivity of the singleplex real-time PCR was 1 copy per reaction for each herpesvirus, while that of the multiplex real-time PCR was 1 copy per reaction for HSV-1 and VZV and 10 copies per reaction for HSV-2, EBV and CMV. Intra and inter-assay variations of the single tube multiplex assay were in the range of 0.02%-3.67% and 0.79%-4.35%, respectively. The assay was evaluated by testing 62 clinical CSF samples and was found to have equivalent sensitivity, specificity and agreement as the routine real-time PCR, but reducing time, cost and amount of used sample. Copyright © 2011 Elsevier Ltd. All rights reserved.

Direct quantification of fungal DNA from soil substrate using real-time PCR.

Science.gov (United States)

Filion, Martin; St-Arnaud, Marc; Jabaji-Hare, Suha H

2003-04-01

Detection and quantification of genomic DNA from two ecologically different fungi, the plant pathogen Fusarium solani f. sp. phaseoli and the arbuscular mycorrhizal fungus Glomus intraradices, was achieved from soil substrate. Specific primers targeting a 362-bp fragment from the SSU rRNA gene region of G. intraradices and a 562-bp fragment from the F. solani f. sp. phaseoli translation elongation factor 1 alpha gene were used in real-time polymerase chain reaction (PCR) assays conjugated with the fluorescent SYBR(R) Green I dye. Standard curves showed a linear relation (r(2)=0.999) between log values of fungal genomic DNA of each species and real-time PCR threshold cycles and were quantitative over 4-5 orders of magnitude. Real-time PCR assays were applied to in vitro-produced fungal structures and sterile and non-sterile soil substrate seeded with known propagule numbers of either fungi. Detection and genomic DNA quantification was obtained from the different treatments, while no amplicon was detected from non-seeded non-sterile soil samples, confirming the absence of cross-reactivity with the soil microflora DNA. A significant correlation (Pgenomic DNA of F. solani f. sp. phaseoli or G. intraradices detected and the number of fungal propagules present in seeded soil substrate. The DNA extraction protocol and real-time PCR quantification assay can be performed in less than 2 h and is adaptable to detect and quantify genomic DNA from other soilborne fungi.

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

Science.gov (United States)

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

2014-05-01

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

Identification of human intestinal parasites affecting an asymptomatic peri-urban Argentinian population using multi-parallel quantitative real-time polymerase chain reaction.

Science.gov (United States)

Cimino, Rubén O; Jeun, Rebecca; Juarez, Marisa; Cajal, Pamela S; Vargas, Paola; Echazú, Adriana; Bryan, Patricia E; Nasser, Julio; Krolewiecki, Alejandro; Mejia, Rojelio

2015-07-17

In resource-limited countries, stool microscopy is the diagnostic test of choice for intestinal parasites (soil-transmitted helminths and/or intestinal protozoa). However, sensitivity and specificity is low. Improved diagnosis of intestinal parasites is especially important for accurate measurements of prevalence and intensity of infections in endemic areas. The study was carried out in Orán, Argentina. A total of 99 stool samples from a local surveillance campaign were analyzed by concentration microscopy and McMaster egg counting technique compared to the analysis by multi-parallel quantitative real-time polymerase chain reaction (qPCR). This study compared the performance of qPCR assay and stool microscopy for 8 common intestinal parasites that infect humans including the helminths Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Trichuris trichiura, and the protozoa Giardia lamblia, Cryptosporidium parvum/hominis, and Entamoeba histolytica, and investigated the prevalence of polyparasitism in an endemic area. qPCR showed higher detection rates for all parasites as compared to stool microscopy except T. trichiura. Species-specific primers and probes were able to distinguish between A. duodenale (19.1%) and N. americanus (36.4%) infections. There were 48.6% of subjects co-infected with both hookworms, and a significant increase in hookworm DNA for A. duodenale versus N. americanus (119.6 fg/μL: 0.63 fg/μL, P parasites in an endemic area that has improved diagnostic accuracy compared to stool microscopy. This first time use of multi-parallel qPCR in Argentina has demonstrated the high prevalence of intestinal parasites in a peri-urban area. These results will contribute to more accurate epidemiological survey, refined treatment strategies on a public scale, and better health outcomes in endemic settings.

Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze)

Science.gov (United States)

Quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used technique for measuring gene expression levels due to its simplicity, specificity, and sensitivity. Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a...

Quantification of organellar DNA and RNA using real-time PCR.

Science.gov (United States)

Weihe, Andreas

2014-01-01

Quantitative (real-time) polymerase chain reaction (PCR) allows the measurement of relative organellar gene copy numbers as well as transcript abundance of individual mitochondrial or plastidial genes. Requiring only minute amounts of total DNA or RNA, the described method can replace traditional analyses like Southern or Northern hybridization which require large amounts of organellar nucleic acids and usually provide only semiquantitative data. Here we describe prerequisites, reaction conditions, and data analysis principles, which should be applicable for a wide range of plant species and experimental situations where comparative and precise determination of gene copy numbers or transcript abundance is requested. Sequences of amplification primers for qPCR of organellar genes from Arabidopsis are provided.

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

Science.gov (United States)

Thornton, Brenda; Basu, Chhandak

2011-01-01

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

STeP: A Tool for the Development of Provably Correct Reactive and Real-Time Systems

National Research Council Canada - National Science Library

Manna, Zohar

1999-01-01

This research is directed towards the implementation of a comprehensive toolkit for the development and verification of high assurance reactive systems, especially concurrent, real time, and hybrid systems...

Assessment of litter prevalence of Mycoplasma hyopneumoniae in preweaned piglets utilizing an antemortem tracheobronchial mucus collection technique and a real-time polymerase chain reaction assay.

Science.gov (United States)

Vangroenweghe, Frédéric; Karriker, Locke; Main, Rodger; Christianson, Eric; Marsteller, Thomas; Hammen, Kristin; Bates, Jessica; Thomas, Paul; Ellingson, Josh; Harmon, Karen; Abate, Sarah; Crawford, Kimberly

2015-09-01

The swine industry currently lacks validated antemortem methods of detecting baseline herd prevalence of Mycoplasma hyopneumoniae. The focus of our study was to evaluate alternative antemortem detection techniques and to determine baseline litter prevalence in preweaned pig populations utilizing the selected technique and a real-time polymerase chain reaction (qPCR) assay. Preliminary data was analyzed on weaned piglets with evidence of respiratory disease (n = 32). Five sample types (antemortem nasal swab, tracheobronchial mucus, postmortem deep airway swab, bronchoalveolar lavage, and lung tissue) were collected from each pig. Individual samples were tested for M. hyopneumoniae using qPCR. Compared to nasal swabs, tracheobronchial mucus demonstrated higher test sensitivity (P hyopneumoniae. Two out of 180 litters revealed a positive result (1.1%). Individual qPCR assays were run on the samples collected from sow farm 4. Five out of 30 samples revealed a positive result (16.7%). Tracheobronchial mucus collection in combination with qPCR is a sensitive antemortem sampling technique that can be used to estimate the prevalence of M. hyopneumoniae in preweaned pigs, thus providing insight into the infection dynamics across the entire farrow-to-finish process. © 2015 The Author(s).

Process algebra with timing : real time and discrete time

NARCIS (Netherlands)

Baeten, J.C.M.; Middelburg, C.A.; Bergstra, J.A.; Ponse, A.J.; Smolka, S.A.

2001-01-01

We present real time and discrete time versions of ACP with absolute timing and relative timing. The starting-point is a new real time version with absolute timing, called ACPsat, featuring urgent actions and a delay operator. The discrete time versions are conservative extensions of the discrete

Process algebra with timing: Real time and discrete time

NARCIS (Netherlands)

Baeten, J.C.M.; Middelburg, C.A.

1999-01-01

We present real time and discrete time versions of ACP with absolute timing and relative timing. The startingpoint is a new real time version with absolute timing, called ACPsat , featuring urgent actions and a delay operator. The discrete time versions are conservative extensions of the discrete

Evaluation of Amplification Targets for the Specific Detection of Bordetella pertussis Using Real-Time Polymerase Chain Reaction

Directory of Open Access Journals (Sweden)

Mohammad Rubayet Hasan

2014-01-01

Full Text Available BACKGROUND: Bordetella pertussis infections continue to be a major public health challenge in Canada. Polymerase chain reaction (PCR assays to detect B pertussis are typically based on the multicopy insertion sequence IS481, which offers high sensitivity but lacks species specificity.

Identification and validation of biomarkers of IgV(H) mutation status in chronic lymphocytic leukemia using microfluidics quantitative real-time polymerase chain reaction technology.

Science.gov (United States)

Abruzzo, Lynne V; Barron, Lynn L; Anderson, Keith; Newman, Rachel J; Wierda, William G; O'brien, Susan; Ferrajoli, Alessandra; Luthra, Madan; Talwalkar, Sameer; Luthra, Rajyalakshmi; Jones, Dan; Keating, Michael J; Coombes, Kevin R

2007-09-01

To develop a model incorporating relevant prognostic biomarkers for untreated chronic lymphocytic leukemia patients, we re-analyzed the raw data from four published gene expression profiling studies. We selected 88 candidate biomarkers linked to immunoglobulin heavy-chain variable region gene (IgV(H)) mutation status and produced a reliable and reproducible microfluidics quantitative real-time polymerase chain reaction array. We applied this array to a training set of 29 purified samples from previously untreated patients. In an unsupervised analysis, the samples clustered into two groups. Using a cutoff point of 2% homology to the germline IgV(H) sequence, one group contained all 14 IgV(H)-unmutated samples; the other contained all 15 mutated samples. We confirmed the differential expression of 37 of the candidate biomarkers using two-sample t-tests. Next, we constructed 16 different models to predict IgV(H) mutation status and evaluated their performance on an independent test set of 20 new samples. Nine models correctly classified 11 of 11 IgV(H)-mutated cases and eight of nine IgV(H)-unmutated cases, with some models using three to seven genes. Thus, we can classify cases with 95% accuracy based on the expression of as few as three genes.

Real-time RPA assay for rapid detection and differentiation of wild-type pseudorabies and gE-deleted vaccine viruses.

Science.gov (United States)

Wang, Jianchang; Liu, Libing; Wang, Jinfeng; Pang, Xiaoyu; Yuan, Wanzhe

2018-02-15

The objective of this study was to develop a dual real-time recombinase polymerase amplification (RPA) assay using exo probes for the detection and differentiation of pseudorabies virus (PRV). Specific RPA primers and probes were designed for gB and gE genes of PRV within the conserved region of viral genome. The reaction process can be completed in 20 min at 39 °C. The dual real-time RPA assay performed in the single tube was capable of specific detecting and differentiating of the wild-type PRV and gE-deleted vaccine strains, without cross-reactions with other non-targeted pig viruses. The analytical sensitivity of the assay was 10 2 copies for gB and gE genes. The dual real-time RPA demonstrated a 100% diagnostic agreement with the real-time PCR on 4 PRV strains and 37 clinical samples. Through the linear regression analysis, the R 2 value of the real-time RPA and the real-time PCR for gB and gE was 0.983 and 0.992, respectively. The dual real-time RPA assay provides an alternative useful tool for rapid, simple, and reliable detection and differentiation of PRV, especially in remote and rural areas. Copyright © 2017 Elsevier Inc. All rights reserved.

Real-time radiography

International Nuclear Information System (INIS)

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

1981-01-01

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

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.

Involvement of DNA polymerase δ in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

International Nuclear Information System (INIS)

Dresler, S.L.; Gowans, B.J.; Robinson-Hill, R.M.; Hunting, D.J.

1988-01-01

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase δ as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, the authors describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, α or δ. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase α several hundred times more strongly than it inhibits DNA polymerase δ. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase δ. It appears that repair synthesis at late time after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase δ

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...

Identification of four squid species by quantitative real-time polymerase chain reaction.

Science.gov (United States)

Ye, Jian; Feng, Junli; Liu, Shasha; Zhang, Yanping; Jiang, Xiaona; Dai, Zhiyuan

2016-02-01

Squids are distributed worldwide, including many species of commercial importance, and they are often made into varieties of flavor foods. The rapid identification methods for squid species especially their processed products, however, have not been well developed. In this study, quantitative real-time PCR (qPCR) systems based on specific primers and TaqMan probes have been established for rapid and accurate identification of four common squid species (Ommastrephes bartramii, Dosidicus gigas, Illex argentinus, Todarodes pacificus) in Chinese domestic market. After analyzing mitochondrial genes reported in GenBank, the mitochondrial cytochrome b (Cytb) gene was selected for O. bartramii detection, cytochrome c oxidase subunit I (COI) gene for D. gigas and T. Pacificus detection, ATPase subunit 6 (ATPase 6) gene for I. Argentinus detection, and 12S ribosomal RNA (12S rDNA) gene for designing Ommastrephidae-specific primers and probe. As a result, all the TaqMan systems are of good performance, and efficiency of each reaction was calculated by making standard curves. This method could detect target species either in single or mixed squid specimen, and it was applied to identify 12 squid processed products successfully. Thus, it would play an important role in fulfilling labeling regulations and squid fishery control. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rapid detection of health-care-associated bloodstream infection in critical care using multipathogen real-time polymerase chain reaction technology: a diagnostic accuracy study and systematic review.

Science.gov (United States)

Warhurst, Geoffrey; Dunn, Graham; Chadwick, Paul; Blackwood, Bronagh; McAuley, Daniel; Perkins, Gavin D; McMullan, Ronan; Gates, Simon; Bentley, Andrew; Young, Duncan; Carlson, Gordon L; Dark, Paul

2015-05-01

There is growing interest in the potential utility of real-time polymerase chain reaction (PCR) in diagnosing bloodstream infection by detecting pathogen deoxyribonucleic acid (DNA) in blood samples within a few hours. SeptiFast (Roche Diagnostics GmBH, Mannheim, Germany) is a multipathogen probe-based system targeting ribosomal DNA sequences of bacteria and fungi. It detects and identifies the commonest pathogens causing bloodstream infection. As background to this study, we report a systematic review of Phase III diagnostic accuracy studies of SeptiFast, which reveals uncertainty about its likely clinical utility based on widespread evidence of deficiencies in study design and reporting with a high risk of bias. Determine the accuracy of SeptiFast real-time PCR for the detection of health-care-associated bloodstream infection, against standard microbiological culture. Prospective multicentre Phase III clinical diagnostic accuracy study using the standards for the reporting of diagnostic accuracy studies criteria. Critical care departments within NHS hospitals in the north-west of England. Adult patients requiring blood culture (BC) when developing new signs of systemic inflammation. SeptiFast real-time PCR results at species/genus level compared with microbiological culture in association with independent adjudication of infection. Metrics of diagnostic accuracy were derived including sensitivity, specificity, likelihood ratios and predictive values, with their 95% confidence intervals (CIs). Latent class analysis was used to explore the diagnostic performance of culture as a reference standard. Of 1006 new patient episodes of systemic inflammation in 853 patients, 922 (92%) met the inclusion criteria and provided sufficient information for analysis. Index test assay failure occurred on 69 (7%) occasions. Adult patients had been exposed to a median of 8 days (interquartile range 4-16 days) of hospital care, had high levels of organ support activities and recent

Development of a Real-Time Microchip PCR System for Portable Plant Disease Diagnosis

Science.gov (United States)

Kim, Hyun Soo; Cifci, Osman S.; Vaughn-Diaz, Vanessa L.; Ma, Bo; Kim, Sungman; Abdel-Raziq, Haron; Ong, Kevin; Jo, Young-Ki; Gross, Dennis C.; Shim, Won-Bo; Han, Arum

2013-01-01

Rapid and accurate detection of plant pathogens in the field is crucial to prevent the proliferation of infected crops. Polymerase chain reaction (PCR) process is the most reliable and accepted method for plant pathogen diagnosis, however current conventional PCR machines are not portable and require additional post-processing steps to detect the amplified DNA (amplicon) of pathogens. Real-time PCR can directly quantify the amplicon during the DNA amplification without the need for post processing, thus more suitable for field operations, however still takes time and require large instruments that are costly and not portable. Microchip PCR systems have emerged in the past decade to miniaturize conventional PCR systems and to reduce operation time and cost. Real-time microchip PCR systems have also emerged, but unfortunately all reported portable real-time microchip PCR systems require various auxiliary instruments. Here we present a stand-alone real-time microchip PCR system composed of a PCR reaction chamber microchip with integrated thin-film heater, a compact fluorescence detector to detect amplified DNA, a microcontroller to control the entire thermocycling operation with data acquisition capability, and a battery. The entire system is 25×16×8 cm3 in size and 843 g in weight. The disposable microchip requires only 8-µl sample volume and a single PCR run consumes 110 mAh of power. A DNA extraction protocol, notably without the use of liquid nitrogen, chemicals, and other large lab equipment, was developed for field operations. The developed real-time microchip PCR system and the DNA extraction protocol were used to successfully detect six different fungal and bacterial plant pathogens with 100% success rate to a detection limit of 5 ng/8 µl sample. PMID:24349341

Development of a real-time microchip PCR system for portable plant disease diagnosis.

Directory of Open Access Journals (Sweden)

Chiwan Koo

Full Text Available Rapid and accurate detection of plant pathogens in the field is crucial to prevent the proliferation of infected crops. Polymerase chain reaction (PCR process is the most reliable and accepted method for plant pathogen diagnosis, however current conventional PCR machines are not portable and require additional post-processing steps to detect the amplified DNA (amplicon of pathogens. Real-time PCR can directly quantify the amplicon during the DNA amplification without the need for post processing, thus more suitable for field operations, however still takes time and require large instruments that are costly and not portable. Microchip PCR systems have emerged in the past decade to miniaturize conventional PCR systems and to reduce operation time and cost. Real-time microchip PCR systems have also emerged, but unfortunately all reported portable real-time microchip PCR systems require various auxiliary instruments. Here we present a stand-alone real-time microchip PCR system composed of a PCR reaction chamber microchip with integrated thin-film heater, a compact fluorescence detector to detect amplified DNA, a microcontroller to control the entire thermocycling operation with data acquisition capability, and a battery. The entire system is 25 × 16 × 8 cm(3 in size and 843 g in weight. The disposable microchip requires only 8-µl sample volume and a single PCR run consumes 110 mAh of power. A DNA extraction protocol, notably without the use of liquid nitrogen, chemicals, and other large lab equipment, was developed for field operations. The developed real-time microchip PCR system and the DNA extraction protocol were used to successfully detect six different fungal and bacterial plant pathogens with 100% success rate to a detection limit of 5 ng/8 µl sample.

An Undergraduate Laboratory Experiment for Upper-Level Forensic Science, Biochemistry, or Molecular Biology Courses: Human DNA Amplification Using STR Single Locus Primers by Real-Time PCR with SYBR Green Detection

Science.gov (United States)

Elkins, Kelly M.; Kadunc, Raelynn E.

2012-01-01

In this laboratory experiment, real-time polymerase chain reaction (real-time PCR) was conducted using published human TPOX single-locus DNA primers for validation and various student-designed short tandem repeat (STR) primers for Combined DNA Index System (CODIS) loci. SYBR Green was used to detect the amplification of the expected amplicons. The…

A TaqMan-based real-time PCR assay for porcine parvovirus 4 detection and quantification in reproductive tissues of sows

Science.gov (United States)

Porcine parvovirus 4 (PPV4) is a DNA virus, and a member of the Parvoviridae family within the Bocavirus genera. It was recently detected in swine, but its epidemiology and pathology remain unclear. A TaqMan-based real-time polymerase chain reaction (qPCR) assay targeting a conserved region of the O...

Detection of Aspergillus flavus and A. fumigatus in Bronchoalveolar Lavage Specimens of Hematopoietic Stem Cell Transplants and Hematological Malignancies Patients by Real-Time Polymerase Chain Reaction, Nested PCR and Mycological Assays

Science.gov (United States)

Zarrinfar, Hossein; Mirhendi, Hossein; Fata, Abdolmajid; Khodadadi, Hossein; Kordbacheh, Parivash

2015-01-01

Background: Pulmonary aspergillosis (PA) is one of the most serious complications in immunocompromised patients, in particular among hematopoietic stem cell transplants (HSCT) and patients with hematological malignancies. Objectives: The current study aimed to evaluate the incidence of PA and utility of molecular methods in HSCT and patients with hematological malignancies, four methods including direct examination, culture, nested polymerase chain reaction (PCR) and real-time PCR were performed on bronchoalveolar lavage (BAL) specimens in Tehran, Iran. Patients and Methods: During 16 months, 46 BAL specimens were obtained from individuals with allogeneic HSCT (n = 18) and patients with hematological malignancies (n = 28). Direct wet mounts with 20% potassium hydroxide (KOH) and culture on mycological media were performed. The molecular detection of Aspergillus fumigatus and A. flavus was done by amplifying the conserved sequences of internal transcribed spacer 1 (ITS1) ribosomal DNA by nested-PCR and the β-tubulin gene by TaqMan real-time PCR. Results: Seven (15.2%) out of 46 specimens were positive in direct examination and showed branched septate hyphae; 11 (23.9%) had positive culture including eight (72.7%) A. flavus and three (27.3%) A. fumigatus; 22 (47.8%) had positive nested-PCR and eight (17.4%) had positive real-time PCR. The incidence of invasive pulmonary aspergillosis (IPA) in these patients included proven IPA in 1 (2.2%), probable IPA in 10 (21.7%), possible IPA in 19 (41.3%) and not IPA in 16 cases (34.8%). Conclusions: The incidence of IPA in allogeneic HSCT and patients with hematological malignancies was relatively high and A. flavus was the most common cause of PA. As molecular methods had higher sensitivity, it may be useful as screening methods in HSCT and patients with hematological malignancies, or to determine when empirical antifungal therapy can be withheld. PMID:25763133

Detection and Quantification of Viable and Nonviable Trypanosoma cruzi Parasites by a Propidium Monoazide Real-Time Polymerase Chain Reaction Assay

Science.gov (United States)

Cancino-Faure, Beatriz; Fisa, Roser; Alcover, M. Magdalena; Jimenez-Marco, Teresa; Riera, Cristina

2016-01-01

Molecular techniques based on real-time polymerase chain reaction (qPCR) allow the detection and quantification of DNA but are unable to distinguish between signals from dead or live cells. Because of the lack of simple techniques to differentiate between viable and nonviable cells, the aim of this study was to optimize and evaluate a straightforward test based on propidium monoazide (PMA) dye action combined with a qPCR assay (PMA-qPCR) for the selective quantification of viable/nonviable epimastigotes of Trypanosoma cruzi. PMA has the ability to penetrate the plasma membrane of dead cells and covalently cross-link to the DNA during exposure to bright visible light, thereby inhibiting PCR amplification. Different concentrations of PMA (50–200 μM) and epimastigotes of the Maracay strain of T. cruzi (1 × 105–10 parasites/mL) were assayed; viable and nonviable parasites were tested and quantified by qPCR with a TaqMan probe specific for T. cruzi. In the PMA-qPCR assay optimized at 100 μM PMA, a significant qPCR signal reduction was observed in the nonviable versus viable epimastigotes treated with PMA, with a mean signal reduction of 2.5 logarithm units and a percentage of signal reduction > 98%, in all concentrations of parasites assayed. This signal reduction was also observed when PMA-qPCR was applied to a mixture of live/dead parasites, which allowed the detection of live cells, except when the concentration of live parasites was low (10 parasites/mL). The PMA-qPCR developed allows differentiation between viable and nonviable epimastigotes of T. cruzi and could thus be a potential method of parasite viability assessment and quantification. PMID:27139452

Optimization and Validation of a Real Time Reverse Transcriptase Polymerase Chain Reaction with RNA Internal Control to Detect Rubella RNA

Directory of Open Access Journals (Sweden)

Winny Xie

2013-12-01

Full Text Available BACKGROUND: According to a report from WHO, cases of rubella infection in Indonesia has increased up to 10-fold from 2007 to 2011. Despite no data of congenital rubella syndrome in the report, there are approximately 45,000 cases of babies born with heart failure and 0.1-0.3% live births with congenital deafness in Indonesia. Allegedly, rubella infection during pregnancy may play a role in this condition. This study aimed to optimize and validate a real-time reverse transcriptase polymerase chain reaction (RT-qPCR method to detect rubella virus RNA as an aid for the diagnosis of congenital rubella infection. METHODS: Method optimization was conducted using nucleic acids extracted from Trimovax Merieux vaccine with the High Pure Viral Nucleic Acid Kit. One step RT-qPCR was performed with Quantifast Multiplex RTPCR+R Kit. Target synthetic DNA was designed and used to determine the sensitivity of the method. RNA internal control was synthesized to control the process of extraction and amplification. RESULTS: The analytical sensitivity of this method was as low as 5 copies target synthetic DNA/μl. The mean Coefficient of Variation (CV % of the critical threshold (Ct obtained were 2.71%, 1.20%, 1.62%, and 1.59% for within run, between run, between kit lots, and between operators, respectively. Recovery of the target synthetic DNA from amniotic fluid was 100.51% (by the log copies/μl at the concentration of 1,000,000 copies/μl. CONCLUSIONS: RT-qPCR is successfully used for the detection of rubella virus RNA in vaccine and synthetic nucleic acid. With its high sensitivity, good precision and recovery, this method offers a means to improve the diagnosis of congenital rubella infection in developing countries like Indonesia. KEYWORDS: congenital rubella, RT-qPCR, prenatal diagnosis, amniotic fluid.

Quantification of viable spray-dried potential probiotic lactobacilli using real-time PCR

Directory of Open Access Journals (Sweden)

Radulović Zorica

2012-01-01

Full Text Available The basic requirement for probiotic bacteria to be able to perform expected positive effects is to be alive. Therefore, appropriate quantification methods are crucial. Bacterial quantification based on nucleic acid detection is increasingly used. Spray-drying (SD is one of the possibilities to improve the survival of probiotic bacteria against negative environmental effects. The aim of this study was to investigate the survival of spray-dried Lactobacillus plantarum 564 and Lactobacillus paracasei Z-8, and to investigate the impact on some probiotic properties caused by SD of both tested strains. Besides the plate count technique, the aim was to examine the possibility of using propidium monoazide (PMA in combination with real-time polymerase chain reaction (PCR for determining spray-dried tested strains. The number of intact cells, Lb. plantarum 564 and Lb. paracasei Z-8, was determined by real-time PCR with PMA, and it was similar to the number of investigated strains obtained by the plate count method. Spray-dried Lb. plantarum 564 and Lb. paracasei Z-8 demonstrated very good probiotic ability. It may be concluded that the PMA real-time PCR determination of the viability of probiotic bacteria could complement the plate count method and SD may be a cost-effective way to produce large quantities of some probiotic cultures. [Projekat Ministarstva nauke Republike Srbije, br. 046010

SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes.

Science.gov (United States)

Tien, Wei-Ping; Lim, Gareth; Yeo, Gladys; Chiang, Suzanna Nicole; Chong, Chee-Seng; Ng, Lee-Ching; Hapuarachchi, Hapuarachchige Chanditha

2017-09-19

The monitoring of vectors is one of the key surveillance measures to assess the risk of arbovirus transmission and the success of control strategies in endemic regions. The recent re-emergence of Zika virus (ZIKV) in the tropics, including Singapore, emphasizes the need to develop cost-effective, rapid and accurate assays to monitor the virus spread by mosquitoes. As ZIKV infections largely remain asymptomatic, early detection of ZIKV in the field-caught mosquitoes enables timely implementation of appropriate mosquito control measures. We developed a rapid, sensitive and specific real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for the detection of ZIKV in field-caught mosquitoes. The primers and PCR cycling conditions were optimized to minimize non-specific amplification due to cross-reactivity with the genomic material of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex tritaeniorhynchus, Culex sitiens and Anopheles sinensis, as well as accompanying microbiota. The performance of the assay was further evaluated with a panel of flaviviruses and alphaviruses as well as in field-caught Ae. aegypti mosquitoes confirmed to be positive for ZIKV. As compared to a probe-based assay, the newly developed assay demonstrated 100% specificity and comparable detection sensitivity for ZIKV in mosquitoes. Being a SYBR Green-based method, the newly-developed assay is cost-effective and easy to adapt, thus is applicable to large-scale vector surveillance activities in endemic countries, including those with limited resources and expertise. The amplicon size (119 bp) also allows sequencing to confirm the virus type. The primers flank relatively conserved regions of ZIKV genome, so that, the assay is able to detect genetically diverse ZIKV strains. Our findings, therefore, testify the potential use of the newly-developed assay in vector surveillance programmes for ZIKV in endemic regions.

Essays in real-time forecasting

OpenAIRE

Liebermann, Joelle

2012-01-01

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

Time-lapse crystallography snapshots of a double-strand break repair polymerase in action.

Science.gov (United States)

Jamsen, Joonas A; Beard, William A; Pedersen, Lars C; Shock, David D; Moon, Andrea F; Krahn, Juno M; Bebenek, Katarzyna; Kunkel, Thomas A; Wilson, Samuel H

2017-08-15

DNA polymerase (pol) μ is a DNA-dependent polymerase that incorporates nucleotides during gap-filling synthesis in the non-homologous end-joining pathway of double-strand break repair. Here we report time-lapse X-ray crystallography snapshots of catalytic events during gap-filling DNA synthesis by pol μ. Unique catalytic intermediates and active site conformational changes that underlie catalysis are uncovered, and a transient third (product) metal ion is observed in the product state. The product manganese coordinates phosphate oxygens of the inserted nucleotide and PP i . The product metal is not observed during DNA synthesis in the presence of magnesium. Kinetic analyses indicate that manganese increases the rate constant for deoxynucleoside 5'-triphosphate insertion compared to magnesium. The likely product stabilization role of the manganese product metal in pol μ is discussed. These observations provide insight on structural attributes of this X-family double-strand break repair polymerase that impact its biological function in genome maintenance.DNA polymerase (pol) μ functions in DNA double-strand break repair. Here the authors use time-lapse X-ray crystallography to capture the states of pol µ during the conversion from pre-catalytic to product complex and observe a third transiently bound metal ion in the product state.

Development of RODOS, a comprehensive real-time on-line decision support system for nuclear emergency management in Europe. Final report

International Nuclear Information System (INIS)

Ehrhardt, J.; Weis, A.

1996-09-01

The development of RODOS, a comprehensive, Real-time, On-line DecisiOn Support system for nuclear emergency management, that would be capable of finding broad application across Europe was included as a major item in the Radiation Protection Research Action of the European Commission's 3rd Framework Programme; it remains an important priority in the 4th Framework programme (1995-1998). When complete, the RODOS system is intended to be applicable from the vicinity of the release and the early phases of an accident to far distant areas and longer time periods. In this way it will be possible to achieve estimates, analyses, and prognoses of accident consequences with and without considering protective actions and countermeasures, which are consistent throughout all accident phases and distance ranges. It will also be possible to evaluate alternative combinations of measures in term of both, feasibility in the given situation, and public acceptability, socio-psychological and political implications. This Final Report summarises the results achieved by the partners of the EC contract FI3P-CT92-0036 within the time period 1992 to 1995, and more generally, gives an overview of the development status of the RODOS system and its functionalities realised by the end of the contract period. (orig.) [de

Development and evaluation of a real-time fluorescent polymerase chain reaction assay for the detection of bovine contaminates in cattle feed.

Science.gov (United States)

Rensen, Gabriel; Smith, Wayne; Ruzante, Juliana; Sawyer, Mary; Osburn, Bennie; Cullor, James

2005-01-01

A real-time fluorescent polymerase chain reaction assay for detecting prohibited ruminant materials such as bovine meat and bone meal (BMBM) in cattle feed using primers and FRET probes targeting the ruminant specific mitochondrial cytochrome b gene was developed and evaluated on two different types of cattle feed. Common problems involved with PCR based testing of cattle feed include the presence of high levels of PCR inhibitors and the need for certain pre-sample processing techniques in order to perform DNA extractions. We have developed a pre-sample processing technique for extracting DNA from cattle feed which does not require the feed sample to be ground to a fine powder and utilizes materials that are disposed of between samples, thus, reducing the potential of cross contamination. The DNA extraction method utilizes Whatman FTA card technology, is adaptable to high sample throughput analysis and allows for room temperature storage with established archiving of samples of up to 14 years. The Whatman FTA cards are subsequently treated with RNAse and undergo a Chelex-100 extraction (BioRad, Hercules, CA), thus removing potential PCR inhibitors and eluting the DNA from the FTA card for downstream PCR analysis. The detection limit was evaluated over a period of 30 trials on calf starter mix and heifer starter ration feed samples spiked with known concentrations of BMBM. The PCR detection assay detected 0.05% wt/wt BMBM contamination with 100% sensitivity, 100% specificity, and 100% confidence. Concentrations of 0.005% and 0.001% wt/wt BMBM contamination were also detected in both feed types but with varying levels of confidence.

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...

Embryonation of Ostertagia ostertagi eggs affects the outcome of real-time quantitative PCR

DEFF Research Database (Denmark)

Drag, Markus; Höglund, Johan; Nejsum, Peter

prior to detection and quantification by real-time quantitative polymerase chain reaction (qPCR). Fresh O. ostertagi eggs were isolated from cattle faeces and stored at 4°C or 25°C under aerobic or anaerobic conditions. Embryonation was monitored by microscopy and the ITS2 copies were determined by q...... the outcome of qPCR analysis for the quantitative determination of O. ostertagi eggs in cattle faeces. Cold storage at 4°C for up to 3 days or anaerobicvacuum packing at 25°C for up to 336 h will entail no undesirable effects on ITS2 copies....

Embryonation of Ostertagia ostertagi eggs affects the outcome of real-time quantitative PCR

DEFF Research Database (Denmark)

Drag, Markus; Höglund, Johan; Nejsum, Peter

prior to detection and quantification by real-time quantitative polymerase chain reaction (qPCR) . Fresh O. ostertagi eggs were isolated from cattle faeces and stored at 4°C or 25°C under aerobic or anaerobic conditions. Embryonation was monitored by microscopy and the ITS2 copies were determined by q...... the outcome of qPCR analysis for the quantitative determination of O. ostertagi eggs in cattle faeces. Cold storage at 4°C for up to 3 days or anaerobic vacuum packing at 25°C for up to 336 h will entail no undesirable effects on ITS2 copies....

The value of molecular expression of KIT and KIT ligand analysed using real-time polymerase chain reaction and immunohistochemistry as a prognostic indicator for canine cutaneous mast cell tumours.

Science.gov (United States)

Costa Casagrande, T A; de Oliveira Barros, L M; Fukumasu, H; Cogliati, B; Chaible, L M; Dagli, M L Z; Matera, J M

2015-03-01

This study investigated the correlation between KIT gene expression determined by immunohistochemistry and real-time polymerase chain reaction (RT-PCR) and the rate of tumour recurrence and tumour-related deaths in dogs affected with mast cell tumour (MCT). Kaplan-Meier curves were constructed to compare tumour recurrence and tumour-related death between patients. The log-rank test was used to check for significant differences between curves. KIT-I, KIT-II and KIT-III staining patterns were observed in 9 (11.11%), 50 (61.73%) and 22 (27.16%) tumours, respectively. Tumour recurrence rates and tumour-related deaths were not associated with KIT staining patterns (P = 0278, P > 0.05), KIT (P = 0.289, P > 0.05) or KIT ligand (P = 0.106, P > 0.05) gene expression. Despite the lack of association between KIT staining pattern and patient survival time, the results suggest a correlation between aberrant KIT localization and increased proliferative activity of MCTs. RT-PCR seems to be a sensible method for quantitative detection of KIT gene expression in canine MCT, although expressions levels are not correlated with prognosis. © 2013 Blackwell Publishing Ltd.

Sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction detecting feline coronavirus mutations in effusion and serum/plasma of cats to diagnose feline infectious peritonitis.

Science.gov (United States)

Felten, Sandra; Leutenegger, Christian M; Balzer, Hans-Joerg; Pantchev, Nikola; Matiasek, Kaspar; Wess, Gerhard; Egberink, Herman; Hartmann, Katrin

2017-08-02

Feline coronavirus (FCoV) exists as two pathotypes, and FCoV spike gene mutations are considered responsible for the pathotypic switch in feline infectious peritonitis (FIP) pathogenesis. The aim of this study was to evaluate sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) specifically designed to detect FCoV spike gene mutations at two nucleotide positions. It was hypothesized that this test would correctly discriminate feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). The study included 63 cats with signs consistent with FIP. FIP was confirmed in 38 cats. Twenty-five control cats were definitively diagnosed with a disease other than FIP. Effusion and/or serum/plasma samples were examined by real-time RT-PCR targeting the two FCoV spike gene fusion peptide mutations M1058 L and S1060A using an allelic discrimination approach. Sensitivity, specificity, negative and positive predictive values including 95% confidence intervals (95% CI) were calculated. FIPV was detected in the effusion of 25/59 cats, one of them being a control cat with chronic kidney disease. A mixed population of FIPV/FECV was detected in the effusion of 2/59 cats; all of them had FIP. RT-PCR was negative or the pathotype could not be determined in 34/59 effusion samples. In effusion, sensitivity was 68.6% (95% CI 50.7-83.2), specificity was 95.8% (95% CI 78.9-99.9). No serum/plasma samples were positive for FIPV. Although specificity of the test in effusions was high, one false positive result occurred. The use of serum/plasma cannot be recommended due to a low viral load in blood.

Real-time PCR assays for detection and quactification of Edwardsiella tarda, Edwardsiella piscicida, Edwardsiella piscicida-like sp. in catfish tissues and pond water

Science.gov (United States)

Researchers have proposed the adoption of 3 distinct genetic taxa among bacteria previously classified as Edwardsiella tarda; namely E. tarda, E. piscicida, and a taxon presently termed E. piscicida–like. Individual real-time polymerase chain reaction (qPCR) assays were developed, based on published...

Use of Nested and Real-Time PCR for the Detection of Ceratocystis fagacearum in the Sapwood of Diseased Oak Species in Minnesota

Science.gov (United States)

A. Yang; J. Juzwik

2017-01-01

Oak wilt caused by Ceratocystis fagacearum is a significant disease of Quercus spp. in the eastern United States. Early and accurate detection of the pathogen is particularly important when disease control is planned. Nested and real-time polymerase chain reaction (PCR) methods utilizing fungal DNA extracted from sapwood drill...

Cost-effective optimization of real-time PCR based detection of Campylobacter and Salmonella with inhibitor tolerant DNA polymerases

DEFF Research Database (Denmark)

Fachmann, Mette Sofie Rousing; Josefsen, Mathilde Hasseldam; Hoorfar, Jeffrey

2015-01-01

bacterial cells in two validated real-time PCR assays for Campylobacter and Salmonella. The five best performing (based on: limit of detection (LOD), maximum fluorescence, shape of amplification curves, and amplification efficiency) were subsequently applied to meat and fecal samples. The VeriQuest q......PCR master mix performed best for both meat and fecal samples (LODs of 102 and 104 CFU ml-1 in the purest and crudest DNA extractions, respectively) compared with Tth (LOD=102 -103 and 105 -106 CFU ml-1 ). AmpliTaqGold and HotMasterTaq both performed well (LOD=102 -104 CFU ml-1 ) with meat samples and poorly...... (LOD=103 -106 CFU ml-1 /not detected) with fecal samples. CONCLUSIONS: Applying the VeriQuest qPCR master mix in the two tested real-time PCR assays could allow for simpler sample preparation and thus a reduction in cost. SIGNIFICANCE AND IMPACT OF STUDY: This work exemplifies a cost-effective strategy...

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

ISTTOK real-time architecture

International Nuclear Information System (INIS)

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

2014-01-01

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

Detection of Balamuthia mandrillaris DNA by real-time PCR targeting the RNase P gene

Directory of Open Access Journals (Sweden)

Lewin Astrid

2008-12-01

Full Text Available Abstract Background The free-living amoeba Balamuthia mandrillaris may cause fatal encephalitis both in immunocompromised and in – apparently – immunocompetent humans and other mammalian species. Rapid, specific, sensitive, and reliable detection requiring little pathogen-specific expertise is an absolute prerequisite for a successful therapy and a welcome tool for both experimental and epidemiological research. Results A real-time polymerase chain reaction assay using TaqMan® probes (real-time PCR was established specifically targeting the RNase P gene of B. mandrillaris amoebae. The assay detected at least 2 (down to 0.5 genomes of B. mandrillaris grown in axenic culture. It did not react with DNA from closely related Acanthamoeba (3 species, nor with DNA from Toxoplasma gondii, Leishmania major, Pneumocystis murina, Mycobacterium bovis (BCG, human brain, various mouse organs, or from human and murine cell lines. The assay efficiently detected B. mandrillaris DNA in spiked cell cultures, spiked murine organ homogenates, B. mandrillaris-infected mice, and CNS tissue-DNA preparations from 2 patients with proven cerebral balamuthiasis. This novel primer set was successfully combined with a published set that targets the B. mandrillaris 18S rRNA gene in a duplex real-time PCR assay to ensure maximum specificity and as a precaution against false negative results. Conclusion A real-time PCR assay for B. mandrillaris amoebae is presented, that is highly specific, sensitive, and reliable and thus suited both for diagnosis and for research.

A real-time hybrid neuron network for highly parallel cognitive systems.

Science.gov (United States)

Christiaanse, Gerrit Jan; Zjajo, Amir; Galuzzi, Carlo; van Leuken, Rene

2016-08-01

For comprehensive understanding of how neurons communicate with each other, new tools need to be developed that can accurately mimic the behaviour of such neurons and neuron networks under `real-time' constraints. In this paper, we propose an easily customisable, highly pipelined, neuron network design, which executes optimally scheduled floating-point operations for maximal amount of biophysically plausible neurons per FPGA family type. To reduce the required amount of resources without adverse effect on the calculation latency, a single exponent instance is used for multiple neuron calculation operations. Experimental results indicate that the proposed network design allows the simulation of up to 1188 neurons on Virtex7 (XC7VX550T) device in brain real-time yielding a speed-up of x12.4 compared to the state-of-the art.

Detection of Mycobacterium avium subsp. paratuberculosis in bovine manure using Whatman FTA card technology and Lightcycler real-time PCR.

Science.gov (United States)

Jaravata, Carmela V; Smith, Wayne L; Rensen, Gabriel J; Ruzante, Juliana M; Cullor, James S

2006-01-01

A modified forensic DNA extraction and real-time fluorescent polymerase chain reaction assay has been evaluated for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine fecal samples using primers and fluorescent resonance energy transfer (FRET) probes targeting the IS900 gene sequence of MAP. DNA was successfully extracted from manure samples by utilizing the Whatman FTA card technology, which allows for simple processing and storage of samples at room temperature. The FTA cards were washed and subjected to a Chelex-100 incubation to remove any remaining polymerase chain reaction (PCR) inhibitors and to elute the DNA from the FTA card. This isolated DNA was then subjected to direct real time fluorescent PCR analysis. Detection of MAP DNA from bovine fecal samples spiked with known concentrations of viable MAP cells was obtained. The detection limits of the assay was consistently found to be between 10(2) and 10(4) colony forming units [CFU]/g, with some samples containing as low as 10 CFU/g, yielding positive assay results. This cost-efficient assay allows reporting of results as early as 4 h after fecal collection, which can be particularly useful in highthroughput herd screening.

The use of newly developed real-time PCR for the rapid identification of bacteria in culture-negative osteomyelitis.

Science.gov (United States)

Kobayashi, Naomi; Bauer, Thomas W; Sakai, Hiroshige; Togawa, Daisuke; Lieberman, Isador H; Fujishiro, Takaaki; Procop, Gary W

2006-12-01

We report a case of a culture-negative osteomyelitis in which our newly developed real-time polymerase chain reaction (PCR) could differentiate Staphylococcus aureus from Staphylococcus epidermidis. This is the first report that described the application of this novel assay to an orthopedics clinical sample. This assay may be useful for other clinical culture-negative cases in a combination with a broad-spectrum assay as a rapid microorganism identification method.

Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructanstructans.

Science.gov (United States)

Muller, Laura K.; Lorch, Jeffrey M.; Lindner, Daniel L.; O'Connor, Michael; Gargas, Andrea; Blehert, David S.

2013-01-01

The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The test is highly sensitive, consistently detecting as little as 3.3 fg of genomic DNA from G. destructans. The real-time PCR test specifically amplified genomic DNA from G. destructans but did not amplify target sequence from 54 closely related fungal isolates (including 43 Geomyces spp. isolates) associated with bats. The test was further qualified by analyzing DNA extracted from 91 bat wing skin samples, and PCR results matched histopathology findings. These data indicate the real-time TaqMan PCR method described herein is a sensitive, specific, and rapid test to detect DNA from G. destructans and provides a valuable tool for WNS diagnostics and research.

Selection of Suitable Reference Genes for Quantitative Real-time PCR in Sapium sebiferum

Directory of Open Access Journals (Sweden)

Xue Chen

2017-05-01

Full Text Available Chinese tallow (Sapium sebiferum L. is a promising landscape and bioenergy plant. Measuring gene expression by quantitative real-time polymerase chain reaction (qRT-PCR can provide valuable information on gene function. Stably expressed reference genes for normalization are a prerequisite for ensuring the accuracy of the target gene expression level among different samples. However, the reference genes in Chinese tallow have not been systematically validated. In this study, 12 candidate reference genes (18S, GAPDH, UBQ, RPS15, SAND, TIP41, 60S, ACT7, PDF2, APT, TBP, and TUB were investigated with qRT-PCR in 18 samples, including those from different tissues, from plants treated with sucrose and cold stresses. The data were calculated with four common algorithms, geNorm, BestKeeper, NormFinder, and the delta cycle threshold (ΔCt. TIP41 and GAPDH were the most stable for the tissue-specific experiment, GAPDH and 60S for cold treatment, and GAPDH and UBQ for sucrose stresses, while the least stable genes were 60S, TIP41, and 18S respectively. The comprehensive results showed APT, GAPDH, and UBQ to be the top-ranked stable genes across all the samples. The stability of 60S was the lowest during all experiments. These selected reference genes were further validated by comparing the expression profiles of the chalcone synthase gene in Chinese tallow in different samples. The results will help to improve the accuracy of gene expression studies in Chinese tallow.

Evaluation of three 5' exonuclease-based real-time polymerase chain reaction assays for detection of pathogenic Leptospira species in canine urine.

Science.gov (United States)

Fink, Jamie M; Moore, George E; Landau, Ruth; Vemulapalli, Ramesh

2015-03-01

Leptospirosis is caused by several pathogenic Leptospira species, and is an important infectious disease of dogs. Early detection of infection is crucial for an effective antibiotic treatment of the disease. Though different polymerase chain reaction (PCR) assays have been developed for detection of pathogenic Leptospira spp., thorough evaluation of the performance of these assays using dog urine samples has not been carried out. In the current study, the performance of 3 real-time PCR (qPCR) assays was assessed, 1 targeting the 16S ribosomal RNA (rRNA) gene and the other 2 targeting the lipL32 gene, a gene for the LipL32 outer membrane protein. With DNA extracted from laboratory-cultured pathogenic Leptospira spp., all 3 qPCR assays showed 100% specificity and had identical lower limits of detection. Compared to a conventional, gel-based PCR assay, all 3 qPCR assays were 100-fold more sensitive. There was a 100% agreement in the results of the 3 assays when tested on urine samples collected aseptically from 30 dogs suspected for leptospirosis. However, when tested on 30 urine samples that were collected by the free-catch method, the 16S rRNA-based assay falsely detected 13.3% of the samples as positive for pathogenic Leptospira spp. Nucleotide sequence analysis of the amplified DNA fragments showed that the assay resulted in false positives because of unrelated bacteria. All urine samples collected from 100 apparently healthy dogs at a local animal shelter tested negative for pathogenic Leptospira spp. These results highlight the importance of sample-specific validation of PCR-based diagnostic assays and the application of appropriately validated assays for more reliable pathogen detection. © 2015 The Author(s).

A fast and easy real-time PCR genotyping method for the HLA-G 14-bp insertion/deletion polymorphism in the 3' untranslated region

DEFF Research Database (Denmark)

Djurisic, S; Sørensen, A E; Hviid, T V F

2012-01-01

and reliable method to screen for the HLA-G 14-bp insertion/deletion polymorphism using an optimized real-time polymerase chain reaction protocol. The genotyping assay has been validated by comparison with conventional methods. As results can be obtained within a few hours, the assay will have a potential...

Rapid detection of Van genes in rectal swabs by real time PCR in Southern Brazil

Directory of Open Access Journals (Sweden)

Vlademir Cantarelli

2011-10-01

Full Text Available INTRODUCTION: Laboratory-based surveillance is an important component in the control of vancomycin resistant enterococci (VRE. METHODS: The study aimed to evaluate real-time polymerase chain reaction (RT-PCR (genes vanA-vanB for VRE detection on 115 swabs from patients included in a surveillance program. RESULTS: Sensitivity of RT-PCR was similar to primary culture (75% and 79.5%, respectively when compared to broth enriched culture, whereas specificity was 83.1%. CONCLUSIONS: RT-PCR provides same day results, however it showed low sensitivity for VRE detection.

A Dynamic Speech Comprehension Test for Assessing Real-World Listening Ability.

Science.gov (United States)

Best, Virginia; Keidser, Gitte; Freeston, Katrina; Buchholz, Jörg M

2016-07-01

Many listeners with hearing loss report particular difficulties with multitalker communication situations, but these difficulties are not well predicted using current clinical and laboratory assessment tools. The overall aim of this work is to create new speech tests that capture key aspects of multitalker communication situations and ultimately provide better predictions of real-world communication abilities and the effect of hearing aids. A test of ongoing speech comprehension introduced previously was extended to include naturalistic conversations between multiple talkers as targets, and a reverberant background environment containing competing conversations. In this article, we describe the development of this test and present a validation study. Thirty listeners with normal hearing participated in this study. Speech comprehension was measured for one-, two-, and three-talker passages at three different signal-to-noise ratios (SNRs), and working memory ability was measured using the reading span test. Analyses were conducted to examine passage equivalence, learning effects, and test-retest reliability, and to characterize the effects of number of talkers and SNR. Although we observed differences in difficulty across passages, it was possible to group the passages into four equivalent sets. Using this grouping, we achieved good test-retest reliability and observed no significant learning effects. Comprehension performance was sensitive to the SNR but did not decrease as the number of talkers increased. Individual performance showed associations with age and reading span score. This new dynamic speech comprehension test appears to be valid and suitable for experimental purposes. Further work will explore its utility as a tool for predicting real-world communication ability and hearing aid benefit. American Academy of Audiology.

A real-time architecture for time-aware agents.

Science.gov (United States)

Prouskas, Konstantinos-Vassileios; Pitt, Jeremy V

2004-06-01

This paper describes the specification and implementation of a new three-layer time-aware agent architecture. This architecture is designed for applications and environments where societies of humans and agents play equally active roles, but interact and operate in completely different time frames. The architecture consists of three layers: the April real-time run-time (ART) layer, the time aware layer (TAL), and the application agents layer (AAL). The ART layer forms the underlying real-time agent platform. An original online, real-time, dynamic priority-based scheduling algorithm is described for scheduling the computation time of agent processes, and it is shown that the algorithm's O(n) complexity and scalable performance are sufficient for application in real-time domains. The TAL layer forms an abstraction layer through which human and agent interactions are temporally unified, that is, handled in a common way irrespective of their temporal representation and scale. A novel O(n2) interaction scheduling algorithm is described for predicting and guaranteeing interactions' initiation and completion times. The time-aware predicting component of a workflow management system is also presented as an instance of the AAL layer. The described time-aware architecture addresses two key challenges in enabling agents to be effectively configured and applied in environments where humans and agents play equally active roles. It provides flexibility and adaptability in its real-time mechanisms while placing them under direct agent control, and it temporally unifies human and agent interactions.

Evaluation of six nucleic acid amplification tests used for diagnosis of Neisseria gonorrhoeae in Russia compared with an international strictly validated real-time porA pseudogene polymerase chain reaction.

Science.gov (United States)

Shipitsyna, E; Zolotoverkhaya, E; Hjelmevoll, S O; Maximova, A; Savicheva, A; Sokolovsky, E; Skogen, V; Domeika, M; Unemo, M

2009-11-01

In Russia, laboratory diagnosis of gonorrhoea has been mainly based on microscopy only and, in some settings, relatively rare suboptimal culturing. In recent years, Russian developed and manufactured nucleic acid amplification tests (NAAT) have been implemented for routine diagnosis of Neisseria gonorrhoeae. However, these NAATs have never been validated to any international well-recognized diagnostic NAAT. This study aims to evaluate the performance characteristics of six Russian NAATs for N. gonorrhoeae diagnostics. In total, 496 symptomatic patients were included. Five polymerase chain reaction (PCR) assays and one real-time nucleic acid sequence based amplification (NASBA) assay, developed by three Russian companies, were evaluated on urogenital samples, i.e. cervical and first voided urine (FVU) samples from females (n = 319), urethral and FVU samples from males (n = 127), and extragenital samples, i.e. rectal and pharyngeal samples, from 50 additional female patients with suspicion of gonorrhoea. As reference method, an international strictly validated real-time porA pseudogene PCR was applied. The prevalence of N. gonorrhoeae was 2.7% and 16% among the patients providing urogenital and extragenital samples, respectively. The Russian NAATs and the reference method displayed high level of concordance (99.4-100%). The sensitivities, specificities, positive predictive values and negative predictive values of the Russian tests in different specimens were 66.7-100%, 100%, 100%, and 99.4-100%, respectively. Russian N. gonorrhoeae diagnostic NAATs comprise relatively good performance characteristics. However, larger studies are crucial and, beneficially, the Russian assays should also be evaluated to other international highly sensitive and specific, and ideally Food and Drug Administration approved, NAATs such as Aptima Combo 2 (Gen-Probe).

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.

The use of real-time polymerase chain reaction with high resolution melting (real-time PCR-HRM) analysis for the detection and discrimination of nematodes Bursaphelenchus xylophilus and Bursaphelenchus mucronatus.

Science.gov (United States)

Filipiak, Anna; Hasiów-Jaroszewska, Beata

2016-04-01

The real-time PCR-HRM analysis was developed for the detection and discrimination of the quarantine nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus. A set of primers was designed to target the ITS region of rDNA. The results have demonstrated that this analysis is a valuable tool for differentiation of these both species. Copyright © 2016 Elsevier Ltd. All rights reserved.

Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers' diarrhea samples.

Science.gov (United States)

Youmans, Bonnie P; Ajami, Nadim J; Jiang, Zhi-Dong; Petrosino, Joseph F; DuPont, Herbert L; Highlander, Sarah K

2014-01-01

Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

DNA Polymerases Drive DNA Sequencing-by-Synthesis Technologies: Both Past and Present

Directory of Open Access Journals (Sweden)

Cheng-Yao eChen

2014-06-01

Full Text Available Next-generation sequencing (NGS technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. E. coli DNA polymerase I proteolytic (Klenow fragment was originally utilized in Sanger's dideoxy chain terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today's standard capillary electrophoresis (CE and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ⱷ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ⱷ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies.

Evaluation of a first mine real time diesel particulate matter (DPM) monitor

Energy Technology Data Exchange (ETDEWEB)

Stewart Gillies; Hsin Wei Wu [Gillies Wu Mining Technology (Australia)

2008-04-15

The objective of the study was to develop, test and prove up under mine conditions a Diesel Particulate Matter (DPM) real time atmospheric monitoring unit. The design for the new instrument, termed the D-PDM, is based on the recently developed real time respirable dust PDM. The project's main activities were to undertake through internationally recognised laboratory testing an evaluation of the new design and to undertake a comprehensive underground series of tests to establish the robustness and reliability of the new approach. The phases of design, the international laboratory testing and the underground mine evaluation in five operating mines proved that the monitor is capable in normal mine atmospheres of accurately measuring DPM levels in real time. The monitor has successfully reported data when used as a static or stationary instrument, when placed within the cab of a moving vehicle and when worn on a person's belt. The outcomes of the project provide the industry access to an enhanced tool for understanding the presence of DPM in the mine atmosphere.

Diagnostic performance of fecal quantitative real-time polymerase chain reaction for detection of Lawsonia intracellularis–associated proliferative enteropathy in nursery pigs

DEFF Research Database (Denmark)

Pedersen, Ken Steen; Stege, Helle; Jensen, Tim Kåre

2013-01-01

Quantitative polymerase chain reaction (qPCR) tests for detection and quantification of Lawsonia intracellularis in feces from pigs have been developed. The objective of the current study was to evaluate the diagnostic performance of a fecal qPCR test for detection of nursery pigs with L. intrace......Quantitative polymerase chain reaction (qPCR) tests for detection and quantification of Lawsonia intracellularis in feces from pigs have been developed. The objective of the current study was to evaluate the diagnostic performance of a fecal qPCR test for detection of nursery pigs with L...

Progress in using real-time GPS for seismic monitoring of the Cascadia megathrust

Science.gov (United States)

Szeliga, W. M.; Melbourne, T. I.; Santillan, V. M.; Scrivner, C.; Webb, F.

2014-12-01

We report on progress in our development of a comprehensive real-time GPS-based seismic monitoring system for the Cascadia subduction zone. This system is based on 1 Hz point position estimates computed in the ITRF08 reference frame. Convergence from phase and range observables to point position estimates is accelerated using a Kalman filter based, on-line stream editor. Positions are estimated using a short-arc approach and algorithms from JPL's GIPSY-OASIS software with satellite clock and orbit products from the International GNSS Service (IGS). The resulting positions show typical RMS scatter of 2.5 cm in the horizontal and 5 cm in the vertical with latencies below 2 seconds. To facilitate the use of these point position streams for applications such as seismic monitoring, we broadcast real-time positions and covariances using custom-built streaming software. This software is capable of buffering 24-hour streams for hundreds of stations and providing them through a REST-ful web interface. To demonstrate the power of this approach, we have developed a Java-based front-end that provides a real-time visual display of time-series, vector displacement, and contoured peak ground displacement. We have also implemented continuous estimation of finite fault slip along the Cascadia megathrust using an NIF approach. The resulting continuous slip distributions are combined with pre-computed tsunami Green's functions to generate real-time tsunami run-up estimates for the entire Cascadia coastal margin. This Java-based front-end is available for download through the PANGA website. We currently analyze 80 PBO and PANGA stations along the Cascadia margin and are gearing up to process all 400+ real-time stations operating in the Pacific Northwest, many of which are currently telemetered in real-time to CWU. These will serve as milestones towards our over-arching goal of extending our processing to include all of the available real-time streams from the Pacific rim. In addition

Real time programming environment for Windows

Energy Technology Data Exchange (ETDEWEB)

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

1998-04-01

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

A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus

OpenAIRE

Kang, Xiao-ping; Jiang, Tao; Li, Yong-qiang; Lin, Fang; Liu, Hong; Chang, Guo-hui; Zhu, Qing-yu; Qin, E-de; Qin, Cheng-feng; Yang, Yin-hui

2010-01-01

Abstract A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009) influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose) for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same a...

Advanced Visualization System for Monitoring the ATLAS TDAQ Network in real-time

CERN Document Server

Batraneanu, S M; The ATLAS collaboration; Martin, B; Savu, D O; Stancu, S N; Leahu, L

2012-01-01

The trigger and data acquisition (TDAQ) system of the ATLAS experiment at CERN comprises approximately 2500 servers interconnected by three separate Ethernet networks, totaling 250 switches. Due to its real-time nature, there are additional requirements in comparison to conventional networks in terms of speed and performance. A comprehensive monitoring framework has been developed for expert use. However, non experts may experience difficulties in using it and interpreting data. Moreover, specific performance issues, such as single component saturation or unbalanced workload, need to be spotted with ease, in real-time, and understood in the context of the full system view. We addressed these issues by developing an innovative visualization system where the users benefit from the advantages of 3D graphics to visualize the large monitoring parameter space associated with our system. This has been done by developing a hierarchical model of the complete system onto which we overlaid geographical, logical and real...

An In-Home Digital Network Architecture for Real-Time and Non-Real-Time Communication

NARCIS (Netherlands)

Scholten, Johan; Jansen, P.G.; Hanssen, F.T.Y.; Hattink, Tjalling

2002-01-01

This paper describes an in-home digital network architecture that supports both real-time and non-real-time communication. The architecture deploys a distributed token mechanism to schedule communication streams and to offer guaranteed quality-ofservice. Essentially, the token mechanism prevents

On the Physiology of Normal Swallowing as Revealed by Magnetic Resonance Imaging in Real Time

Directory of Open Access Journals (Sweden)

Arno Olthoff

2014-01-01

Full Text Available The aim of this study was to assess the physiology of normal swallowing using recent advances in real-time magnetic resonance imaging (MRI. Therefore ten young healthy subjects underwent real-time MRI and flexible endoscopic evaluations of swallowing (FEES with thickened pineapple juice as oral contrast bolus. MRI movies were recorded in sagittal, coronal, and axial orientations during successive swallows at about 25 frames per second. Intermeasurement variation was analyzed and comparisons between real-time MRI and FEES were performed. Twelve distinct swallowing events could be quantified by real-time MRI (start time, end time, and duration. These included five valve functions: oro-velar opening, velo-pharyngeal closure, glottal closure, epiglottic retroflexion, and esophageal opening; three bolus transports: oro-velar transit, pharyngeal delay, pharyngeal transit; and four additional events: laryngeal ascent, laryngeal descent, vallecular, and piriform sinus filling and pharyngeal constriction. Repetitive measurements confirmed the general reliability of the MRI method with only two significant differences for the start times of the velo-pharyngeal closure (t(8=-2.4, P≤0.046 and laryngeal ascent (t(8=-2.6, P≤0.031. The duration of the velo-pharyngeal closure was significantly longer in real-time MRI compared to FEES (t(8=-3.3, P≤0.011. Real-time MRI emerges as a simple, robust, and reliable tool for obtaining comprehensive functional and anatomical information about the swallowing process.

MARTe: A Multiplatform Real-Time Framework

Science.gov (United States)

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

2010-04-01

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

Real-Time and Real-Fast Performance of General-Purpose and Real-Time Operating Systems in Multithreaded Physical Simulation of Complex Mechanical Systems

Directory of Open Access Journals (Sweden)

Carlos Garre

2014-01-01

Full Text Available Physical simulation is a valuable tool in many fields of engineering for the tasks of design, prototyping, and testing. General-purpose operating systems (GPOS are designed for real-fast tasks, such as offline simulation of complex physical models that should finish as soon as possible. Interfacing hardware at a given rate (as in a hardware-in-the-loop test requires instead maximizing time determinism, for which real-time operating systems (RTOS are designed. In this paper, real-fast and real-time performance of RTOS and GPOS are compared when simulating models of high complexity with large time steps. This type of applications is usually present in the automotive industry and requires a good trade-off between real-fast and real-time performance. The performance of an RTOS and a GPOS is compared by running a tire model scalable on the number of degrees-of-freedom and parallel threads. The benchmark shows that the GPOS present better performance in real-fast runs but worse in real-time due to nonexplicit task switches and to the latency associated with interprocess communication (IPC and task switch.

Selective detection of viable seed-borne Acidovorax citrulli by real-time PCR with propidium monoazide.

Science.gov (United States)

Tian, Qian; Feng, Jian-Jun; Hu, Jie; Zhao, Wen-Jun

2016-10-14

In recent years, use of the DNA-intercalating dye propidium monoazide (PMA) in real-time PCR has been reported as a novel method to detect viable bacteria in different types of samples, such as food, environmental, and microbiological samples. In this study, viable cells of Acidovorax citrulli, the causal agent of bacterial seedling blight and fruit blotch, were selectively detected and differentiated from dead cells by real-time fluorescent polymerase chain reaction amplification after the bacterial solution was treated with the DNA-binding dye PMA. The primers and TaqMan probe were based on the A. citrulli genome (Aave_1909, Gene ID: 4669443) and were highly specific for A. citrulli. The detection threshold of this assay was 10 3 colony-forming units per mL (CFU/mL) in pure cell suspensions containing viable and dead cells and infected watermelon seeds. Application of this assay enables the selective detection of viable cells of A. citrulli and facilitates monitoring of the pathogen in watermelon and melon seeds.

Scalable Real-Time Negotiation Toolkit

National Research Council Canada - National Science Library

Lesser, Victor

2004-01-01

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

Validation of Reference Genes for Quantitative Expression Analysis by Real-Time RT-PCR in Four Lepidopteran Insects

OpenAIRE

Teng, Xiaolu; Zhang, Zan; He, Guiling; Yang, Liwen; Li, Fei

2012-01-01

Quantitative real-time polymerase chain reaction (qPCR) is an efficient and widely used technique to monitor gene expression. Housekeeping genes (HKGs) are often empirically selected as the reference genes for data normalization. However, the suitability of HKGs used as the reference genes has been seldom validated. Here, six HKGs were chosen (actin A3, actin A1, GAPDH, G3PDH, E2F, rp49) in four lepidopteran insects Bombyx mori L. (Lepidoptera: Bombycidae), Plutella xylostella L. (Plutellidae...

Model Checking Real-Time Systems

DEFF Research Database (Denmark)

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

2018-01-01

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

Modular specification of real-time systems

DEFF Research Database (Denmark)

Inal, Recep

1994-01-01

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

Hard Real-Time Networking on Firewire

NARCIS (Netherlands)

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

2005-01-01

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

Assessing HER2 amplification by IHC, FISH, and real-time polymerase chain reaction analysis (real-time PCR) following LCM in formalin-fixed paraffin embedded tissue from 40 women with ovarian cancer

DEFF Research Database (Denmark)

Hillig, Thore; Thode, Jørgen; Breinholt, Ellen Marie

2012-01-01

. Only few ovarian cancer patients were HER2 overexpressed measured by IHC or FISH and thus could be eligible for antibody-based therapy with trastuzumab (Herceptin). Interestingly, we find an increased number of HER2 positive patients by real-time PCR analysis on microdissected cancer cells, suggesting...

Comparison of Abbott and Da-an real-time PCR for quantitating serum HBV DNA.

Science.gov (United States)

Qiu, Ning; Li, Rui; Yu, Jian-Guo; Yang, Wen; Zhang, Wei; An, Yong; Li, Tong; Liu, Xue-En; Zhuang, Hui

2014-09-07

To compare the performance of the Da-an real-time hepatitis B virus (HBV) DNA assay and Abbott RealTime HBV assay. HBV DNA standards as well as a total of 180 clinical serum samples from patients with chronic hepatitis B were measured using the Abbott and Da-an real-time polymerase chain reaction (PCR) assays. Correlation and Bland-Altman plot analysis was used to compare the performance of the Abbott and Da-an assays. The HBV DNA levels were logarithmically transformed for analysis. All statistical analyses were performed using SPSS for Windows version 18.0. The correlation between the two assays was analyzed by Pearson's correlation and linear regression. The Bland-Altman plots were used for the analysis of agreement between the two assays. A P value of Da-an assay were significantly correlated with the expected values of HBV DNA standards (r = 0.999, for Abbott; r = 0.987, for Da-an, P Da-an assay. Moreover, HBV DNA levels measured by the Abbott assay were significantly higher than those of the Da-an assay (6.23 ± 1.76 log IU/mL vs 5.46 ± 1.55 log IU/mL, P Da-an assay presented lower sensitivity and a narrower linear range as compared to the Abbott assay, suggesting the need to be improved.

Using Real-Time PCR as a tool for monitoring the authenticity of commercial coffees.

Science.gov (United States)

Ferreira, Thiago; Farah, Adriana; Oliveira, Tatiane C; Lima, Ivanilda S; Vitório, Felipe; Oliveira, Edna M M

2016-05-15

Coffee is one of the main food products commercialized in the world. Its considerable market value among food products makes it susceptible to adulteration, especially with cereals. Therefore, the objective of this study was to develop a method based on Real-Time Polymerase Chain Reaction (PCR) for detection of cereals in commercial ground roast and soluble coffees. After comparison with standard curves obtained by serial dilution of DNA extracted from barley, corn and rice, the method was sensitive and specific to quantify down to 0.6 pg, 14 pg and 16 pg of barley, corn and rice DNA, respectively. To verify the applicability of the method, 30 commercial samples obtained in different countries were evaluated and those classified as gourmets or superior did not present the tested cereals DNA. However, barley was detected in various traditional (cheaper) samples from South America. In addition, corn and rice were also detected in different samples. Real-Time PCR showed to be suitable for detection of food adulterants in commercial ground roast and soluble coffees. Copyright © 2015 Elsevier Ltd. All rights reserved.

Prototyping real-time systems

OpenAIRE

Clynch, Gary

1994-01-01

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

Software Design Methods for Real-Time Systems

Science.gov (United States)

1989-12-01

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

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...

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...

Distributed, Embedded and Real-time Java Systems

CERN Document Server

Wellings, Andy

2012-01-01

Research on real-time Java technology has been prolific over the past decade, leading to a large number of corresponding hardware and software solutions, and frameworks for distributed and embedded real-time Java systems.  This book is aimed primarily at researchers in real-time embedded systems, particularly those who wish to understand the current state of the art in using Java in this domain.  Much of the work in real-time distributed, embedded and real-time Java has focused on the Real-time Specification for Java (RTSJ) as the underlying base technology, and consequently many of the Chapters in this book address issues with, or solve problems using, this framework. Describes innovative techniques in: scheduling, memory management, quality of service and communication systems supporting real-time Java applications; Includes coverage of multiprocessor embedded systems and parallel programming; Discusses state-of-the-art resource management for embedded systems, including Java’s real-time garbage collect...

Research of real-time communication software

Science.gov (United States)

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

2003-11-01

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

ASDEX Upgrade Discharge Control System—A real-time plasma control framework

International Nuclear Information System (INIS)

Treutterer, W.; Cole, R.; Lüddecke, K.; Neu, G.; Rapson, C.; Raupp, G.; Zasche, D.; Zehetbauer, T.

2014-01-01

Highlights: • The ASDEX Upgrade Discharge Control System (DCS) is a comprehensive control system to conduct fusion experiments. • DCS supports real-time diagnostic integration, adaptable feedback schemes, actuator management and exception handling. • DCS offers workflow management, logging and archiving, self-monitoring and inter-process communication. • DCS is based on a distributed, modular software framework architecture designed for real-time operation. • DCS is composed of re-usable generic but highly customisable components. - Abstract: ASDEX Upgrade is a fusion experiment with a size and complexity to allow extrapolation of technical and physical conditions and requirements to devices like ITER and even beyond. In addressing advanced physics topics it makes extensive use of sophisticated real-time control methods. It comprises real-time diagnostic integration, dynamically adaptable multivariable feedback schemes, actuator management including load distribution schemes and a powerful monitoring and pulse supervision concept based on segment scheduling and exception handling. The Discharge Control System (DCS) supplies all this functionality on base of a modular software framework architecture designed for real-time operation. It provides system-wide services like workflow management, logging and archiving, self-monitoring and inter-process communication on Linux, VxWorks and Solaris operating systems. By default DCS supports distributed computing, and a communication layer allows multi-directional signal transfer and data-driven process synchronisation over shared memory as well as over a number of real-time networks. The entire system is built following the same common design concept combining a rich set of re-usable generic but highly customisable components with a configuration-driven component deployment method. We will give an overview on the architectural concepts as well as on the outstanding capabilities of DCS in the domains of inter

ASDEX Upgrade Discharge Control System—A real-time plasma control framework

Energy Technology Data Exchange (ETDEWEB)

Treutterer, W., E-mail: Wolfgang.Treutterer@ipp.mpg.de [Max-Planck-Institut für Plasmaphysik, EURATOM Association, Boltzmannstraße 2, 85748 Garching (Germany); Cole, R.; Lüddecke, K. [Unlimited Computer Systems GmbH, Iffeldorf (Germany); Neu, G.; Rapson, C.; Raupp, G.; Zasche, D.; Zehetbauer, T. [Max-Planck-Institut für Plasmaphysik, EURATOM Association, Boltzmannstraße 2, 85748 Garching (Germany)

2014-03-15

Highlights: • The ASDEX Upgrade Discharge Control System (DCS) is a comprehensive control system to conduct fusion experiments. • DCS supports real-time diagnostic integration, adaptable feedback schemes, actuator management and exception handling. • DCS offers workflow management, logging and archiving, self-monitoring and inter-process communication. • DCS is based on a distributed, modular software framework architecture designed for real-time operation. • DCS is composed of re-usable generic but highly customisable components. - Abstract: ASDEX Upgrade is a fusion experiment with a size and complexity to allow extrapolation of technical and physical conditions and requirements to devices like ITER and even beyond. In addressing advanced physics topics it makes extensive use of sophisticated real-time control methods. It comprises real-time diagnostic integration, dynamically adaptable multivariable feedback schemes, actuator management including load distribution schemes and a powerful monitoring and pulse supervision concept based on segment scheduling and exception handling. The Discharge Control System (DCS) supplies all this functionality on base of a modular software framework architecture designed for real-time operation. It provides system-wide services like workflow management, logging and archiving, self-monitoring and inter-process communication on Linux, VxWorks and Solaris operating systems. By default DCS supports distributed computing, and a communication layer allows multi-directional signal transfer and data-driven process synchronisation over shared memory as well as over a number of real-time networks. The entire system is built following the same common design concept combining a rich set of re-usable generic but highly customisable components with a configuration-driven component deployment method. We will give an overview on the architectural concepts as well as on the outstanding capabilities of DCS in the domains of inter

Development and evaluation of the quantitative real-time PCR assay in detection and typing of herpes simplex virus in swab specimens from patients with genital herpes.

Science.gov (United States)

Liu, Junlian; Yi, Yong; Chen, Wei; Si, Shaoyan; Yin, Mengmeng; Jin, Hua; Liu, Jianjun; Zhou, Jinlian; Zhang, Jianzhong

2015-01-01

Genital herpes (GH), which is caused mainly by herpes simplex virus (HSV)-2 and HSV-1, remains a worldwide problem. Laboratory confirmation of GH is important, particularly as there are other conditions which present similarly to GH, while atypical presentations of GH also occur. Currently, virus culture is the classical method for diagnosis of GH, but it is time consuming and with low sensitivity. A major advance for diagnosis of GH is to use Real-time polymerase chain reaction (PCR). In this study, to evaluate the significance of the real-time PCR method in diagnosis and typing of genital HSV, the primers and probes targeted at HSV-1 DNA polymerase gene and HSV-2 glycoprotein D gene fraction were designed and applied to amplify DNA from HSV-1 or HSV-2 by employing the real-time PCR technique. Then the PCR reaction system was optimized and evaluated. HSV in swab specimens from patients with genital herpes was detected by real-time PCR. The real-time PCR assay showed good specificity for detection and typing of HSV, with good linear range (5×10(2)~5×10(8) copies/ml, r=0.997), a sensitivity of 5×10(2) copies/ml, and good reproducibility (intra-assay coefficients of variation 2.29% and inter-assay coefficients of variation 4.76%). 186 swab specimens were tested for HSV by real-time PCR, and the positive rate was 23.7% (44/186). Among the 44 positive specimens, 8 (18.2%) were positive for HSV-1 with a viral load of 8.5546×10(6) copies/ml and 36 (81.2%) were positive for HSV-2 with a viral load of 1.9861×10(6) copies/ml. It is concluded that the real-time PCR is a specific, sensitive and rapid method for the detection and typing of HSV, which can be widely used in clinical diagnosis of GH.

Development of RODOS (Real-Time, On-Line Decision Support System), a comprehensive decision support system for nuclear emergencies in Europe - an overview

International Nuclear Information System (INIS)

Ehrhardt, J.; Paesler-Sauer, J.; Schuele, O.

1993-01-01

Within its Radiation Protection Research Programme, the Commission of the European Communities has embarked on a major project aiming at the development of an integrated and comprehensive real-time on-line decision support system (RODOS) for nuclear emergencies in Europe, applicable for the vicinity of the release and the early phase up to far distant areas and the later stages of an accident. At present, the project is organised as a joint venture of some 18 European institutes closely cooperating with several Russian and Ukrainian laboratories. The main purpose of the project is to provide the methodological basis, develop models and data and install the hardware and software framework of RODOS. The paper describes the key features, the overall design and the software structure broadly accepted by the contractors. The paper sketches the present status of RODOS and gives an overview of the important tasks of R and D work for the next project phases. (author)

Integration of MDSplus in real-time systems

International Nuclear Information System (INIS)

Luchetta, A.; Manduchi, G.; Taliercio, C.

2006-01-01

RFX-mod makes extensive usage of real-time systems for feedback control and uses MDSplus to interface them to the main Data Acquisition system. For this purpose, the core of MDSplus has been ported to VxWorks, the operating system used for real-time control in RFX. Using this approach, it is possible to integrate real-time systems, but MDSplus is used only for non-real-time tasks, i.e. those tasks which are executed before and after the pulse and whose performance does not affect the system time constraints. More extensive use of MDSplus in real-time systems is foreseen, and a real-time layer for MDSplus is under development, which will provide access to memory-mapped pulse files, shared by the tasks running on the same CPU. Real-time communication will also be integrated in the MDSplus core to provide support for distributed memory-mapped pulse files

Real analysis a comprehensive course in analysis, part 1

CERN Document Server

Simon, Barry

2015-01-01

A Comprehensive Course in Analysis by Poincaré Prize winner Barry Simon is a five-volume set that can serve as a graduate-level analysis textbook with a lot of additional bonus information, including hundreds of problems and numerous notes that extend the text and provide important historical background. Depth and breadth of exposition make this set a valuable reference source for almost all areas of classical analysis. Part 1 is devoted to real analysis. From one point of view, it presents the infinitesimal calculus of the twentieth century with the ultimate integral calculus (measure theory)

Using the rate of bacterial clearance determined by real-time polymerase chain reaction as a timely surrogate marker to evaluate the appropriateness of antibiotic usage in critical patients with Acinetobacter baumannii bacteremia.

Science.gov (United States)

Chuang, Yu-Chung; Chang, Shan-Chwen; Wang, Wei-Kung

2012-08-01

Bacteremia caused by Acinetobacter baumannii is becoming more frequent among critically ill patients, and has been associated with high mortality and prolonged hospital stay. Multidrug resistance and delay in blood culture have been shown to be significant barriers to appropriate antibiotic treatment. Quantitative polymerase chain reaction assays were recently used to monitor bacterial loads; we hypothesized that the rate of bacterial clearance determined by quantitative polymerase chain reaction can be used as a timely surrogate marker to evaluate the appropriateness of antibiotic usage. Prospective observational study. University hospital and research laboratory. Patients with culture-proven A. baumannii bacteremia in the intensive care units were prospectively enrolled from April 2008 to February 2009. Plasmid Oxa-51/pCRII-TOPO, which contained a 431-bp fragment of the A. baumannii-specific Oxa-51 gene in a pCRII-TOPO vector, was used as the standard. Sequential bacterial DNA loads in the blood were measured by a quantitative polymerase chain reaction assay. We enrolled 51 patients with A. baumannii bacteremia, and examined 318 sequential whole blood samples. The initial mean bacterial load was 2.15 log copies/mL, and the rate of bacterial clearance was 0.088 log copies/mL/day. Multivariate linear regression using the generalized estimation equation approach revealed that the use of immunosuppressants was an independent predictor for slower bacterial clearance (coefficient, 1.116; pcritical patients. These findings highlight the importance of appropriate antibiotic usage and development of effective antibiotics against A. baumannii in an era of emerging antibiotic resistance. The rate of bacterial clearance could serve as a timely surrogate marker for evaluating the appropriateness of antibiotics.

Role of Text and Student Characteristics in Real-Time Reading Processes across the Primary Grades

Science.gov (United States)

de Leeuw, Linda; Segers, Eliane; Verhoeven, Ludo

2016-01-01

Although much is known about beginning readers using behavioural measures, real-time processes are still less clear. The present study examined eye movements (skipping rate, gaze, look back and second-pass duration) as a function of text-related (difficulty and word class) and student-related characteristics (word decoding, reading comprehension,…

Dense time discretization technique for verification of real time systems

International Nuclear Information System (INIS)

Makackas, Dalius; Miseviciene, Regina

2016-01-01

Verifying the real-time system there are two different models to control the time: discrete and dense time based models. This paper argues a novel verification technique, which calculates discrete time intervals from dense time in order to create all the system states that can be reached from the initial system state. The technique is designed for real-time systems specified by a piece-linear aggregate approach. Key words: real-time system, dense time, verification, model checking, piece-linear aggregate

Quantitative assessment of hematopoietic chimerism by quantitative real-time polymerase chain reaction of sequence polymorphism systems after hematopoietic stem cell transplantation.

Science.gov (United States)

Qin, Xiao-ying; Li, Guo-xuan; Qin, Ya-zhen; Wang, Yu; Wang, Feng-rong; Liu, Dai-hong; Xu, Lan-ping; Chen, Huan; Han, Wei; Wang, Jing-zhi; Zhang, Xiao-hui; Li, Jin-lan; Li, Ling-di; Liu, Kai-yan; Huang, Xiao-jun

2011-08-01

Analysis of changes in recipient and donor hematopoietic cell origin is extremely useful to monitor the effect of hematopoietic stem cell transplantation (HSCT) and sequential adoptive immunotherapy by donor lymphocyte infusions. We developed a sensitive, reliable and rapid real-time PCR method based on sequence polymorphism systems to quantitatively assess the hematopoietic chimerism after HSCT. A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time PCR in 101 HSCT patients with leukemia and other hematological diseases. The chimerism kinetics of bone marrow samples of 8 HSCT patients in remission and relapse situations were followed longitudinally. Recipient genotype discrimination was possible in 97.0% (98 of 101) with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Using serial dilutions of plasmids containing specific SP markers, the linear correlation (r) of 0.99, the slope between -3.2 and -3.7 and the sensitivity of 0.1% were proved reproducible. By this method, it was possible to very accurately detect autologous signals in the range from 0.1% to 30%. The accuracy of the method in the very important range of autologous signals below 5% was extraordinarily high (standard deviation real-time PCR method over short tandem repeat PCR chimerism assays is the absence of PCR competition and plateau biases, with demonstrated greater sensitivity and linearity. Finally, we prospectively analyzed bone marrow samples of 8 patients who received allografts and presented the chimerism kinetics of remission and relapse situations that illustrated the sensitivity level and the promising clinical application of this method. This SP-based real-time PCR assay provides a rapid, sensitive, and accurate quantitative assessment of mixed chimerism that can be useful in predicting graft rejection and early relapse.

Identification and quantification of genetically modified Moonshade carnation lines using conventional and TaqMan real-time polymerase chain reaction methods.

Science.gov (United States)

Li, Peng; Jia, Junwei; Bai, Lan; Pan, Aihu; Tang, Xueming

2013-07-01

Genetically modified carnation (Dianthus caryophyllus L.) Moonshade was approved for planting and commercialization in several countries from 2004. Developing methods for analyzing Moonshade is necessary for implementing genetically modified organism labeling regulations. In this study, the 5'-transgene integration sequence was isolated using thermal asymmetric interlaced (TAIL)-PCR. Based upon the 5'-transgene integration sequence, conventional and TaqMan real-time PCR assays were established. The relative limit of detection for the conventional PCR assay was 0.05 % for Moonshade using 100 ng total carnation genomic DNA, corresponding to approximately 79 copies of the carnation haploid genome, and the limits of detection and quantification of the TaqMan real-time PCR assay were estimated to be 51 and 254 copies of haploid carnation genomic DNA, respectively. These results are useful for identifying and quantifying Moonshade and its derivatives.

Whole blood Nested PCR and Real-time PCR amplification of Talaromyces marneffei specific DNA for diagnosis.

Science.gov (United States)

Lu, Sha; Li, Xiqing; Calderone, Richard; Zhang, Jing; Ma, Jianchi; Cai, Wenying; Xi, Liyan

2016-02-01

Talaromyces marneffei is a dimorphic pathogenic fungus, which is a life-threatening invasive mycosis in the immunocompromised host. Prompt diagnosis of T. marneffei infection remains difficult although there has been progress in attempts to expedite the diagnosis of this infection. We previously demonstrated the value of nested polymerase chain reaction (PCR) to detect T. marneffei in paraffin embedded tissue samples with high sensitivity and specificity. In this study, this assay was used to detect the DNA of T. marneffei in whole blood samples. Real-time PCR assay was also evaluated to identify T. marneffei in the same samples. Twenty out of 30 whole blood samples (67%) collected from 23 patients were found positive by using the nested PCR assay, while 23/30 (77%) samples were found positive by using the real-time PCR assay. In order to express accurately the fungal loads, we used a normalized linearized plasmid as an internal control for real-time PCR. The assay results were correlated as the initial quantity (copies/μl) with fungal burden. These data indicate that combination of nested PCR and real-time PCR assay provides an attractive alternative for identification of T. marneffei DNA in whole blood samples of HIV-infected patients. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Lactobacillus rhamnosus R11 consumed in a food supplement survived human digestive transit without modifying microbiota equilibrium as assessed by real-time polymerase chain reaction.

Science.gov (United States)

Firmesse, Olivier; Mogenet, Agnès; Bresson, Jean-Louis; Corthier, Gérard; Furet, Jean-Pierre

2008-01-01

The aim of this study was to evaluate the survival of Lactobacillus rhamnosus R11 and Lactobacillus acidophilus R52 in the human digestive tract and their effects on the microbiota homeostasis. We designed an open human trial including 14 healthy volunteers. A 3-week exclusion period of fermented products was followed by a 12-day consumption period of 4 capsules daily containing 2 x 10(9)L. rhamnosus R11 and 1 x 10(8)L. acidophilus R52, and a 12-day wash-out period. The 2 strains and dominant bacterial groups of the microbiota were quantified by real-time polymerase chain reaction. At the end of the capsule consumption period, high levels of L. rhamnosus R11 were detected in faecal samples from all volunteers, reaching a mean value of 7.1 log(10) colony-forming unit (CFU) equivalents/g of stool. L. acidophilus R52 was detected in the stools of only 1 volunteer, reaching a maximum level of 6.1 log(10) CFU equivalents/g of stool. Dilution plating enumerations performed in parallel provided less consistent and generally lower levels. No significant effect of capsule consumption was observed on microbiota homeostasis for the dominant faecal populations. Mean values of 8.8, 9.2, 9.9 and 10.6 log(10) CFU equivalents/g of stool were obtained for the Clostridium coccoides, Bifidobacterium sp., Bacteroides sp. and Clostridium leptum groups, respectively.

Detection of Babesia canis vogeli and Hepatozoon canis in canine blood by a single-tube real-time fluorescence resonance energy transfer polymerase chain reaction assay and melting curve analysis.

Science.gov (United States)

Kongklieng, Amornmas; Intapan, Pewpan M; Boonmars, Thidarut; Thanchomnang, Tongjit; Janwan, Penchom; Sanpool, Oranuch; Lulitanond, Viraphong; Taweethavonsawat, Piyanan; Chungpivat, Sudchit; Maleewong, Wanchai

2015-03-01

A real-time fluorescence resonance energy transfer polymerase chain reaction (qFRET PCR) coupled with melting curve analysis was developed for detection of Babesia canis vogeli and Hepatozoon canis infections in canine blood samples in a single tube assay. The target of the assay was a region within the 18S ribosomal RNA gene amplified in either species by a single pair of primers. Following amplification from the DNA of infected dog blood, a fluorescence melting curve analysis was done. The 2 species, B. canis vogeli and H. canis, could be detected and differentiated in infected dog blood samples (n = 37) with high sensitivity (100%). The detection limit for B. canis vogeli was 15 copies of a positive control plasmid, and for H. canis, it was 150 copies of a positive control plasmid. The assay could simultaneously distinguish the DNA of both parasites from the DNA of controls. Blood samples from 5 noninfected dogs were negative, indicating high specificity. Several samples can be run at the same time. The assay can reduce misdiagnosis and the time associated with microscopic examination, and is not prone to the carryover contamination associated with the agarose gel electrophoresis step of conventional PCR. In addition, this qFRET PCR method would be useful to accurately determine the range of endemic areas or to discover those areas where the 2 parasites co-circulate. © 2015 The Author(s).

Sustained Reduction of Ventilator-Associated Pneumonia Rates Using Real-Time Course Correction With a Ventilator Bundle Compliance Dashboard.

Science.gov (United States)

Talbot, Thomas R; Carr, Devin; Parmley, C Lee; Martin, Barbara J; Gray, Barbara; Ambrose, Anna; Starmer, Jack

2015-11-01

The effectiveness of practice bundles on reducing ventilator-associated pneumonia (VAP) has been questioned. To implement a comprehensive program that included a real-time bundle compliance dashboard to improve compliance and reduce ventilator-associated complications. DESIGN Before-and-after quasi-experimental study with interrupted time-series analysis. SETTING Academic medical center. In 2007 a comprehensive institutional ventilator bundle program was developed. To assess bundle compliance and stimulate instant course correction of noncompliant parameters, a real-time computerized dashboard was developed. Program impact in 6 adult intensive care units (ICUs) was assessed. Bundle compliance was noted as an overall cumulative bundle adherence assessment, reflecting the percentage of time all elements were concurrently in compliance for all patients. The VAP rate in all ICUs combined decreased from 19.5 to 9.2 VAPs per 1,000 ventilator-days following program implementation (Pdashboard was associated with significant sustained decreases in VAP rates and an increase in bundle compliance among adult ICU patients.

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.

A FRET-based real-time PCR assay to identify the main causal agents of New World tegumentary leishmaniasis.

Directory of Open Access Journals (Sweden)

Pablo Tsukayama

Full Text Available In South America, various species of Leishmania are endemic and cause New World tegumentary leishmaniasis (NWTL. The correct identification of these species is critical for adequate clinical management and surveillance activities. We developed a real-time polymerase chain reaction (PCR assay and evaluated its diagnostic performance using 64 archived parasite isolates and 192 prospectively identified samples collected from individuals with suspected leishmaniasis enrolled at two reference clinics in Lima, Peru. The real-time PCR assay was able to detect a single parasite and provided unambiguous melting peaks for five Leishmania species of the Viannia subgenus that are highly prevalent in South America: L. (V. braziliensis, L. (V. panamensis, L. (V. guyanensis, L. (V. peruviana and L. (V. lainsoni. Using kinetoplastid DNA-based PCR as a gold standard, the real-time PCR had sensitivity and specificity values of 92% and 77%, respectively, which were significantly higher than those of conventional tests such as microscopy, culture and the leishmanin skin test (LST. In addition, the real-time PCR identified 147 different clinical samples at the species level, providing an overall agreement of 100% when compared to multilocus sequence typing (MLST data performed on a subset of these samples. Furthermore, the real-time PCR was three times faster and five times less expensive when compared to PCR - MLST for species identification from clinical specimens. In summary, this new assay represents a cost-effective and reliable alternative for the identification of the main species causing NWTL in South America.

Mixed - mode Operating System for Real - time Performance

Directory of Open Access Journals (Sweden)

Hasan M. M.

2017-11-01

Full Text Available The purpose of the mixed-mode system research is to handle devices with the accuracy of real-time systems and at the same time, having all the benefits and facilities of a matured Graphic User Interface(GUIoperating system which is typicallynon-real-time. This mixed-mode operating system comprising of a real-time portion and a non-real-time portion was studied and implemented to identify the feasibilities and performances in practical applications (in the context of scheduled the real-time events. In this research an i8751 microcontroller-based hardware was used to measure the performance of the system in real-time-only as well as non-real-time-only configurations. The real-time portion is an 486DX-40 IBM PC system running under DOS-based real-time kernel and the non-real-time portion is a Pentium IIIbased system running under Windows NT. It was found that mixed-mode systems performed as good as a typical real-time system and in fact, gave many additional benefits such as simplified/modular programming and load tolerance.

Real-time polymerase chain reaction for diagnosing infectious mononucleosis in pediatric patients: A systematic review and meta-analysis.

Science.gov (United States)

Jiang, Sha-Yi; Yang, Jing-Wei; Shao, Jing-Bo; Liao, Xue-Lian; Lu, Zheng-Hua; Jiang, Hui

2016-05-01

In this meta-analysis, we evaluated the diagnostic role of Epstein-Barr virus deoxyribonucleic acid detection and quantitation in the serum of pediatric and young adult patients with infectious mononucleosis. The primary outcome of this meta-analysis was the sensitivity and specificity of Epstein-Barr virus (EBV) deoxyribonucleic acid (DNA) detection and quantitation using polymerase chain reaction (PCR). A systematic review and meta-analysis was performed by searching for articles that were published through September 24, 2014 in the following databases: Medline, Cochrane, EMBASE, and Google Scholar. The following keywords were used for the search: "Epstein-Barr virus," "infectious mononucleosis," "children/young adults/infant/pediatric," and "polymerase chain reaction or PCR." Three were included in this analysis. We found that for detection by PCR, the pooled sensitivity for detecting EBV DNA was 77% (95%CI, 66-86%) and the pooled specificity for was 98% (95%CI, 93-100%). Our findings indicate that this PCR-based assay has high specificity and good sensitivity for detecting of EBV DNA, indicating it may useful for identifying patients with infectious mononucleosis. This assay may also be helpful to identify young athletic patients or highly physically active pediatric patients who are at risk for a splenic rupture due to acute infectious mononucleosis. © 2015 Wiley Periodicals, Inc.

Real-Time Simulation Technique of a Microgrid Model for DER Penetration

Directory of Open Access Journals (Sweden)

Konstantina Mentesidi

2014-12-01

Full Text Available Comprehensive analysis of Distributed Energy Resources (DER integration requires tools that provide computational power and flexibility. In this context, throughout this paper PHIL simulations are performed to emulate the energy management system of a real microgrid including a diesel synchronous machine and inverter-based sources. Moreover, conventional frequency and voltage droops were incorporated into the respective inverters. The results were verified at the real microgrid installation in the Centre for Renewable Energy Sources (CRES premises. This research work is divided into two steps: A Real time in RSCAD/RTDS and Power Hardware-in-the-Loop (PHIL simulations where the diesel generator´s active power droop control is evaluated, the battery inverter´s droop curves are simulated and the load sharing for parallel operation of the system´s generation units is examined. B microgrid experiments during which various tests were executed concerning the diesel generator and the battery inverters in order to examine their dynamic operation within the LV islanded power system.

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.

Real-time data access layer for MDSplus

International Nuclear Information System (INIS)

Manduchi, G.; Luchetta, A.; Taliercio, C.; Fredian, T.; Stillerman, J.

2008-01-01

Recent extensions to MDSplus allow data handling in long discharges and provide a real-time data access and communication layer. The real-time data access layer is an additional component of MDSplus: it is possible to use the traditional MDSplus API during normal operation, and to select a subset of data items to be used in real time. Real-time notification is provided by a communication layer using a publish-subscribe pattern. The notification covers processes sharing the same data items even running on different machines, thus allowing the implementation of distributed control systems. The real-time data access layer has been developed for Windows, Linux, and VxWorks; it is currently being ported to Linux RTAI. In order to quantify the fingerprint of the presented system, the performance of the real-time access layer approach is compared with that of an ad hoc, manually optimized program in a sample real-time application

A Real-Time Systems Symposium Preprint.

Science.gov (United States)

1983-09-01

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

Benefits of real-time gas management

International Nuclear Information System (INIS)

Nolty, R.; Dolezalek, D. Jr.

1994-01-01

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

A system for EPID-based real-time treatment delivery verification during dynamic IMRT treatment.

Science.gov (United States)

Fuangrod, Todsaporn; Woodruff, Henry C; van Uytven, Eric; McCurdy, Boyd M C; Kuncic, Zdenka; O'Connor, Daryl J; Greer, Peter B

2013-09-01

To design and develop a real-time electronic portal imaging device (EPID)-based delivery verification system for dynamic intensity modulated radiation therapy (IMRT) which enables detection of gross treatment delivery errors before delivery of substantial radiation to the patient. The system utilizes a comprehensive physics-based model to generate a series of predicted transit EPID image frames as a reference dataset and compares these to measured EPID frames acquired during treatment. The two datasets are using MLC aperture comparison and cumulative signal checking techniques. The system operation in real-time was simulated offline using previously acquired images for 19 IMRT patient deliveries with both frame-by-frame comparison and cumulative frame comparison. Simulated error case studies were used to demonstrate the system sensitivity and performance. The accuracy of the synchronization method was shown to agree within two control points which corresponds to approximately ∼1% of the total MU to be delivered for dynamic IMRT. The system achieved mean real-time gamma results for frame-by-frame analysis of 86.6% and 89.0% for 3%, 3 mm and 4%, 4 mm criteria, respectively, and 97.9% and 98.6% for cumulative gamma analysis. The system can detect a 10% MU error using 3%, 3 mm criteria within approximately 10 s. The EPID-based real-time delivery verification system successfully detected simulated gross errors introduced into patient plan deliveries in near real-time (within 0.1 s). A real-time radiation delivery verification system for dynamic IMRT has been demonstrated that is designed to prevent major mistreatments in modern radiation therapy.

Making real-time reactive systems reliable

Science.gov (United States)

Marzullo, Keith; Wood, Mark

1990-01-01

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

Comprehensive RNA Polymerase II Interactomes Reveal Distinct and Varied Roles for Each Phospho-CTD Residue

Directory of Open Access Journals (Sweden)

Kevin M. Harlen

2016-06-01

Full Text Available Transcription controls splicing and other gene regulatory processes, yet mechanisms remain obscure due to our fragmented knowledge of the molecular connections between the dynamically phosphorylated RNA polymerase II (Pol II C-terminal domain (CTD and regulatory factors. By systematically isolating phosphorylation states of the CTD heptapeptide repeat (Y1S2P3T4S5P6S7, we identify hundreds of protein factors that are differentially enriched, revealing unappreciated connections between the Pol II CTD and co-transcriptional processes. These data uncover a role for threonine-4 in 3′ end processing through control of the transition between cleavage and termination. Furthermore, serine-5 phosphorylation seeds spliceosomal assembly immediately downstream of 3′ splice sites through a direct interaction with spliceosomal subcomplex U1. Strikingly, threonine-4 phosphorylation also impacts splicing by serving as a mark of co-transcriptional spliceosome release and ensuring efficient post-transcriptional splicing genome-wide. Thus, comprehensive Pol II interactomes identify the complex and functional connections between transcription machinery and other gene regulatory complexes.

[Usefulness of a real-time quantitative polymerase-chain reaction (PCR) assay for the diagnosis of congenital and postnatal cytomegalovirus infection].

Science.gov (United States)

Reina, J; Weber, I; Riera, E; Busquets, M; Morales, C

2014-05-01

Cytomegalovirus (CMV) is the main virus causing congenital and postnatal infections in the pediatric population. The aim of this study is to evaluate the usefulness of a quantitative real-time PCR in the diagnosis of these infections using urine as a single sample. We studied all the urine samples of newborns (< 7 days) with suspected congenital infection, and urine of patients with suspected postnatal infection (urine negative at birth). Urines were simultaneously studied by cell culture, qualitative PCR (PCRc), and quantitative real-time PCR (PCRq). We analyzed 332 urine samples (270 to rule out congenital infection and 62 postnatal infections). Of the first, 22 were positive in the PCRq, 19 in the PCRc, and 17 in the culture. PCRq had a sensitivity of 100%, on comparing the culture with the rest of the techniques. Using the PCRq as a reference method, culture had a sensitivity of 77.2%, and PCRc 86.3%. In cases of postnatal infection, PCRq detected 16 positive urines, the PCRq 12, and the cell culture 10. The urines showed viral loads ranging from 2,178 to 116,641 copies/ml. The genomic amplification technique PCRq in real time was more sensitive than the other techniques evaluated. This technique should be considered as a reference (gold standard), leaving the cell culture as a second diagnostic level. The low cost and the automation of PCRq would enable the screening for CMV infection in large neonatal and postnatal populations. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

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.

Research Directions in Real-Time Systems.

Science.gov (United States)

1996-09-01

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

Development of a real-time PCR to detect Demodex canis DNA in different tissue samples.

Science.gov (United States)

Ravera, Ivan; Altet, Laura; Francino, Olga; Bardagí, Mar; Sánchez, Armand; Ferrer, Lluís

2011-02-01

The present study reports the development of a real-time polymerase chain reaction (PCR) to detect Demodex canis DNA on different tissue samples. The technique amplifies a 166 bp of D. canis chitin synthase gene (AB 080667) and it has been successfully tested on hairs extracted with their roots and on formalin-fixed paraffin embedded skin biopsies. The real-time PCR amplified on the hairs of all 14 dogs with a firm diagnosis of demodicosis and consistently failed to amplify on negative controls. Eleven of 12 skin biopsies with a morphologic diagnosis of canine demodicosis were also positive. Sampling hairs on two skin points (lateral face and interdigital skin), D. canis DNA was detected on nine of 51 healthy dogs (17.6%) a much higher percentage than previously reported with microscopic studies. Furthermore, it is foreseen that if the number of samples were increased, the percentage of positive dogs would probably also grow. Moreover, in four of the six dogs with demodicosis, the samples taken from non-lesioned skin were positive. This finding, if confirmed in further studies, suggests that demodicosis is a generalized phenomenon in canine skin, due to proliferation of local mite populations, even though macroscopic lesions only appear in certain areas. The real-time PCR technique to detect D. canis DNA described in this work is a useful tool to advance our understanding of canine demodicosis.

Evaluation of Reference Genes for Real-Time Quantitative PCR Analysis in Larvae of Spodoptera litura Exposed to Azadirachtin Stress Conditions

OpenAIRE

Benshui Shu; Jingjing Zhang; Gaofeng Cui; Ranran Sun; Veeran Sethuraman; Xin Yi; Guohua Zhong

2018-01-01

Azadirachtin is an efficient and broad-spectrum botanical insecticide against more than 150 kinds of agricultural pests with the effects of mortality, antifeedant and growth regulation. Real-time quantitative polymerase chain reaction (RT-qPCR) could be one of the powerful tools to analyze the gene expression level and investigate the mechanism of azadirachtin at transcriptional level, however, the ideal reference genes are needed to normalize the expression profiling of target genes. In this...

Principles and applications of polymerase chain reaction in medical diagnostic fields: a review.

Science.gov (United States)

Valones, Marcela Agne Alves; Guimarães, Rafael Lima; Brandão, Lucas André Cavalcanti; de Souza, Paulo Roberto Eleutério; de Albuquerque Tavares Carvalho, Alessandra; Crovela, Sergio

2009-01-01

Recent developments in molecular methods have revolutionized the detection and characterization of microorganisms in a broad range of medical diagnostic fields, including virology, mycology, parasitology, microbiology and dentistry. Among these methods, Polymerase Chain Reaction (PCR) has generated great benefits and allowed scientific advancements. PCR is an excellent technique for the rapid detection of pathogens, including those difficult to culture. Along with conventional PCR techniques, Real-Time PCR has emerged as a technological innovation and is playing an ever-increasing role in clinical diagnostics and research laboratories. Due to its capacity to generate both qualitative and quantitative results, Real-Time PCR is considered a fast and accurate platform. The aim of the present literature review is to explore the clinical usefulness and potential of both conventional PCR and Real-Time PCR assays in diverse medical fields, addressing its main uses and advances.

Real-Time MENTAT programming language and architecture

Science.gov (United States)

Grimshaw, Andrew S.; Silberman, Ami; Liu, Jane W. S.

1989-01-01

Real-time MENTAT, a programming environment designed to simplify the task of programming real-time applications in distributed and parallel environments, is described. It is based on the same data-driven computation model and object-oriented programming paradigm as MENTAT. It provides an easy-to-use mechanism to exploit parallelism, language constructs for the expression and enforcement of timing constraints, and run-time support for scheduling and exciting real-time programs. The real-time MENTAT programming language is an extended C++. The extensions are added to facilitate automatic detection of data flow and generation of data flow graphs, to express the timing constraints of individual granules of computation, and to provide scheduling directives for the runtime system. A high-level view of the real-time MENTAT system architecture and programming language constructs is provided.

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.

Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

Science.gov (United States)

Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

2013-01-01

Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.

Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

Directory of Open Access Journals (Sweden)

Huali Huang

Full Text Available Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L. DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.

Evaluation of Four Endogenous Reference Genes and Their Real-Time PCR Assays for Common Wheat Quantification in GMOs Detection

Science.gov (United States)

Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

2013-01-01

Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat. PMID:24098735

Run-time middleware to support real-time system scenarios

NARCIS (Netherlands)

Goossens, K.; Koedam, M.; Sinha, S.; Nelson, A.; Geilen, M.

2015-01-01

Systems on Chip (SOC) are powerful multiprocessor systems capable of running multiple independent applications, often with both real-time and non-real-time requirements. Scenarios exist at two levels: first, combinations of independent applications, and second, different states of a single

Advanced real-time manipulation of video streams

CERN Document Server

Herling, Jan

2014-01-01

Diminished Reality is a new fascinating technology that removes real-world content from live video streams. This sensational live video manipulation actually removes real objects and generates a coherent video stream in real-time. Viewers cannot detect modified content. Existing approaches are restricted to moving objects and static or almost static cameras and do not allow real-time manipulation of video content. Jan Herling presents a new and innovative approach for real-time object removal with arbitrary camera movements.

Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA.

Science.gov (United States)

Majid, Farjana; Jahan, Munira; Lutful Moben, Ahmed; Tabassum, Shahina

2014-01-01

Both real-time-polymerase chain reaction (PCR) and hybrid capture 2 (HC2) assay can detect and quantify hepatitis B virus (HBV) DNA. However, real-time-PCR can detect a wide range of HBV DNA, while HC2 assay could not detect lower levels of viremia. The present study was designed to detect and quantify HBV DNA by real-time-PCR and HC2 assay and compare the quantitative data of these two assays. A cross-sectional study was conducted in between July 2010 and June 2011. A total of 66 serologically diagnosed chronic hepatitis B (CHB) patients were selected for the study. Real-time-PCR and HC2 assay was done to detect HBV DNA. Data were analyzed by statistical Package for the social sciences (SPSS). Among 66 serologically diagnosed chronic hepatitis B patients 40 (60.61%) patients had detectable and 26 (39.39%) had undetectable HBV DNA by HC2 assay. Concordant results were obtained for 40 (60.61%) out of these 66 patients by real-time-PCR and HC2 assay with mean viral load of 7.06 ± 1.13 log 10 copies/ml and 6.95 ± 1.08 log 10 copies/ml, respectively. In the remaining 26 patients, HBV DNA was detectable by real-time-PCR in 20 patients (mean HBV DNA level was 3.67 ± 0.72 log 10 copies/ml. However, HBV DNA could not be detectable in six cases by the both assays. The study showed strong correlation (r = 0.915) between real-time-PCR and HC2 assay for the detection and quantification of HBV DNA. HC2 assay may be used as an alternative to real-time-PCR for CHB patients. How to cite this article: Majid F, Jahan M, Moben AL, Tabassum S. Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA. Euroasian J Hepato-Gastroenterol 2014;4(1):31-35.

Archtecture of distributed real-time systems

OpenAIRE

Wing Leung, Cheuk

2013-01-01

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

Quantitative analysis of waterfowl parvoviruses in geese and Muscovy ducks by real-time polymerase chain reaction: correlation between age, clinical symptoms and DNA copy number of waterfowl parvoviruses

Directory of Open Access Journals (Sweden)

Woźniakowski Grzegorz

2012-03-01

Full Text Available Abstract Background Waterfowl parvoviruses cause serious loss in geese and ducks production. Goose parvovirus (GPV is infectious for geese and ducks while Muscovy duck parvovirus (MDPV infects Muscovy ducks only. So far, for these viruses' sensitive detection polymerase chain reaction (PCR and loop-mediated isothermal amplification (LAMP were applied. However, there was no molecular biology method for both waterfowl parvoviruses detection and quantification which could unify the laboratory procedures. The level of GPV and MDPV replication and distribution plays a significant role in the parvoviral infection progress and is strictly correlated to clinical symptoms. Meanwhile, experiments conducted previously on GPV distribution in geese, performed as animal trial, did not involve epidemiological data from the disease field cases. The study on the correlation between age, clinical symptoms and viral DNA copy number may be benefitable in understanding the GPV and MDPV infection. Such data may also aid in determination of the stage and severity of the infection with parvoviruses. Therefore the aim of this study was to develop quantitative real-time PCR for parallel detection of GPV and MDPV in geese and Muscovy ducks and to determine the correlation between the age of the infected birds, clinical symptoms and DNA copy number for the estimation of the disease stage or severity. Results In order to develop quantitative real-time PCR the viral material was collected from 13 farms of geese and 3 farms of Muscovy ducks. The designed primers and Taqman probe for real-time PCR were complementary to GPV and MDPV inverted terminal repeats region. The pITR plasmid was constructed, purified and used to prepare dilutions for standard curve preparation and DNA quantification. The applied method detected both GPV and MDPV in all the examined samples extracted from the heart and liver of the infected birds. The conducted correlation tests have shown relationship

The real-time price elasticity of electricity

NARCIS (Netherlands)

Lijesen, M.G.

2007-01-01

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

Implementing Run-Time Evaluation of Distributed Timing Constraints in a Real-Time Environment

DEFF Research Database (Denmark)

Kristensen, C. H.; Drejer, N.

1994-01-01

In this paper we describe a solution to the problem of implementing run-time evaluation of timing constraints in distributed real-time environments......In this paper we describe a solution to the problem of implementing run-time evaluation of timing constraints in distributed real-time environments...

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.

A study of real-time content marketing : formulating real-time content marketing based on content, search and social media

OpenAIRE

Nguyen, Thi Kim Duyen

2015-01-01

The primary objective of this research is to understand profoundly the new concept of content marketing – real-time content marketing on the aspect of the digital marketing experts. Particularly, the research will focus on the real-time content marketing theories and how to build real-time content marketing strategy based on content, search and social media. It also finds out how marketers measure and keep track of conversion rates of their real-time content marketing plan. Practically, th...

A system for EPID-based real-time treatment delivery verification during dynamic IMRT treatment

Energy Technology Data Exchange (ETDEWEB)

Fuangrod, Todsaporn [Faculty of Engineering and Built Environment, School of Electrical Engineering and Computer Science, the University of Newcastle, NSW 2308 (Australia); Woodruff, Henry C.; O’Connor, Daryl J. [Faculty of Science and IT, School of Mathematical and Physical Sciences, the University of Newcastle, NSW 2308 (Australia); Uytven, Eric van; McCurdy, Boyd M. C. [Division of Medical Physics, CancerCare Manitoba, 675 McDermot Avenue, Winnipeg, Manitoba R3E 0V9 (Canada); Department of Physics and Astronomy, University of Manitoba, Winnipeg, Manitoba R3T 2N2 (Canada); Department of Radiology, University of Manitoba, Winnipeg, Manitoba R3T 2N2 (Canada); Kuncic, Zdenka [School of Physics, University of Sydney, Sydney, NSW 2006 (Australia); Greer, Peter B. [Faculty of Science and IT, School of Mathematical and Physical Sciences, the University of Newcastle, NSW 2308, Australia and Department of Radiation Oncology, Calvary Mater Newcastle Hospital, Locked Bag 7, Hunter region Mail Centre, Newcastle, NSW 2310 (Australia)

2013-09-15

Purpose: To design and develop a real-time electronic portal imaging device (EPID)-based delivery verification system for dynamic intensity modulated radiation therapy (IMRT) which enables detection of gross treatment delivery errors before delivery of substantial radiation to the patient.Methods: The system utilizes a comprehensive physics-based model to generate a series of predicted transit EPID image frames as a reference dataset and compares these to measured EPID frames acquired during treatment. The two datasets are using MLC aperture comparison and cumulative signal checking techniques. The system operation in real-time was simulated offline using previously acquired images for 19 IMRT patient deliveries with both frame-by-frame comparison and cumulative frame comparison. Simulated error case studies were used to demonstrate the system sensitivity and performance.Results: The accuracy of the synchronization method was shown to agree within two control points which corresponds to approximately ∼1% of the total MU to be delivered for dynamic IMRT. The system achieved mean real-time gamma results for frame-by-frame analysis of 86.6% and 89.0% for 3%, 3 mm and 4%, 4 mm criteria, respectively, and 97.9% and 98.6% for cumulative gamma analysis. The system can detect a 10% MU error using 3%, 3 mm criteria within approximately 10 s. The EPID-based real-time delivery verification system successfully detected simulated gross errors introduced into patient plan deliveries in near real-time (within 0.1 s).Conclusions: A real-time radiation delivery verification system for dynamic IMRT has been demonstrated that is designed to prevent major mistreatments in modern radiation therapy.

A system for EPID-based real-time treatment delivery verification during dynamic IMRT treatment

International Nuclear Information System (INIS)

Fuangrod, Todsaporn; Woodruff, Henry C.; O’Connor, Daryl J.; Uytven, Eric van; McCurdy, Boyd M. C.; Kuncic, Zdenka; Greer, Peter B.

2013-01-01

Purpose: To design and develop a real-time electronic portal imaging device (EPID)-based delivery verification system for dynamic intensity modulated radiation therapy (IMRT) which enables detection of gross treatment delivery errors before delivery of substantial radiation to the patient.Methods: The system utilizes a comprehensive physics-based model to generate a series of predicted transit EPID image frames as a reference dataset and compares these to measured EPID frames acquired during treatment. The two datasets are using MLC aperture comparison and cumulative signal checking techniques. The system operation in real-time was simulated offline using previously acquired images for 19 IMRT patient deliveries with both frame-by-frame comparison and cumulative frame comparison. Simulated error case studies were used to demonstrate the system sensitivity and performance.Results: The accuracy of the synchronization method was shown to agree within two control points which corresponds to approximately ∼1% of the total MU to be delivered for dynamic IMRT. The system achieved mean real-time gamma results for frame-by-frame analysis of 86.6% and 89.0% for 3%, 3 mm and 4%, 4 mm criteria, respectively, and 97.9% and 98.6% for cumulative gamma analysis. The system can detect a 10% MU error using 3%, 3 mm criteria within approximately 10 s. The EPID-based real-time delivery verification system successfully detected simulated gross errors introduced into patient plan deliveries in near real-time (within 0.1 s).Conclusions: A real-time radiation delivery verification system for dynamic IMRT has been demonstrated that is designed to prevent major mistreatments in modern radiation therapy

Application of XML in real-time data warehouse

Science.gov (United States)

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

2009-07-01

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

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

Science.gov (United States)

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

2013-01-01

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

Global conformational dynamics of a Y-family DNA polymerase during catalysis.

Directory of Open Access Journals (Sweden)

Cuiling Xu

2009-10-01

Full Text Available Replicative DNA polymerases are stalled by damaged DNA while the newly discovered Y-family DNA polymerases are recruited to rescue these stalled replication forks, thereby enhancing cell survival. The Y-family DNA polymerases, characterized by low fidelity and processivity, are able to bypass different classes of DNA lesions. A variety of kinetic and structural studies have established a minimal reaction pathway common to all DNA polymerases, although the conformational intermediates are not well defined. Furthermore, the identification of the rate-limiting step of nucleotide incorporation catalyzed by any DNA polymerase has been a matter of long debate. By monitoring time-dependent fluorescence resonance energy transfer (FRET signal changes at multiple sites in each domain and DNA during catalysis, we present here a real-time picture of the global conformational transitions of a model Y-family enzyme: DNA polymerase IV (Dpo4 from Sulfolobus solfataricus. Our results provide evidence for a hypothetical DNA translocation event followed by a rapid protein conformational change prior to catalysis and a subsequent slow, post-chemistry protein conformational change. Surprisingly, the DNA translocation step was induced by the binding of a correct nucleotide. Moreover, we have determined the directions, rates, and activation energy barriers of the protein conformational transitions, which indicated that the four domains of Dpo4 moved in a synchronized manner. These results showed conclusively that a pre-chemistry conformational change associated with domain movements was too fast to be the rate-limiting step. Rather, the rearrangement of active site residues limited the rate of correct nucleotide incorporation. Collectively, the conformational dynamics of Dpo4 offer insights into how the inter-domain movements are related to enzymatic function and their concerted interactions with other proteins at the replication fork.

The developmental stage of strongyle eggs affects the outcome of real-time PCR analysis

DEFF Research Database (Denmark)

Roust, Tina; Haakansson, Ida T.; Rhod, Maria

Several molecular diagnostic tests are based upon measuring and quantifying DNA obtained from parasite eggs. It is well-known that such eggs undergo development during storage, but it remains unknown to which extent the stage of development can affect the diagnostic test result. This project...... investigated whether the developmental stage of strongyle eggs affects real-time polymerase chain reaction (PCR) results. Mixed species strongyle eggs were obtained from the faeces of a naturally infected horse. Eggs were isolated and placed in microtiter plates with demineralised water. A total of 25 wells...... containing 100 eggs each were set up and kept refrigerated for up to five days. Once daily, five wells were microscopied on an inverted microscope, the developmental stages of the eggs were noted, and the eggs harvested for DNA extraction. The protocol was repeated three times. Genomic DNA was extracted...

Mixed - mode Operating System for Real - time Performance

OpenAIRE

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

2017-01-01

The purpose of the mixed-mode system research is to handle devices with the accuracy of real-time systems and at the same time, having all the benefits and facilities of a matured Graphic User Interface(GUI)operating system which is typicallynon-real-time. This mixed-mode operating system comprising of a real-time portion and a non-real-time portion was studied and implemented to identify the feasibilities and performances in practical applications (in the context of scheduled the real-time e...

Novel real-time polymerase chain reaction assay for simultaneous detection of recurrent fusion genes in acute myeloid leukemia.

Science.gov (United States)

Dolz, Sandra; Barragán, Eva; Fuster, Óscar; Llop, Marta; Cervera, José; Such, Esperanza; De Juan, Inmaculada; Palanca, Sarai; Murria, Rosa; Bolufer, Pascual; Luna, Irene; Gómez, Inés; López, María; Ibáñez, Mariam; Sanz, Miguel A

2013-09-01

The recent World Health Organization classification recognizes different subtypes of acute myeloid leukemia (AML) according to the presence of several recurrent genetic abnormalities. Detection of these abnormalities and other molecular changes is of increasing interest because it contributes to a refined diagnosis and prognostic assessment in AML and enables monitoring of minimal residual disease. These genetic abnormalities can be detected using single RT-PCR, although the screening is still labor intensive and costly. We have developed a novel real-time RT-PCR assay to simultaneously detect 15 AML-associated rearrangements that is a simple and easily applicable method for use in clinical diagnostic laboratories. This method showed 100% specificity and sensitivity (95% confidence interval, 91% to 100% and 92% to 100%, respectively). The procedure was validated in a series of 105 patients with AML. The method confirmed all translocations detected using standard cytogenetics and fluorescence in situ hybridization and some additional undetected rearrangements. Two patients demonstrated two molecular rearrangements simultaneously, with BCR-ABL1 implicated in both, in addition to RUNX1-MECOM in one patient and PML-RARA in another. In conclusion, this novel real-time RT-PCR assay for simultaneous detection of multiple AML-associated fusion genes is a versatile and sensitive method for reliable screening of recurrent rearrangements in AML. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Mixed-mode Operating System for Real-time Performance

Directory of Open Access Journals (Sweden)

M.M. Hasan

2017-11-01

Full Text Available The purpose of the mixed-mode system research is to handle devices with the accuracy of real-time systems and at the same time, having all the benefits and facilities of a matured Graphic User Interface (GUI operating system which is typically nonreal-time. This mixed-mode operating system comprising of a real-time portion and a non-real-time portion was studied and implemented to identify the feasibilities and performances in practical applications (in the context of scheduled the real-time events. In this research an i8751 microcontroller-based hardware was used to measure the performance of the system in real-time-only as well as non-real-time-only configurations. The real-time portion is an 486DX-40 IBM PC system running under DOS-based realtime kernel and the non-real-time portion is a Pentium III based system running under Windows NT. It was found that mixed-mode systems performed as good as a typical realtime system and in fact, gave many additional benefits such as simplified/modular programming and load tolerance.

Quality of bulk tank milk samples from Danish dairy herds based on real-time polymerase chain reaction identification of mastitis pathogens

DEFF Research Database (Denmark)

Katholm, Jørgen; Bennedsgaard, T.W.; Koskinen, M.T.

2012-01-01

Results of a commercial real-time PCR analysis for 11 mastitis pathogens from bulk tank milk (BTM) samples from all 4,258 Danish dairy herds in November 2009 to January 2010 were compared with somatic cell count (SCC) and total bacteria count (TBC) estimates in BTM. For Streptococcus agalactiae......, Streptococcus dysgalactiae, and Streptococcus uberis, a low real-time PCR cycle threshold (Ct) value (corresponding to high bacterial DNA quantity) was correlated with higher SCC and higher TBC. For Staphylococcus aureus, low Ct values were correlated only with higher SCC. For the environmental mastitis...... pathogens Klebsiella spp., Enterococcus spp., and Escherichia coli, low Ct values had a correlation with higher TBC. Staphylococcus spp. were found in the BTM from all herds, Strep. uberis in 95%, Staph. aureus in 91%, and Strep. dysgalactiae in 86%, whereas E. coli, Klebsiella, and Strep. agalactiae were...

Testing of real-time-software

International Nuclear Information System (INIS)

Friesland, G.; Ovenhausen, H.

1975-05-01

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

Validation and Assessment of Multi-GNSS Real-Time Precise Point Positioning in Simulated Kinematic Mode Using IGS Real-Time Service

Directory of Open Access Journals (Sweden)

Liang Wang

2018-02-01

Full Text Available Precise Point Positioning (PPP is a popular technology for precise applications based on the Global Navigation Satellite System (GNSS. Multi-GNSS combined PPP has become a hot topic in recent years with the development of multiple GNSSs. Meanwhile, with the operation of the real-time service (RTS of the International GNSS Service (IGS agency that provides satellite orbit and clock corrections to broadcast ephemeris, it is possible to obtain the real-time precise products of satellite orbits and clocks and to conduct real-time PPP. In this contribution, the real-time multi-GNSS orbit and clock corrections of the CLK93 product are applied for real-time multi-GNSS PPP processing, and its orbit and clock qualities are investigated, first with a seven-day experiment by comparing them with the final multi-GNSS precise product ‘GBM’ from GFZ. Then, an experiment involving real-time PPP processing for three stations in the Multi-GNSS Experiment (MGEX network with a testing period of two weeks is conducted in order to evaluate the convergence performance of real-time PPP in a simulated kinematic mode. The experimental result shows that real-time PPP can achieve a convergence performance of less than 15 min for an accuracy level of 20 cm. Finally, the real-time data streams from 12 globally distributed IGS/MGEX stations for one month are used to assess and validate the positioning accuracy of real-time multi-GNSS PPP. The results show that the simulated kinematic positioning accuracy achieved by real-time PPP on different stations is about 3.0 to 4.0 cm for the horizontal direction and 5.0 to 7.0 cm for the three-dimensional (3D direction.

The FERMI-Elettra distributed real-time framework

International Nuclear Information System (INIS)

Pivetta, L.; Gaio, G.; Passuello, R.; Scalamera, G.

2012-01-01

FERMI-Elettra is a Free Electron Laser (FEL) based on a 1.5 GeV linac. The pulsed operation of the accelerator and the necessity to characterize and control each electron bunch requires synchronous acquisition of the beam diagnostics together with the ability to drive actuators in real-time at the linac repetition rate. The Adeos/Xenomai real-time extensions have been adopted in order to add real-time capabilities to the Linux based control system computers running the Tango software. A software communication protocol based on Gigabit Ethernet and known as Network Reflective Memory (NRM) has been developed to implement a shared memory across the whole control system, allowing computers to communicate in real-time. The NRM architecture, the real-time performance and the integration in the control system are described. (authors)

Real-time video quality monitoring

Science.gov (United States)

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

2011-12-01

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

Assessing reference genes for accurate transcript normalization using quantitative real-time PCR in pearl millet [Pennisetum glaucum (L. R. Br].

Directory of Open Access Journals (Sweden)

Prasenjit Saha

Full Text Available Pearl millet [Pennisetum glaucum (L. R.Br.], a close relative of Panicoideae food crops and bioenergy grasses, offers an ideal system to perform functional genomics studies related to C4 photosynthesis and abiotic stress tolerance. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR provides a sensitive platform to conduct such gene expression analyses. However, the lack of suitable internal control reference genes for accurate transcript normalization during qRT-PCR analysis in pearl millet is the major limitation. Here, we conducted a comprehensive assessment of 18 reference genes on 234 samples which included an array of different developmental tissues, hormone treatments and abiotic stress conditions from three genotypes to determine appropriate reference genes for accurate normalization of qRT-PCR data. Analyses of Ct values using Stability Index, BestKeeper, ΔCt, Normfinder, geNorm and RefFinder programs ranked PP2A, TIP41, UBC2, UBQ5 and ACT as the most reliable reference genes for accurate transcript normalization under different experimental conditions. Furthermore, we validated the specificity of these genes for precise quantification of relative gene expression and provided evidence that a combination of the best reference genes are required to obtain optimal expression patterns for both endogeneous genes as well as transgenes in pearl millet.

Design considerations for computationally constrained two-way real-time video communication

Science.gov (United States)

Bivolarski, Lazar M.; Saunders, Steven E.; Ralston, John D.

2009-08-01

Today's video codecs have evolved primarily to meet the requirements of the motion picture and broadcast industries, where high-complexity studio encoding can be utilized to create highly-compressed master copies that are then broadcast one-way for playback using less-expensive, lower-complexity consumer devices for decoding and playback. Related standards activities have largely ignored the computational complexity and bandwidth constraints of wireless or Internet based real-time video communications using devices such as cell phones or webcams. Telecommunications industry efforts to develop and standardize video codecs for applications such as video telephony and video conferencing have not yielded image size, quality, and frame-rate performance that match today's consumer expectations and market requirements for Internet and mobile video services. This paper reviews the constraints and the corresponding video codec requirements imposed by real-time, 2-way mobile video applications. Several promising elements of a new mobile video codec architecture are identified, and more comprehensive computational complexity metrics and video quality metrics are proposed in order to support the design, testing, and standardization of these new mobile video codecs.

A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus

Directory of Open Access Journals (Sweden)

Qin E-de

2010-06-01

Full Text Available Abstract A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009 influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same as that of each single-target RT-PCR for pandemic H1N1 and even more sensitive for H5N1 with the same primers and probes. No cross reactivity of detecting other subtype influenza viruses or respiratory tract viruses was observed. Two hundred and thirty-six clinical specimens were tested by comparing with single real-time RT-PCR and result from the duplex assay was 100% consistent with the results of single real-time RT-PCR and sequence analysis.

Rapid and accurate identification by real-time PCR of biotoxin-producing dinoflagellates from the family gymnodiniaceae.

Science.gov (United States)

Smith, Kirsty F; de Salas, Miguel; Adamson, Janet; Rhodes, Lesley L

2014-03-07

The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR) assays targeting the large subunit ribosomal RNA (LSU rRNA) gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.

Heterogeneous Embedded Real-Time Systems Environment

Science.gov (United States)

2003-12-01

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

Temporal Proof Methodologies for Real-Time Systems,

Science.gov (United States)

1990-09-01

real time systems that communicate either through shared variables or by message passing and real time issues such as time-outs, process priorities (interrupts) and process scheduling. The authors exhibit two styles for the specification of real - time systems . While the first approach uses bounded versions of temporal operators the second approach allows explicit references to time through a special clock variable. Corresponding to two styles of specification the authors present and compare two fundamentally different proof

Real-time communication protocols: an overview

NARCIS (Netherlands)

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

2003-01-01

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

Self-Organization in Embedded Real-Time Systems

CERN Document Server

Brinkschulte, Uwe; Rettberg, Achim

2013-01-01

This book describes the emerging field of self-organizing, multicore, distributed and real-time embedded systems.  Self-organization of both hardware and software can be a key technique to handle the growing complexity of modern computing systems. Distributed systems running hundreds of tasks on dozens of processors, each equipped with multiple cores, requires self-organization principles to ensure efficient and reliable operation. This book addresses various, so-called Self-X features such as self-configuration, self-optimization, self-adaptation, self-healing and self-protection. Presents open components for embedded real-time adaptive and self-organizing applications; Describes innovative techniques in: scheduling, memory management, quality of service, communications supporting organic real-time applications; Covers multi-/many-core embedded systems supporting real-time adaptive systems and power-aware, adaptive hardware and software systems; Includes case studies of open embedded real-time self-organizi...

A GIS-driven integrated real-time surveillance pilot system for national West Nile virus dead bird surveillance in Canada

Directory of Open Access Journals (Sweden)

Aramini Jeff

2006-04-01

Full Text Available Abstract Background An extensive West Nile virus surveillance program of dead birds, mosquitoes, horses, and human infection has been launched as a result of West Nile virus first being reported in Canada in 2001. Some desktop and web GIS have been applied to West Nile virus dead bird surveillance. There have been urgent needs for a comprehensive GIS services and real-time surveillance. Results A pilot system was developed to integrate real-time surveillance, real-time GIS, and Open GIS technology in order to enhance West Nile virus dead bird surveillance in Canada. Driven and linked by the newly developed real-time web GIS technology, this integrated real-time surveillance system includes conventional real-time web-based surveillance components, integrated real-time GIS components, and integrated Open GIS components. The pilot system identified the major GIS functions and capacities that may be important to public health surveillance. The six web GIS clients provide a wide range of GIS tools for public health surveillance. The pilot system has been serving Canadian national West Nile virus dead bird surveillance since 2005 and is adaptable to serve other disease surveillance. Conclusion This pilot system has streamlined, enriched and enhanced national West Nile virus dead bird surveillance in Canada, improved productivity, and reduced operation cost. Its real-time GIS technology, static map technology, WMS integration, and its integration with non-GIS real-time surveillance system made this pilot system unique in surveillance and public health GIS.

Detection of human papillomavirus by hybrid capture and real time PCR methods in patients with chronic cervicitis and cervical intraepithelial

Directory of Open Access Journals (Sweden)

Elisha Khandker

2016-07-01

Full Text Available Background and objectives:Cervical cancer due to Human papillomavirus (HPV is one of the leading causes of morbidity and mortality in women. Testing of HPV can identify women who are at risk of cervical cancer. Nowadays, molecular methods like real time polymerase chain reaction (PCR and hybrid capture technique are applied for detecting HPV in cervical specimens. The objective of the present study was to determine the rate of HPV infection in women with chronic cervicitis and cervical intraepithelial neoplasia (CIN by a commercial real time polymerase chain reaction test kit and by a hybrid capture HPV DNA test. Methods:Women aged between 20 to 55 years with chronic cervicitis and CIN were enrolled in the study after obtaining informed consent. Cervical specimen was collected by using cervical brush and stored in transport medium until used. HPV was detected by High Risk Screen Real-TM Quant 2x (Sacace, Biotechnologies SrI, Italy real time PCR kit (HR RT-PCR and by Hybrid Capture-2 High-Risk HPV DNA (Hc-2; Digene Corporation, USA test. Results: Total 72 women with chronic cervicitis and CIN of different grades were included in the study. Out of this, HPV infection detected by HR RT-PCR was 31 (43% and by Hc-2 was 14 (19.4%. Both the tests were able to detect HPV infection in all the CIN 3 cases and in most of the CIN 2 cases. However, HR RT-PCR detected higher number of HPV in chronic cervicitis and CIN1 cases. Conclusion:The study has shown that HR RT-PCR and Hc-2 tests are equally effective in detecting HPV infection in patients with CIN 2 and CIN 3 lesions. However, HR RT-PCR is more sensitive test for detecting HPV in chronic cervicitis and early CIN lesions and, therefore can be used in epidemiological study to detect presence of HPV in general population. IMC J Med Sci 2016; 10(2: 45-48

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.

Real-time specifications

DEFF Research Database (Denmark)

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

2015-01-01

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

Development of a real-time PCR assay for detection of planktonic red king crab (Paralithodes camtschaticus (Tilesius 1815)) larvae

Science.gov (United States)

Jensen, Pamela C.; Purcell, Maureen K.; Morado, J. Frank; Eckert, Ginny L.

2012-01-01

The Alaskan red king crab (Paralithodes camtschaticus) fishery was once one of the most economically important single-species fisheries in the world, but is currently depressed. This fishery would benefit from improved stock assessment capabilities. Larval crab distribution is patchy temporally and spatially, requiring extensive sampling efforts to locate and track larval dispersal. Large-scale plankton surveys are generally cost prohibitive because of the effort required for collection and the time and taxonomic expertise required to sort samples to identify plankton individually via light microscopy. Here, we report the development of primers and a dual-labeled probe for use in a DNA-based real-time polymerase chain reaction assay targeting the red king crab, mitochondrial gene cytochrome oxidase I for the detection of red king crab larvae DNA in plankton samples. The assay allows identification of plankton samples containing crab larvae DNA and provides an estimate of DNA copy number present in a sample without sorting the plankton sample visually. The assay was tested on DNA extracted from whole red king crab larvae and plankton samples seeded with whole larvae, and it detected DNA copies equivalent to 1/10,000th of a larva and 1 crab larva/5mL sieved plankton, respectively. The real-time polymerase chain reaction assay can be used to screen plankton samples for larvae in a fraction of the time required for traditional microscopial methods, which offers advantages for stock assessment methodologies for red king crab as well as a rapid and reliable method to assess abundance of red king crab larvae as needed to improve the understanding of life history and population processes, including larval population dynamics.

Bayesian estimation for quantification by real-time polymerase chain reaction under a branching process model of the DNA molecules amplification process

NARCIS (Netherlands)

Lalam, N.; Jacob, C.

2007-01-01

The aim of Quantitative Polymerase Chain Reaction is to determine the initial amount X0 of specific nucleic acids from an observed trajectory of the amplification process, the amplification being achieved through successive replication cycles. This process depends on the efficiency fpngn of

Expression of Leptin and Visfatin in Gingival Tissues of Chronic Periodontitis With and Without Type 2 Diabetes Mellitus: A Study Using Enzyme-Linked Immunosorbent Assay and Real-Time Polymerase Chain Reaction.

Science.gov (United States)

Ghallab, Noha A; Amr, Eman M; Shaker, Olfat G

2015-07-01

The aim of this study is to investigate the protein and gene expression of leptin and visfatin in gingival tissue from patients with chronic periodontitis (CP), patients with CP and type 2 diabetes mellitus (T2DM), and healthy individuals. The study includes 50 individuals: 10 healthy individuals, 20 patients with CP, and 20 patients with CP and T2DM. Plaque index, gingival index, probing depth, and clinical attachment loss were measured, and gingival biopsies were obtained. Leptin and visfatin protein expression in gingival tissues was determined using enzyme-linked immunosorbent assay, and messenger RNA (mRNA) expression was measured via real-time polymerase chain reaction. The highest leptin mRNA and protein expression was observed in the control group and was significantly (P ≤0.05) different from the CP and CP+T2DM groups. Gingival tissues from patients with CP and T2DM had a significant increase in visfatin and a decrease in leptin gene and protein expression (P <0.05) compared with both controls and patients with CP. Expression of leptin and visfatin in the gingival tissues suggests a possible role for these adipokines in the pathogenesis of CP and T2DM.

Hard Real-Time Task Scheduling in Cloud Computing Using an Adaptive Genetic Algorithm

Directory of Open Access Journals (Sweden)

Amjad Mahmood

2017-04-01

Full Text Available In the Infrastructure-as-a-Service cloud computing model, virtualized computing resources in the form of virtual machines are provided over the Internet. A user can rent an arbitrary number of computing resources to meet their requirements, making cloud computing an attractive choice for executing real-time tasks. Economical task allocation and scheduling on a set of leased virtual machines is an important problem in the cloud computing environment. This paper proposes a greedy and a genetic algorithm with an adaptive selection of suitable crossover and mutation operations (named as AGA to allocate and schedule real-time tasks with precedence constraint on heterogamous virtual machines. A comprehensive simulation study has been done to evaluate the performance of the proposed algorithms in terms of their solution quality and efficiency. The simulation results show that AGA outperforms the greedy algorithm and non-adaptive genetic algorithm in terms of solution quality.

Effectiveness of real-time polymerase chain reaction assay for the detection of Mycobacterium tuberculosis in pathological samples: a systematic review and meta-analysis.

Science.gov (United States)

Babafemi, Emmanuel O; Cherian, Benny P; Banting, Lee; Mills, Graham A; Ngianga, Kandala

2017-10-25

Rapid and accurate diagnosis of tuberculosis (TB) is key to manage the disease and to control and prevent its transmission. Many established diagnostic methods suffer from low sensitivity or delay of timely results and are inadequate for rapid detection of Mycobacterium tuberculosis (MTB) in pulmonary and extra-pulmonary clinical samples. This study examined whether a real-time polymerase chain reaction (RT-PCR) assay, with a turn-a-round time of 2 h, would prove effective for routine detection of MTB by clinical microbiology laboratories. A systematic literature search was performed for publications in any language on the detection of MTB in pathological samples by RT-PCR assay. The following sources were used MEDLINE via PubMed, EMBASE, BIOSIS Citation Index, Web of Science, SCOPUS, ISI Web of Knowledge and Cochrane Infectious Diseases Group Specialised Register, grey literature, World Health Organization and Centres for Disease Control and Prevention websites. Forty-six studies met set inclusion criteria. Generated pooled summary estimates (95% CIs) were calculated for overall accuracy and bivariate meta-regression model was used for meta-analysis. Summary estimates for pulmonary TB (31 studies) were as follows: sensitivity 0.82 (95% CI 0.81-0.83), specificity 0.99 (95% CI 0.99-0.99), positive likelihood ratio 43.00 (28.23-64.81), negative likelihood ratio 0.16 (0.12-0.20), diagnostic odds ratio 324.26 (95% CI 189.08-556.09) and area under curve 0.99. Summary estimates for extra-pulmonary TB (25 studies) were as follows: sensitivity 0.70 (95% CI 0.67-0.72), specificity 0.99 (95% CI 0.99-0.99), positive likelihood ratio 29.82 (17.86-49.78), negative likelihood ratio 0.33 (0.26-0.42), diagnostic odds ratio 125.20 (95% CI 65.75-238.36) and area under curve 0.96. RT-PCR assay demonstrated a high degree of sensitivity for pulmonary TB and good sensitivity for extra-pulmonary TB. It indicated a high degree of specificity for ruling in TB infection from sampling

Fluorescence probes for real-time remote cyanobacteria monitoring: A review of challenges and opportunities.

Science.gov (United States)

Bertone, Edoardo; Burford, Michele A; Hamilton, David P

2018-05-10

In recent years, there has been a widespread deployment of submersible fluorescence sensors by water utilities. They are used to measure diagnostic pigments and estimate algae and cyanobacteria abundance in near real-time. Despite being useful and promising tools, operators and decision-makers often rely on the data provided by these probes without a full understanding of their limitations. As a result, this may lead to wrong and misleading estimations which, in turn, means that researchers and technicians distrust these sensors. In this review paper, we list and discuss the main limitations of such probes, as well as identifying the effect of environmental factors on pigment production, and in turn, the conversion to cyanobacteria abundance estimation. We argue that a comprehensive calibration approach to obtain reliable readings goes well beyond manufacturers' recommendations, and should involve several context-specific experiments. We also believe that if such a comprehensive set of experiments is conducted, the data collected from fluorescence sensors could be used in artificial intelligence modelling approaches to reliably predict, in near real-time, the presence and abundance of different cyanobacteria species. This would have significant benefits for both drinking and recreational water management, given that cyanobacterial toxicity, and taste and odour compounds production, are species-dependent. Copyright © 2018 Elsevier Ltd. All rights reserved.

On Real-Time Systems Using Local Area Networks.

Science.gov (United States)

1987-07-01

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

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

Science.gov (United States)

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

2015-04-15

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

Mixed-mode Operating System for Real-time Performance

OpenAIRE

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

2017-01-01

The purpose of the mixed-mode system research is to handle devices with the accuracy of real-time systems and at the same time, having all the benefits and facilities of a matured Graphic User Interface (GUI) operating system which is typically nonreal-time. This mixed-mode operating system comprising of a real-time portion and a non-real-time portion was studied and implemented to identify the feasibilities and performances in practical applications (in the context of scheduled the real-time...

Linux real-time framework for fusion devices

Energy Technology Data Exchange (ETDEWEB)

Neto, Andre [Associacao Euratom-IST, Instituto de Plasmas e Fusao Nuclear, Av. Rovisco Pais, 1049-001 Lisboa (Portugal)], E-mail: andre.neto@cfn.ist.utl.pt; Sartori, Filippo; Piccolo, Fabio [Euratom-UKAEA, Culham Science Centre, Abingdon, Oxon OX14 3DB (United Kingdom); Barbalace, Antonio [Euratom-ENEA Association, Consorzio RFX, 35127 Padova (Italy); Vitelli, Riccardo [Dipartimento di Informatica, Sistemi e Produzione, Universita di Roma, Tor Vergata, Via del Politecnico 1-00133, Roma (Italy); Fernandes, Horacio [Associacao Euratom-IST, Instituto de Plasmas e Fusao Nuclear, Av. Rovisco Pais, 1049-001 Lisboa (Portugal)

2009-06-15

A new framework for the development and execution of real-time codes is currently being developed and commissioned at JET. The foundations of the system are Linux, the Real Time Application Interface (RTAI) and a wise exploitation of the new i386 multi-core processors technology. The driving motivation was the need to find a real-time operating system for the i386 platform able to satisfy JET Vertical Stabilisation Enhancement project requirements: 50 {mu}s cycle time. Even if the initial choice was the VxWorks operating system, it was decided to explore an open source alternative, mostly because of the costs involved in the commercial product. The work started with the definition of a precise set of requirements and milestones to achieve: Linux distribution and kernel versions to be used for the real-time operating system; complete characterization of the Linux/RTAI real-time capabilities; exploitation of the multi-core technology; implementation of all the required and missing features; commissioning of the system. Latency and jitter measurements were compared for Linux and RTAI in both user and kernel-space. The best results were attained using the RTAI kernel solution where the time to reschedule a real-time task after an external interrupt is of 2.35 {+-} 0.35 {mu}s. In order to run the real-time codes in the kernel-space, a solution to provide user-space functionalities to the kernel modules had to be designed. This novel work provided the most common functions from the standard C library and transparent interaction with files and sockets to the kernel real-time modules. Kernel C++ support was also tested, further developed and integrated in the framework. The work has produced very convincing results so far: complete isolation of the processors assigned to real-time from the Linux non real-time activities, high level of stability over several days of benchmarking operations and values well below 3 {mu}s for task rescheduling after external interrupt. From

Linux real-time framework for fusion devices

International Nuclear Information System (INIS)

Neto, Andre; Sartori, Filippo; Piccolo, Fabio; Barbalace, Antonio; Vitelli, Riccardo; Fernandes, Horacio

2009-01-01

A new framework for the development and execution of real-time codes is currently being developed and commissioned at JET. The foundations of the system are Linux, the Real Time Application Interface (RTAI) and a wise exploitation of the new i386 multi-core processors technology. The driving motivation was the need to find a real-time operating system for the i386 platform able to satisfy JET Vertical Stabilisation Enhancement project requirements: 50 μs cycle time. Even if the initial choice was the VxWorks operating system, it was decided to explore an open source alternative, mostly because of the costs involved in the commercial product. The work started with the definition of a precise set of requirements and milestones to achieve: Linux distribution and kernel versions to be used for the real-time operating system; complete characterization of the Linux/RTAI real-time capabilities; exploitation of the multi-core technology; implementation of all the required and missing features; commissioning of the system. Latency and jitter measurements were compared for Linux and RTAI in both user and kernel-space. The best results were attained using the RTAI kernel solution where the time to reschedule a real-time task after an external interrupt is of 2.35 ± 0.35 μs. In order to run the real-time codes in the kernel-space, a solution to provide user-space functionalities to the kernel modules had to be designed. This novel work provided the most common functions from the standard C library and transparent interaction with files and sockets to the kernel real-time modules. Kernel C++ support was also tested, further developed and integrated in the framework. The work has produced very convincing results so far: complete isolation of the processors assigned to real-time from the Linux non real-time activities, high level of stability over several days of benchmarking operations and values well below 3 μs for task rescheduling after external interrupt. From being the

Static Schedulers for Embedded Real-Time Systems

Science.gov (United States)

1989-12-01

Because of the need for having efficient scheduling algorithms in large scale real time systems , software engineers put a lot of effort on developing...provide static schedulers for he Embedded Real Time Systems with single processor using Ada programming language. The independent nonpreemptable...support the Computer Aided Rapid Prototyping for Embedded Real Time Systems so that we determine whether the system, as designed, meets the required

Real-time motional Stark effect in jet

International Nuclear Information System (INIS)

Alves, D.; Stephen, A.; Hawkes, N.; Dalley, S.; Goodyear, A.; Felton, R.; Joffrin, E.; Fernandes, H.

2004-01-01

The increasing importance of real-time measurements and control systems in JET experiments, regarding e.g. Internal Transport Barrier (ITB) and q-profile control, has motivated the development of a real-time motional Stark effect (MSE) system. The MSE diagnostic allows the measurement of local magnetic fields in different locations along the neutral beam path providing, therefore, local measurement of the current and q-profiles. Recently in JET, an upgrade of the MSE diagnostic has been implemented, incorporating a totally new system which allows the use of this diagnostic as a real-time control tool as well as an extended data source for off-line analysis. This paper will briefly describe the technical features of the real-time diagnostic with main focus on the system architecture, which consists of a VME crate hosting three PowerPC processor boards and a fast ADC, all connected via Front Panel Data Port (FPDP). The DSP algorithm implements a lockin-amplifier required to demodulate the JET MSE signals. Some applications for the system will be covered such as: feeding the real-time equilibrium reconstruction code (EQUINOX) and allowing the full coverage analysis of the Neutral Beam time window. A brief comparison between the real-time MSE analysis and the off-line analysis will also be presented

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....

Frequency of Pathogenic Paediatric Bacterial Meningitis in Mozambique: The Critical Role of Multiplex Real-Time Polymerase Chain Reaction to Estimate the Burden of Disease.

Science.gov (United States)

Nhantumbo, Aquino Albino; Cantarelli, Vlademir Vicente; Caireão, Juliana; Munguambe, Alcides Moniz; Comé, Charlotte Elizabeth; Pinto, Gabriela do Carmo; Zimba, Tomás Francisco; Mandomando, Inácio; Semá, Cynthia Baltazar; Dias, Cícero; Moraes, Milton Ozório; Gudo, Eduardo Samo

2015-01-01

In Sub-Saharan Africa, including Mozambique, acute bacterial meningitis (ABM) represents a main cause of childhood mortality. The burden of ABM is seriously underestimated because of the poor performance of culture sampling, the primary method of ABM surveillance in the region. Low quality cerebrospinal fluid (CSF) samples and frequent consumption of antibiotics prior to sample collection lead to a high rate of false-negative results. To our knowledge, this study is the first to determine the frequency of ABM in Mozambique using real-time polymerase chain reaction (qPCR) and to compare results to those of culture sampling. Between March 2013 and March 2014, CSF samples were collected at 3 regional hospitals from patients under 5 years of age, who met World Health Organization case definition criteria for ABM. Macroscopic examination, cytochemical study, culture, and qPCR were performed on all samples. A total of 369 CSF samples were collected from children clinically suspected of ABM. qPCR showed a significantly higher detection rate of ABM-causing pathogens when compared to culture (52.3% [193/369] versus 7.3% [27/369], p = 0.000). The frequency of Streptococcus pneumoniae, Haemophilus influenzae, group B Streptococci, and Neisseria meningitidis were 32.8% (121⁄369), 12.2%, (45⁄369), 3.0% (16⁄369) and 4.3% (11⁄369), respectively, significantly higher compared to that obtained on culture (p < 0.001 for each). Our findings demonstrate that culture is less effective for the diagnosis of ABM than qPCR. The common use of culture rather than qPCR to identify ABM results in serious underestimation of the burden of the disease, and our findings strongly suggest that qPCR should be incorporated into surveillance activities for ABM. In addition, our data showed that S. pneumoniae represents the most common cause of ABM in children under 5 years of age.

Towards exascale real-time RFI mitigation

NARCIS (Netherlands)

van Nieuwpoort, R.V.

2016-01-01

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

Time-Optimal Real-Time Test Case Generation using UPPAAL

DEFF Research Database (Denmark)

Hessel, Anders; Larsen, Kim Guldstrand; Nielsen, Brian

2004-01-01

Testing is the primary software validation technique used by industry today, but remains ad hoc, error prone, and very expensive. A promising improvement is to automatically generate test cases from formal models of the system under test. We demonstrate how to automatically generate real...... test purposes or generated automatically from various coverage criteria of the model.......-time conformance test cases from timed automata specifications. Specifically we demonstrate how to fficiently generate real-time test cases with optimal execution time i.e test cases that are the fastest possible to execute. Our technique allows time optimal test cases to be generated using manually formulated...

Real-time video streaming in mobile cloud over heterogeneous wireless networks

Science.gov (United States)

Abdallah-Saleh, Saleh; Wang, Qi; Grecos, Christos

2012-06-01

are captured for analytical purposes on the mobile user node. Experimental results are obtained and analysed. Future work is identified towards further improvement of the current design and implementation. With this new mobile video networking concept and paradigm implemented and evaluated, results and observations obtained from this study would form the basis of a more in-depth, comprehensive understanding of various challenges and opportunities in supporting high-quality real-time video streaming in mobile cloud over heterogeneous wireless networks.

Performance evaluation of near-real-time accounting systems

International Nuclear Information System (INIS)

Anon.

1981-01-01

Examples are given illustrating the application of near-real-time accounting concepts and principles to actual nuclear facilities. Experience with prototypical systems at the AGNS reprocessing plant and the Los Alamos plutonium facility is described using examples of actual data to illustrate the performance and effectiveness of near-real-time systems. The purpose of the session is to enable participants to: (1) identify the major components of near-real-time accounting systems; (2) describe qualitatively the advantages, limitations, and performance of such systems in real nuclear facilities; (3) identify process and facility design characteristics that affect the performance of near-real-time systems; and (4) describe qualitatively the steps necessary to implement a near-real-time accounting and control system in a nuclear facility

HRT-UML: a design method for hard real-time systems based on the UML notation

Science.gov (United States)

D'Alessandro, Massimo; Mazzini, Silvia; di Natale, Marco; Lipari, Giuseppe

2002-07-01

The Hard Real-Time-Unified Modelling Language (HRT-UML) method aims at providing a comprehensive solution to the modeling of Hard Real Time systems. The experience shows that the design of Hard Real-Time systems needs methodologies suitable for the modeling and analysis of aspects related to time, schedulability and performance. In the context of the European Aerospace community a reference method for design is Hierarchical Object Oriented Design (HOOD) and in particular its extension for the modeling of hard real time systems, Hard Real-Time-Hierarchical Object Oriented Design (HRT-HOOD), recommended by the European Space Agency (ESA) for the development of on-board systems. On the other hand in recent years the Unified Modelling Language (UML) has been gaining a very large acceptance in a wide range of domains, all over the world, becoming a de-facto international standard. Tool vendors are very active in this potentially big market. In the Aerospace domain the common opinion is that UML, as a general notation, is not suitable for Hard Real Time systems, even if its importance is recognized as a standard and as a technological trend in the near future. These considerations suggest the possibility of replacing the HRT-HOOD method with a customized version of UML, that incorporates the advantages of both standards and complements the weak points. This approach has the clear advantage of making HRT-HOOD converge on a more powerful and expressive modeling notation. The paper identifies a mapping of the HRT-HOOD semantics into the UML one, and proposes a UML extension profile, that we call HRT-UML, based on the UML standard extension mechanisms, to fully represent HRT-HOOD design concepts. Finally it discusses the relationships between our profile and the UML profile for schedulability, performance and time, adopted by OMG in November 2001.

General misincorporation frequency: Re-evaluation of the fidelity of DNA polymerases.

Science.gov (United States)

Yang, Jie; Li, Bianbian; Liu, Xiaoying; Tang, Hong; Zhuang, Xiyao; Yang, Mingqi; Xu, Ying; Zhang, Huidong; Yang, Chun

2018-02-19

DNA replication in cells is performed in the presence of four dNTPs and four rNTPs. In this study, we re-evaluated the fidelity of DNA polymerases using the general misincorporation frequency consisting of three incorrect dNTPs and four rNTPs but not using the traditional special misincorporation frequency with only the three incorrect dNTPs. We analyzed both the general and special misincorporation frequencies of nucleotide incorporation opposite dG, rG, or 8-oxoG by Pseudomonas aeruginosa phage 1 (PaP1) DNA polymerase Gp90 or Sulfolobus solfataricus DNA polymerase Dpo4. Both misincorporation frequencies of other DNA polymerases published were also summarized and analyzed. The general misincorporation frequency is obviously higher than the special misincorporation frequency for many DNA polymerases, indicating the real fidelity of a DNA polymerase should be evaluated using the general misincorporation frequency. Copyright © 2018 Elsevier Inc. All rights reserved.

Distributed Issues for Ada Real-Time Systems

Science.gov (United States)

1990-07-23

NUMBERS Distributed Issues for Ada Real - Time Systems MDA 903-87- C- 0056 S. AUTHOR(S) Thomas E. Griest 7. PERFORMING ORGANiZATION NAME(S) AND ADORESS(ES) 8...considerations. I Adding to the problem of distributed real - time systems is the issue of maintaining a common sense of time among all of the processors...because -omeone is waiting for the final output of a very large set of computations. However in real - time systems , consistent meeting of short-term

Design Specifications for Adaptive Real-Time Systems

Science.gov (United States)

1991-12-01

TICfl \\ E CT E Design Specifications for JAN’\\ 1992 Adaptive Real - Time Systems fl Randall W. Lichota U, Alice H. Muntz - December 1991 \\ \\\\/ 0 / r...268-2056 Technical Report CMU/SEI-91-TR-20 ESD-91-TR-20 December 1991 Design Specifications for Adaptive Real - Time Systems Randall W. Lichota Hughes...Design Specifications for Adaptive Real - Time Systems Abstract: The design specification method described in this report treats a software

Design Recovery Technology for Real-Time Systems.

Science.gov (United States)

1995-10-01

RL-TR-95-208 Final Technical Report October 1995 DESIGN RECOVERY TECHNOLOGY FOR REAL TIME SYSTEMS The MITRE Corporation Lester J. Holtzblatt...92 - Jan 95 4. TTTLE AND SUBTITLE DESIGN RECOVERY TECHNOLOGY FOR REAL - TIME SYSTEMS 6. AUTHOR(S) Lester J. Holtzblatt, Richard Piazza, and Susan...behavior of real - time systems in general, our initial efforts have centered on recovering this information from one system in particular, the Modular

[Real time 3D echocardiography

Science.gov (United States)

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

2001-01-01

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

Real-time communication for distributed plasma control systems

Energy Technology Data Exchange (ETDEWEB)

Luchetta, A. [Consorzio RFX, Associazione Euratom-ENEA sulla Fusione, Corso Stati Uniti 4, Padova 35127 (Italy)], E-mail: adriano.luchetta@igi.cnr.it; Barbalace, A.; Manduchi, G.; Soppelsa, A.; Taliercio, C. [Consorzio RFX, Associazione Euratom-ENEA sulla Fusione, Corso Stati Uniti 4, Padova 35127 (Italy)

2008-04-15

Real-time control applications will benefit in the near future from the enhanced performance provided by multi-core processor architectures. Nevertheless real-time communication will continue to be critical in distributed plasma control systems where the plant under control typically is distributed over a wide area. At RFX-mod real-time communication is crucial for hard real-time plasma control, due to the distributed architecture of the system, which consists of several VMEbus stations. The system runs under VxWorks and uses Gigabit Ethernet for sub-millisecond real-time communication. To optimize communication in the system, a set of detailed measurements has been carried out on the target platforms (Motorola MVME5100 and MVME5500) using either the VxWorks User Datagram Protocol (UDP) stack or raw communication based on the data link layer. Measurements have been carried out also under Linux, using its UDP stack or, in alternative, RTnet, an open source hard real-time network protocol stack. RTnet runs under Xenomai or RTAI, two popular real-time extensions based on the Linux kernel. The paper reports on the measurements carried out and compares the results, showing that the performance obtained by using open source code is suitable for sub-millisecond real-time communication in plasma control.

Flood and Weather Monitoring Using Real-time Twitter Data Streams

Science.gov (United States)

Demir, I.; Sit, M. A.; Sermet, M. Y.

2016-12-01

Social media data is a widely used source to making inference within public crisis periods and events in disaster times. Specifically, since Twitter provides large-scale data publicly in real-time, it is one of the most extensive resources with location information. This abstract provides an overview of a real-time Twitter analysis system to support flood preparedness and response using a comprehensive information-centric flood ontology and natural language processing. Within the scope of this project, we deal with acquisition and processing of real-time Twitter data streams. System fetches the tweets with specified keywords and classifies them as related to flooding or heavy weather conditions. The system uses machine learning algorithms to discover patterns using the correlation between tweets and Iowa Flood Information System's (IFIS) extensive resources. The system uses these patterns to forecast the formation and progress of a potential future flood event. While fetching tweets, predefined hashtags are used for filtering and enhancing the relevancy for selected tweets. With this project, tweets can also be used as an alternative data source where other data sources are not sufficient for specific tasks. During the disasters, the photos that people upload alongside their tweets can be collected and placed to appropriate locations on a mapping system. This allows decision making authorities and communities to see the most recent outlook of the disaster interactively. In case of an emergency, concentration of tweets can help the authorities to determine a strategy on how to reach people most efficiently while providing them the supplies they need. Thanks to the extendable nature of the flood ontology and framework, results from this project will be a guide for other natural disasters, and will be shared with the community.

Software Development Technologies for Reactive, Real-Time, and Hybrid Systems

Science.gov (United States)

Manna, Zohar

1996-01-01

The research is directed towards the design and implementation of a comprehensive deductive environment for the development of high-assurance systems, especially reactive (concurrent, real-time, and hybrid) systems. Reactive systems maintain an ongoing interaction with their environment, and are among the most difficult to design and verify. The project aims to provide engineers with a wide variety of tools within a single, general, formal framework in which the tools will be most effective. The entire development process is considered, including the construction, transformation, validation, verification, debugging, and maintenance of computer systems. The goal is to automate the process as much as possible and reduce the errors that pervade hardware and software development.

Real-time quasi-3D tomographic reconstruction

Science.gov (United States)

Buurlage, Jan-Willem; Kohr, Holger; Palenstijn, Willem Jan; Joost Batenburg, K.

2018-06-01

Developments in acquisition technology and a growing need for time-resolved experiments pose great computational challenges in tomography. In addition, access to reconstructions in real time is a highly demanded feature but has so far been out of reach. We show that by exploiting the mathematical properties of filtered backprojection-type methods, having access to real-time reconstructions of arbitrarily oriented slices becomes feasible. Furthermore, we present , software for visualization and on-demand reconstruction of slices. A user of can interactively shift and rotate slices in a GUI, while the software updates the slice in real time. For certain use cases, the possibility to study arbitrarily oriented slices in real time directly from the measured data provides sufficient visual and quantitative insight. Two such applications are discussed in this article.

Alpha: A real-time decentralized operating system for mission-oriented system integration and operation

Science.gov (United States)

Jensen, E. Douglas

1988-01-01

Alpha is a new kind of operating system that is unique in two highly significant ways. First, it is decentralized transparently providing reliable resource management across physically dispersed nodes, so that distributed applications programming can be done largely as though it were centralized. And second, it provides comprehensive, high technology support for real-time system integration and operation, an application area which consists predominately of aperiodic activities having critical time constraints such as deadlines. Alpha is extremely adaptable so that it can be easily optimized for a wide range of problem-specific functionality, performance, and cost. Alpha is the first systems effort of the Archons Project, and the prototype was created at Carnegie-Mellon University directly on modified Sun multiprocessor workstation hardware. It has been demonstrated with a real-time C(sup 2) application. Continuing research is leading to a series of enhanced follow-ons to Alpha; these are portable but initially hosted on Concurrent's MASSCOMP line of multiprocessor products.

An algorithm for learning real-time automata

NARCIS (Netherlands)

Verwer, S.E.; De Weerdt, M.M.; Witteveen, C.

2007-01-01

We describe an algorithm for learning simple timed automata, known as real-time automata. The transitions of real-time automata can have a temporal constraint on the time of occurrence of the current symbol relative to the previous symbol. The learning algorithm is similar to the redblue fringe

De toekomst van Real Time Intelligence

NARCIS (Netherlands)

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

2013-01-01

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

Real-Time Parameter Identification

Data.gov (United States)

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

Real time process algebra with time-dependent conditions

NARCIS (Netherlands)

Baeten, J.C.M.; Middelburg, C.A.

We extend the main real time version of ACP presented in [6] with conditionals in which the condition depends on time. This extension facilitates flexible dependence of proccess behaviour on initialization time. We show that the conditions concerned generalize the conditions introduced earlier

Application of a molecular beacon based real-time isothermal amplification (MBRTIA) technology for simultaneous detection of Bacillus cereus and Staphylococcus aureus.

Science.gov (United States)

Mandappa, I M; Joglekar, Prasanna; Manonmani, H K

2015-07-01

A multiplex real-time isothermal amplification assay was developed using molecular beacons for the detection of Bacillus cereus and Staphylococcus aureus by targeting four important virulence genes. A correlation between targeting highly accessible DNA sequences and isothermal amplification based molecular beacon efficiency and sensitivity was demonstrated using phi(Φ)29 DNA polymerase at a constant isothermal temperature of 30 °C. It was very selective and consistently detected down to 10(1) copies of DNA. The specificity and sensitivity of this assay, when tested with pure culture were high, surpassing those of currently used PCR assays for the detection of these organisms. The molecular beacon based real-time isothermal amplification (MBRTIA) assay could be carried out entirely in 96 well plates or well strips, enabling a rapid and high-throughput detection of food borne pathogens.

Compiling models into real-time systems

International Nuclear Information System (INIS)

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

1992-08-01

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

Compiling models into real-time systems

International Nuclear Information System (INIS)

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

1992-08-01

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

Comparative study of internet cloud and cloudlet over wireless mesh networks for real-time applications

Science.gov (United States)

Khan, Kashif A.; Wang, Qi; Luo, Chunbo; Wang, Xinheng; Grecos, Christos

2014-05-01

Mobile cloud computing is receiving world-wide momentum for ubiquitous on-demand cloud services for mobile users provided by Amazon, Google etc. with low capital cost. However, Internet-centric clouds introduce wide area network (WAN) delays that are often intolerable for real-time applications such as video streaming. One promising approach to addressing this challenge is to deploy decentralized mini-cloud facility known as cloudlets to enable localized cloud services. When supported by local wireless connectivity, a wireless cloudlet is expected to offer low cost and high performance cloud services for the users. In this work, we implement a realistic framework that comprises both a popular Internet cloud (Amazon Cloud) and a real-world cloudlet (based on Ubuntu Enterprise Cloud (UEC)) for mobile cloud users in a wireless mesh network. We focus on real-time video streaming over the HTTP standard and implement a typical application. We further perform a comprehensive comparative analysis and empirical evaluation of the application's performance when it is delivered over the Internet cloud and the cloudlet respectively. The study quantifies the influence of the two different cloud networking architectures on supporting real-time video streaming. We also enable movement of the users in the wireless mesh network and investigate the effect of user's mobility on mobile cloud computing over the cloudlet and Amazon cloud respectively. Our experimental results demonstrate the advantages of the cloudlet paradigm over its Internet cloud counterpart in supporting the quality of service of real-time applications.

Real Time Linux - The RTOS for Astronomy?

Science.gov (United States)

Daly, P. N.

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

The real-time price elasticity of electricity

International Nuclear Information System (INIS)

Lijesen, Mark G.

2007-01-01

The real-time price elasticity of electricity contains important information on the demand response of consumers to the volatility of peak prices. Despite the importance, empirical estimates of the real-time elasticity are hardly available. This paper provides a quantification of the real-time relationship between total peak demand and spot market prices. We find a low value for the real-time price elasticity, which may partly be explained from the fact that not all users observe the spot market price. If we correct for this phenomenon, we find the elasticity to be fairly low for consumers currently active in the spot market. If this conclusion applies to all users, this would imply a limited scope for government intervention in supply security issues. (Author)

Real-time 3-D SAFT-UT system evaluation and validation

International Nuclear Information System (INIS)

Doctor, S.R.; Schuster, G.J.; Reid, L.D.; Hall, T.E.

1996-09-01

SAFT-UT technology is shown to provide significant enhancements to the inspection of materials used in US nuclear power plants. This report provides guidelines for the implementation of SAFT-UT technology and shows the results from its application. An overview of the development of SAFT-UT is provided so that the reader may become familiar with the technology. Then the basic fundamentals are presented with an extensive list of references. A comprehensive operating procedure, which is used in conjunction with the SAFT-UT field system developed by Pacific Northwest Laboratory (PNL), provides the recipe for both SAFT data acquisition and analysis. The specification for the hardware implementation is provided for the SAFT-UT system along with a description of the subsequent developments and improvements. One development of technical interest is the SAFT real time processor. Performance of the real-time processor is impressive and comparison is made of this dedicated parallel processor to a conventional computer and to the newer high-speed computer architectures designed for image processing. Descriptions of other improvements, including a robotic scanner, are provided. Laboratory parametric and application studies, performed by PNL and not previously reported, are discussed followed by a section on field application work in which SAFT was used during inservice inspections of operating reactors

Real-time 3-D SAFT-UT system evaluation and validation

Energy Technology Data Exchange (ETDEWEB)

Doctor, S.R.; Schuster, G.J.; Reid, L.D.; Hall, T.E. [Pacific Northwest National Lab., Richland, WA (United States)

1996-09-01

SAFT-UT technology is shown to provide significant enhancements to the inspection of materials used in US nuclear power plants. This report provides guidelines for the implementation of SAFT-UT technology and shows the results from its application. An overview of the development of SAFT-UT is provided so that the reader may become familiar with the technology. Then the basic fundamentals are presented with an extensive list of references. A comprehensive operating procedure, which is used in conjunction with the SAFT-UT field system developed by Pacific Northwest Laboratory (PNL), provides the recipe for both SAFT data acquisition and analysis. The specification for the hardware implementation is provided for the SAFT-UT system along with a description of the subsequent developments and improvements. One development of technical interest is the SAFT real time processor. Performance of the real-time processor is impressive and comparison is made of this dedicated parallel processor to a conventional computer and to the newer high-speed computer architectures designed for image processing. Descriptions of other improvements, including a robotic scanner, are provided. Laboratory parametric and application studies, performed by PNL and not previously reported, are discussed followed by a section on field application work in which SAFT was used during inservice inspections of operating reactors.

A Programmable Microkernel for Real-Time Systems

Science.gov (United States)

2003-06-01

A Programmable Microkernel for Real - Time Systems Christoph M. Kirsch Thomas A. Henzinger Marco A.A. Sanvido Report No. UCB/CSD-3-1250 June 2003...TITLE AND SUBTITLE A Programmable Microkernel for Real - Time Systems 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...THIS PAGE unclassified Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 A Programmable Microkernel for Real - Time Systems ∗ Christoph M

Real-time object-oriented programming: studies and proposals

International Nuclear Information System (INIS)

Fouquier, Gilles

1996-01-01

This thesis contributes to the introduction of real-time features in object-oriented programming. Object-oriented programming favours modularity and reusability. Therefore, its application to real-time introduces many theoretical and conceptual problems. To deal with these problems, a new real-time object-oriented programming model is presented. This model is based on the active object model which allows concurrence and maintains the encapsulation property. The real-time aspect is treated by replacing the concept of task by the concept of method processing and by associating a real-time constraint to each message (priority or deadline). The set of all the running methods is scheduled. This model, called ATOME, contains several sub-models to deal with the usual concurrence control integrating their priority and deadline processing. The classical HPF and EDF scheduling avoid priority or deadline inversion. This model and its variants are new proposals to program real-time applications in the object-oriented way, therefore easing reusability and code writing. The feasibility of this approach is demonstrated by extending and existing active object-based language to real-time, in using the rules defined in the ATOME model. (author) [fr

Real-time simulation of large-scale floods

Science.gov (United States)

Liu, Q.; Qin, Y.; Li, G. D.; Liu, Z.; Cheng, D. J.; Zhao, Y. H.

2016-08-01

According to the complex real-time water situation, the real-time simulation of large-scale floods is very important for flood prevention practice. Model robustness and running efficiency are two critical factors in successful real-time flood simulation. This paper proposed a robust, two-dimensional, shallow water model based on the unstructured Godunov- type finite volume method. A robust wet/dry front method is used to enhance the numerical stability. An adaptive method is proposed to improve the running efficiency. The proposed model is used for large-scale flood simulation on real topography. Results compared to those of MIKE21 show the strong performance of the proposed model.

Variants of sequence family B Thermococcus kodakaraensis DNA polymerase with increased mismatch extension selectivity.

Directory of Open Access Journals (Sweden)

Claudia Huber

Full Text Available Fidelity and selectivity of DNA polymerases are critical determinants for the biology of life, as well as important tools for biotechnological applications. DNA polymerases catalyze the formation of DNA strands by adding deoxynucleotides to a primer, which is complementarily bound to a template. To ensure the integrity of the genome, DNA polymerases select the correct nucleotide and further extend the nascent DNA strand. Thus, DNA polymerase fidelity is pivotal for ensuring that cells can replicate their genome with minimal error. DNA polymerases are, however, further optimized for more specific biotechnological or diagnostic applications. Here we report on the semi-rational design of mutant libraries derived by saturation mutagenesis at single sites of a 3'-5'-exonuclease deficient variant of Thermococcus kodakaraensis DNA polymerase (KOD pol and the discovery for variants with enhanced mismatch extension selectivity by screening. Sites of potential interest for saturation mutagenesis were selected by their proximity to primer or template strands. The resulting libraries were screened via quantitative real-time PCR. We identified three variants with single amino acid exchanges-R501C, R606Q, and R606W-which exhibited increased mismatch extension selectivity. These variants were further characterized towards their potential in mismatch discrimination. Additionally, the identified enzymes were also able to differentiate between cytosine and 5-methylcytosine. Our results demonstrate the potential in characterizing and developing DNA polymerases for specific PCR based applications in DNA biotechnology and diagnostics.

Mechatronic modeling of real-time wheel-rail contact

CERN Document Server

Bosso, Nicola; Gugliotta, Antonio; Somà, Aurelio

2013-01-01

Real-time simulations of the behaviour of a rail vehicle require realistic solutions of the wheel-rail contact problem which can work in a real-time mode. Examples of such solutions for the online mode have been well known and are implemented within standard and commercial tools for the simulation codes for rail vehicle dynamics. This book is the result of the research activities carried out by the Railway Technology Lab of the Department of Mechanical and Aerospace Engineering at Politecnico di Torino. This book presents work on the project for the development of a real-time wheel-rail contact model and provides the simulation results obtained with dSpace real-time hardware. Besides this, the implementation of the contact model for the development of a real-time model for the complex mechatronic system of a scaled test rig is presented in this book and may be useful for the further validation of the real-time contact model with experiments on a full scale test rig.

Internet-accessible real-time weather information system

Digital Repository Service at National Institute of Oceanography (India)

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

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

Automated real-time software development

Science.gov (United States)

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

1993-01-01

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

Geomagnetic Observatory Data for Real-Time Applications

Science.gov (United States)

Love, J. J.; Finn, C. A.; Rigler, E. J.; Kelbert, A.; Bedrosian, P.

2015-12-01

The global network of magnetic observatories represents a unique collective asset for the scientific community. Historically, magnetic observatories have supported global magnetic-field mapping projects and fundamental research of the Earth's interior and surrounding space environment. More recently, real-time data streams from magnetic observatories have become an important contributor to multi-sensor, operational monitoring of evolving space weather conditions, especially during magnetic storms. In this context, the U.S. Geological Survey (1) provides real-time observatory data to allied space weather monitoring projects, including those of NOAA, the U.S. Air Force, NASA, several international agencies, and private industry, (2) collaborates with Schlumberger to provide real-time geomagnetic data needed for directional drilling for oil and gas in Alaska, (3) develops products for real-time evaluation of hazards for the electric-power grid industry that are associated with the storm-time induction of geoelectric fields in the Earth's conducting lithosphere. In order to implement strategic priorities established by the USGS Natural Hazards Mission Area and the National Science and Technology Council, and with a focus on developing new real-time products, the USGS is (1) leveraging data management protocols already developed by the USGS Earthquake Program, (2) developing algorithms for mapping geomagnetic activity, a collaboration with NASA and NOAA, (3) supporting magnetotelluric surveys and developing Earth conductivity models, a collaboration with Oregon State University and the NSF's EarthScope Program, (4) studying the use of geomagnetic activity maps and Earth conductivity models for real-time estimation of geoelectric fields, (5) initiating geoelectric monitoring at several observatories, (6) validating real-time estimation algorithms against historical geomagnetic and geoelectric data. The success of these long-term projects is subject to funding constraints

Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR.

Science.gov (United States)

Sidstedt, Maja; Hedman, Johannes; Romsos, Erica L; Waitara, Leticia; Wadsö, Lars; Steffen, Carolyn R; Vallone, Peter M; Rådström, Peter

2018-04-01

Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles. Graphical abstract PCR inhibition mechanisms of hemoglobin and immunoglobulin G (IgG). Cq quantification cycle, dsDNA double-stranded DNA, ssDNA single-stranded DNA.

Real-time control strategy to maximize hybrid electric vehicle powertrain efficiency

International Nuclear Information System (INIS)

Shabbir, Wassif; Evangelou, Simos A.

2014-01-01

Highlights: • An off-line local control is proposed for real-time HEV energy management. • Powertrain efficiencies are studied to produce a unified objective function. • Penalty function is designed to ensure charge sustaining operation. • Implementation by storing optimal power share in a two-dimensional control map. • Proposed control improved fuel economy by up to 20% compared to conventional control. - Abstract: The proposed supervisory control system (SCS) uses a control map to maximize the powertrain efficiency of a hybrid electric vehicle (HEV) in real-time. The paper presents the methodology and structure of the control, including a novel, comprehensive and unified expression for the overall powertrain efficiency that considers the engine-generator set and the battery in depth as well as the power electronics. A control map is then produced with instructions for the optimal power share between the engine branch and battery branch of the vehicle such that the powertrain efficiency is maximized. This map is computed off-line and can thereafter be operated in real-time at very low computational cost. A charge sustaining factor is also developed and introduced to ensure the SCS operates the vehicle within desired SOC bounds. This SCS is then tested and benchmarked against two conventional control strategies in a high-fidelity vehicle model, representing a series HEV. Extensive simulation results are presented for repeated cycles of a diverse range of standard driving cycles, showing significant improvements in fuel economy (up to 20%) and less aggressive use of the battery

Real-time earthquake data feasible

Science.gov (United States)

Bush, Susan

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

Verifying real-time systems against scenario-based requirements

DEFF Research Database (Denmark)

Larsen, Kim Guldstrand; Li, Shuhao; Nielsen, Brian

2009-01-01

We propose an approach to automatic verification of real-time systems against scenario-based requirements. A real-time system is modeled as a network of Timed Automata (TA), and a scenario-based requirement is specified as a Live Sequence Chart (LSC). We define a trace-based semantics for a kernel...... subset of the LSC language. By equivalently translating an LSC chart into an observer TA and then non-intrusively composing this observer with the original system model, the problem of verifying a real-time system against a scenario-based requirement reduces to a classical real-time model checking...

Real-time UNIX in HEP data acquisition

International Nuclear Information System (INIS)

Buono, S.; Gaponenko, I.; Jones, R.; Mapelli, L.; Mornacchi, G.; Prigent, D.; Sanchez-Corral, E.; Skiadelli, M.; Toppers, A.; Duval, P.Y.; Ferrato, D.; Le Van Suu, A.; Qian, Z.; Rondot, C.; Ambrosini, G.; Fumagalli, G.; Aguer, M.; Huet, M.

1994-01-01

Today's experimentation in high energy physics is characterized by an increasing need for sensitivity to rare phenomena and complex physics signatures, which require the use of huge and sophisticated detectors and consequently a high performance readout and data acquisition. Multi-level triggering, hierarchical data collection and an always increasing amount of processing power, distributed throughout the data acquisition layers, will impose a number of features on the software environment, especially the need for a high level of standardization. Real-time UNIX seems, today, the best solution for the platform independence, operating system interface standards and real-time features necessary for data acquisition in HEP experiments. We present the results of the evaluation, in a realistic application environment, of a Real-Time UNIX operating system: the EP/LX real-time UNIX system. ((orig.))

Implementation of polymerase chain reaction (PCR and Real-Time PCR in quick identification of bovine herpesvirus 1

Directory of Open Access Journals (Sweden)

Milić Nenad

2010-01-01

Full Text Available Examinations were performed on 65 samples of nasal smeas taken from calves and young cows with clinical symptoms of respiratory infection to determine the presence of the bovine herpes virus 1 using parallel implementation of molecular and standard methods of virological diagnostics. The appearance of a cytopathogenic effect (CPE was not established in inoculated cell lines 24h, 48h and 72h following inoculation, or after two successive passages of the examined material sample through these cell lines. The application of polymerize chain reaction (PCR using a primer for glucoprotein B and thymidine - kinasis, established the presence of bovine herpes virus 1 nucleic acid in one sample of a bovine nasal smear, while the presence of this virus was established in three samples in an examination of the nasal smear samples using the Real-Time PCR method.

Temporal Specification and Verification of Real-Time Systems.

Science.gov (United States)

1991-08-30

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

ClockWork: a Real-Time Feasibility Analysis Tool

NARCIS (Netherlands)

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

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

Failure analysis of real-time systems

International Nuclear Information System (INIS)

Jalashgar, A.; Stoelen, K.

1998-01-01

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

Can Real-Time Data Also Be Climate Quality?

Science.gov (United States)

Brewer, M.; Wentz, F. J.

2015-12-01

GMI, AMSR-2 and WindSat herald a new era of highly accurate and timely microwave data products. Traditionally, there has been a large divide between real-time and re-analysis data products. What if these completely separate processing systems could be merged? Through advanced modeling and physically based algorithms, Remote Sensing Systems (RSS) has narrowed the gap between real-time and research-quality. Satellite microwave ocean products have proven useful for a wide array of timely Earth science applications. Through cloud SST capabilities have enormously benefited tropical cyclone forecasting and day to day fisheries management, to name a few. Oceanic wind vectors enhance operational safety of shipping and recreational boating. Atmospheric rivers are of import to many human endeavors, as are cloud cover and knowledge of precipitation events. Some activities benefit from both climate and real-time operational data used in conjunction. RSS has been consistently improving microwave Earth Science Data Records (ESDRs) for several decades, while making near real-time data publicly available for semi-operational use. These data streams have often been produced in 2 stages: near real-time, followed by research quality final files. Over the years, we have seen this time delay shrink from months or weeks to mere hours. As well, we have seen the quality of near real-time data improve to the point where the distinction starts to blur. We continue to work towards better and faster RFI filtering, adaptive algorithms and improved real-time validation statistics for earlier detection of problems. Can it be possible to produce climate quality data in real-time, and what would the advantages be? We will try to answer these questions…

Academic Training: Real Time Process Control - Lecture series

CERN Multimedia

Françoise Benz

2004-01-01

ACADEMIC TRAINING LECTURE REGULAR PROGRAMME 7, 8 and 9 June From 11:00 hrs to 12:00 hrs - Main Auditorium bldg. 500 Real Time Process Control T. Riesco / CERN-TS What exactly is meant by Real-time? There are several definitions of real-time, most of them contradictory. Unfortunately the topic is controversial, and there does not seem to be 100% agreement over the terminology. Real-time applications are becoming increasingly important in our daily lives and can be found in diverse environments such as the automatic braking system on an automobile, a lottery ticket system, or robotic environmental samplers on a space station. These lectures will introduce concepts and theory like basic concepts timing constraints, task scheduling, periodic server mechanisms, hard and soft real-time.ENSEIGNEMENT ACADEMIQUE ACADEMIC TRAINING Françoise Benz 73127 academic.training@cern.ch

Rapid and Accurate Identification by Real-Time PCR of Biotoxin-Producing Dinoflagellates from the Family Gymnodiniaceae

Directory of Open Access Journals (Sweden)

Kirsty F. Smith

2014-03-01

Full Text Available The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR assays targeting the large subunit ribosomal RNA (LSU rRNA gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.

Detection of Histoplasma capsulatum from clinical specimens by cycling probe-based real-time PCR and nested real-time PCR.

Science.gov (United States)

Muraosa, Yasunori; Toyotome, Takahito; Yahiro, Maki; Watanabe, Akira; Shikanai-Yasuda, Maria Aparecida; Kamei, Katsuhiko

2016-05-01

We developed new cycling probe-based real-time PCR and nested real-time PCR assays for the detection of Histoplasma capsulatum that were designed to detect the gene encoding N-acetylated α-linked acidic dipeptidase (NAALADase), which we previously identified as an H. capsulatum antigen reacting with sera from patients with histoplasmosis. Both assays specifically detected the DNAs of all H. capsulatum strains but not those of other fungi or human DNA. The limited of detection (LOD) of the real-time PCR assay was 10 DNA copies when using 10-fold serial dilutions of the standard plasmid DNA and 50 DNA copies when using human serum spiked with standard plasmid DNA. The nested real-time PCR improved the LOD to 5 DNA copies when using human serum spiked with standard plasmid DNA, which represents a 10-fold higher than that observed with the real-time PCR assay. To assess the ability of the two assays to diagnose histoplasmosis, we analyzed a small number of clinical specimens collected from five patients with histoplasmosis, such as sera (n = 4), formalin-fixed paraffin-embedded (FFPE) tissue (n = 4), and bronchoalveolar lavage fluid (BALF) (n = 1). Although clinical sensitivity of the real-time PCR assay was insufficiently sensitive (33%), the nested real-time PCR assay increased the clinical sensitivity (77%), suggesting it has a potential to be a useful method for detecting H. capsulatum DNA in clinical specimens. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Reviewing real-time performance of nuclear reactor safety systems

International Nuclear Information System (INIS)

Preckshot, G.G.

1993-08-01

The purpose of this paper is to recommend regulatory guidance for reviewers examining real-time performance of computer-based safety systems used in nuclear power plants. Three areas of guidance are covered in this report. The first area covers how to determine if, when, and what prototypes should be required of developers to make a convincing demonstration that specific problems have been solved or that performance goals have been met. The second area has recommendations for timing analyses that will prove that the real-time system will meet its safety-imposed deadlines. The third area has description of means for assessing expected or actual real-time performance before, during, and after development is completed. To ensure that the delivered real-time software product meets performance goals, the paper recommends certain types of code-execution and communications scheduling. Technical background is provided in the appendix on methods of timing analysis, scheduling real-time computations, prototyping, real-time software development approaches, modeling and measurement, and real-time operating systems

Reviewing real-time performance of nuclear reactor safety systems

Energy Technology Data Exchange (ETDEWEB)

Preckshot, G.G. [Lawrence Livermore National Lab., CA (United States)

1993-08-01

The purpose of this paper is to recommend regulatory guidance for reviewers examining real-time performance of computer-based safety systems used in nuclear power plants. Three areas of guidance are covered in this report. The first area covers how to determine if, when, and what prototypes should be required of developers to make a convincing demonstration that specific problems have been solved or that performance goals have been met. The second area has recommendations for timing analyses that will prove that the real-time system will meet its safety-imposed deadlines. The third area has description of means for assessing expected or actual real-time performance before, during, and after development is completed. To ensure that the delivered real-time software product meets performance goals, the paper recommends certain types of code-execution and communications scheduling. Technical background is provided in the appendix on methods of timing analysis, scheduling real-time computations, prototyping, real-time software development approaches, modeling and measurement, and real-time operating systems.

Connecting real-time data to algorithms and databases: EarthCube's Cloud-Hosted Real-time Data Services for the Geosciences (CHORDS)

Science.gov (United States)

Daniels, M. D.; Graves, S. J.; Kerkez, B.; Chandrasekar, V.; Vernon, F.; Martin, C. L.; Maskey, M.; Keiser, K.; Dye, M. J.

2015-12-01

The Cloud-Hosted Real-time Data Services for the Geosciences (CHORDS) project was funded under the National Science Foundation's EarthCube initiative. CHORDS addresses the ever-increasing importance of real-time scientific data in the geosciences, particularly in mission critical scenarios, where informed decisions must be made rapidly. Access to constant streams of real-time data also allow many new transient phenomena in space-time to be observed, however, much of these streaming data are either completely inaccessible or only available to proprietary in-house tools or displays. Small research teams do not have the resources to develop tools for the broad dissemination of their unique real-time data and require an easy to use, scalable, cloud-based solution to facilitate this access. CHORDS will make these diverse streams of real-time data available to the broader geosciences community. This talk will highlight a recently developed CHORDS portal tools and processing systems which address some of the gaps in handling real-time data, particularly in the provisioning of data from the "long-tail" scientific community through a simple interface that is deployed in the cloud, is scalable and is able to be customized by research teams. A running portal, with operational data feeds from across the nation, will be presented. The processing within the CHORDS system will expose these real-time streams via standard services from the Open Geospatial Consortium (OGC) in a way that is simple and transparent to the data provider, while maximizing the usage of these investments. The ingestion of high velocity, high volume and diverse data has allowed the project to explore a NoSQL database implementation. Broad use of the CHORDS framework by geoscientists will help to facilitate adaptive experimentation, model assimilation and real-time hypothesis testing.

The Abbott RealTime High Risk HPV test is a clinically validated human papillomavirus assay for triage in the referral population and use in primary cervical cancer screening in women 30 years and older: a review of validation studies.

Science.gov (United States)

Poljak, Mario; Oštrbenk, Anja

2013-01-01

Human papillomavirus (HPV) testing has become an essential part of current clinical practice in the management of cervical cancer and precancerous lesions. We reviewed the most important validation studies of a next-generation real-time polymerase chain reaction-based assay, the RealTime High Risk HPV test (RealTime)(Abbott Molecular, Des Plaines, IL, USA), for triage in referral population settings and for use in primary cervical cancer screening in women 30 years and older published in peer-reviewed journals from 2009 to 2013. RealTime is designed to detect 14 high-risk HPV genotypes with concurrent distinction of HPV-16 and HPV-18 from 12 other HPV genotypes. The test was launched on the European market in January 2009 and is currently used in many laboratories worldwide for routine detection of HPV. We concisely reviewed validation studies of a next-generation real-time polymerase chain reaction (PCR)-based assay: the Abbott RealTime High Risk HPV test. Eight validation studies of RealTime in referral settings showed its consistently high absolute clinical sensitivity for both CIN2+ (range 88.3-100%) and CIN3+ (range 93.0-100%), as well as comparative clinical sensitivity relative to the currently most widely used HPV test: the Qiagen/Digene Hybrid Capture 2 HPV DNA Test (HC2). Due to the significantly different composition of the referral populations, RealTime absolute clinical specificity for CIN2+ and CIN3+ varied greatly across studies, but was comparable relative to HC2. Four validation studies of RealTime performance in cervical cancer screening settings showed its consistently high absolute clinical sensitivity for both CIN2+ and CIN3+, as well as comparative clinical sensitivity and specificity relative to HC2 and GP5+/6+ PCR. RealTime has been extensively evaluated in the last 4 years. RealTime can be considered clinically validated for triage in referral population settings and for use in primary cervical cancer screening in women 30 years and older.

Virtual timers in hierarchical real-time systems

NARCIS (Netherlands)

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

2009-01-01

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

Comparison of culture versus quantitative real-time polymerase chain reaction for the detection of Taylorella equigenitalis in field samples from naturally infected horses in Canada and Germany.

Science.gov (United States)

Nadin-Davis, Susan; Knowles, Margaret K; Burke, Teresa; Böse, Reinhard; Devenish, John

2015-07-01

A quantitative real-time polymerase chain reaction method (qPCR) was developed and tested for the detection of Taylorella equigenitalis. It was shown to have an analytical sensitivity of 5 colony-forming units (CFU) of T. equigenitalis when applied to the testing of culture swabs that mimicked field samples, and a high analytical specificity in not reacting to 8 other commensal bacterial species associated with horses. As designed, it could also differentiate specifically between T. equigenitalis and T. asinigenitalis. The qPCR was compared to standard culture in a study that included 45 swab samples from 6 horses (1 stallion, 5 mares) naturally infected with T. equigenitalis in Canada, 39 swab samples from 5 naturally infected stallions in Germany, and 311 swab samples from 87 culture negative horses in Canada. When the comparison was conducted on an individual sample swab basis, the qPCR had a statistical sensitivity and specificity of 100% and 96.4%, respectively, and 100% and 99.1% when the comparison was conducted on a sample set basis. A comparison was also made on 203 sample swabs from the 5 German stallions taken over a span of 4 to 9 mo following antibiotic treatment. The qPCR was found to be highly sensitive and at least as good as culture in detecting the presence of T. equigenitalis in post-treatment samples. The work demonstrates that the qPCR assay described here can potentially be used to detect the presence of T. equigenitalis directly from submitted sample swabs taken from infected horses and also for determining T. equigenitalis freedom following treatment.

Human parvovirus B19, varicella zoster virus, and human herpesvirus-6 in mesenchymal stem cells of patients with osteoarthritis: analysis with quantitative real-time polymerase chain reaction.

Science.gov (United States)

Rollín, R; Alvarez-Lafuente, R; Marco, F; Jover, J A; Hernández-García, C; Rodríguez-Navas, C; López-Durán, L; Fernández-Gutiérrez, B

2007-04-01

To investigate whether there is a possible viral transmission using mesenchymal stem cells (MSCs) in autologous or allogeneic transplantation in the context of osteoarthritis (OA) patients. The presence of parvovirus B19 (B19), varicella zoster virus (VZV), and human herpesvirus-6 (HHV-6) was studied in MSCs from bone marrow of patients with OA and healthy controls. MSCs were prepared from bone marrow aspirates obtained from 18 patients undergoing joint replacement as a result of OA and from 10 healthy controls. DNA was extracted from primary MSCs' culture established from these cells and quantitative real-time polymerase chain reaction was performed to analyse the prevalence and viral load of B19, VZV and HHV-6. The prevalence of total viral DNA among patients with OA was 16.7% (3/18), with a mean viral load of 29.7 copies/microg of DNA. One out of 18 was positive for B19 (viral load, 61.2 copies/microg of DNA), two for VZV (mean viral load, 14.4 copies/microg of DNA), and none for HHV-6. The prevalence of total viral DNA in the control group was 20% (2/10), with a mean viral load of 13.4 copies/microg of DNA. Both positive results were of B19 parvoviruses. There were no statistically significant differences among patients and controls. This first approach to the viral prevalence in MSCs of bone marrow in OA patients and healthy controls seems to show a very low risk of viral transmission or reactivation in a possible MSCs' transplantation.

Real-time video compressing under DSP/BIOS

Science.gov (United States)

Chen, Qiu-ping; Li, Gui-ju

2009-10-01

This paper presents real-time MPEG-4 Simple Profile video compressing based on the DSP processor. The programming framework of video compressing is constructed using TMS320C6416 Microprocessor, TDS510 simulator and PC. It uses embedded real-time operating system DSP/BIOS and the API functions to build periodic function, tasks and interruptions etcs. Realize real-time video compressing. To the questions of data transferring among the system. Based on the architecture of the C64x DSP, utilized double buffer switched and EDMA data transfer controller to transit data from external memory to internal, and realize data transition and processing at the same time; the architecture level optimizations are used to improve software pipeline. The system used DSP/BIOS to realize multi-thread scheduling. The whole system realizes high speed transition of a great deal of data. Experimental results show the encoder can realize real-time encoding of 768*576, 25 frame/s video images.

A MIQE-compliant real-time PCR assay for Aspergillus detection.

Directory of Open Access Journals (Sweden)

Gemma L Johnson

Full Text Available The polymerase chain reaction (PCR is widely used as a diagnostic tool in clinical laboratories and is particularly effective for detecting and identifying infectious agents for which routine culture and microscopy methods are inadequate. Invasive fungal disease (IFD is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA, variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published "Minimum Information for the publication of real-time Quantitative PCR Experiments" (MIQE guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95-107%, a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness

A Simultaneous Analytical Method for Duplex Identification of Porcine and Horse in the Meat Products by EvaGreen based Real-time PCR

OpenAIRE

Sakalar, Erg?n; Erg?n, Seyma ?z?irak; Akar, Emine

2015-01-01

A duplex real-time polymerase chain reaction (PCR) based assay for the detection of porcine and horse meat in sausages was designed by using EvaGreen fluorescent dye. Primers were selected from mitochondrial 12S rRNA and 16S rRNA genes which are powerful regions for identification of horse and porcine meat. DNA from reference samples and industrial products was successfully extracted using the GIDAGEN? Multi-Fast DNA Isolation Kit. Genomes were identified based on their specific melting peaks...

Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature

DEFF Research Database (Denmark)

Guðnason, Haukur; Dufva, Hans Martin; Bang, Dang Duong

2007-01-01

investigate 15 different intercalating DNA dyes for their inhibitory effects on PCR, effects on DNA melting temperature and possible preferential binding to GC-rich sequences. Our results demonstrated that in contrast to the results of SYBR Green I, two intercalating dyes SYTO-13 and SYTO-82 do not inhibit......The importance of real-time polymerase chain reaction (PCR) has increased steadily in clinical applications over the last decade. Many applications utilize SYBR Green I dye to follow the accumulation of amplicons in real time. SYBR Green I has, however, a number of limitations that include...... the inhibition of PCR, preferential binding to GC-rich sequences and effects on melting curve analysis. Although a few alternative dyes without some of these limitations have been recently proposed, no large-scale investigation into the properties of intercalating dyes has been performed. In this study, we...

Real-time ISEE data system

Science.gov (United States)

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

1979-01-01

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

Fetal RHD Genotyping Using Real-Time Polymerase Chain Reaction Analysis of Cell-Free Fetal DNA in Pregnancy of RhD Negative Women in South of Iran

Directory of Open Access Journals (Sweden)

Leili Moezzi

2016-05-01

Full Text Available Background: Maternal-fetal RhD antigen incompatibility causes approximately 50% of clinically significant alloimmunization cases. The routine use of prophylactic anti-D immunoglobulin has dramatically reduced hemolytic disease of the fetus and newborn. Recently, fetal RHD genotyping in RhD negative pregnant women has been suggested for appropriate use of anti-D immunoglobulin antenatal prophylaxis and decrease unnecessary prenatal interventions. Materials and Methods: In this prospective cohort study, in order to develop a reliable and non-invasive method for fetal RHD genotyping, cell free fetal DNA (cffDNA was extracted from maternal plasma. Real-time quantitative polymerase chain reaction (qPCR for detection of RHD exons 7, 5, 10 and intron 4 was performed and the results were compared to the serological results of cord blood cells as the gold standard method. SRY gene and hypermethylated Ras-association domain family member 1 (RASSF1A gene were used to confirm the presence of fetal DNA in male and female fetuses, respectively. Results: Out of 48 fetuses between 8 and 32 weeks (wks of gestational age (GA, we correctly diagnosed 45 cases (93.75% of RHD positive fetuses and 2 cases (4.16% of the RHD negative one. Exon 7 was amplified in one sample, while three other RHD gene sequences were not detected; the sample was classified as inconclusive, and the RhD serology result after birth showed that the fetus was RhD-negative. Conclusion: Our results showed high accuracy of the qPCR method using cffDNA for fetal RHD genotyping and implicate on the efficiency of this technique to predict the competence of anti-D immunoglobulin administration.

Effectiveness of real-time polymerase chain reaction assay for the detection of Mycobacterium tuberculosis in pathological samples: a systematic review and meta-analysis

Directory of Open Access Journals (Sweden)

Emmanuel O. Babafemi

2017-10-01

Full Text Available Abstract Background Rapid and accurate diagnosis of tuberculosis (TB is key to manage the disease and to control and prevent its transmission. Many established diagnostic methods suffer from low sensitivity or delay of timely results and are inadequate for rapid detection of Mycobacterium tuberculosis (MTB in pulmonary and extra-pulmonary clinical samples. This study examined whether a real-time polymerase chain reaction (RT-PCR assay, with a turn-a-round time of 2 h, would prove effective for routine detection of MTB by clinical microbiology laboratories. Methods A systematic literature search was performed for publications in any language on the detection of MTB in pathological samples by RT-PCR assay. The following sources were used MEDLINE via PubMed, EMBASE, BIOSIS Citation Index, Web of Science, SCOPUS, ISI Web of Knowledge and Cochrane Infectious Diseases Group Specialised Register, grey literature, World Health Organization and Centres for Disease Control and Prevention websites. Forty-six studies met set inclusion criteria. Generated pooled summary estimates (95% CIs were calculated for overall accuracy and bivariate meta-regression model was used for meta-analysis. Results Summary estimates for pulmonary TB (31 studies were as follows: sensitivity 0.82 (95% CI 0.81–0.83, specificity 0.99 (95% CI 0.99–0.99, positive likelihood ratio 43.00 (28.23–64.81, negative likelihood ratio 0.16 (0.12–0.20, diagnostic odds ratio 324.26 (95% CI 189.08–556.09 and area under curve 0.99. Summary estimates for extra-pulmonary TB (25 studies were as follows: sensitivity 0.70 (95% CI 0.67–0.72, specificity 0.99 (95% CI 0.99–0.99, positive likelihood ratio 29.82 (17.86–49.78, negative likelihood ratio 0.33 (0.26–0.42, diagnostic odds ratio 125.20 (95% CI 65.75–238.36 and area under curve 0.96. Conclusions RT-PCR assay demonstrated a high degree of sensitivity for pulmonary TB and good sensitivity for extra-pulmonary TB. It indicated a

Comprehensive Cost Minimization in Distribution Networks Using Segmented-time Feeder Reconfiguration and Reactive Power Control of Distributed Generators

DEFF Research Database (Denmark)

Chen, Shuheng; Hu, Weihao; Chen, Zhe

2016-01-01

In this paper, an efficient methodology is proposed to deal with segmented-time reconfiguration problem of distribution networks coupled with segmented-time reactive power control of distributed generators. The target is to find the optimal dispatching schedule of all controllable switches...... and distributed generators’ reactive powers in order to minimize comprehensive cost. Corresponding constraints, including voltage profile, maximum allowable daily switching operation numbers (MADSON), reactive power limits, and so on, are considered. The strategy of grouping branches is used to simplify...... (FAHPSO) is implemented in VC++ 6.0 program language. A modified version of the typical 70-node distribution network and several real distribution networks are used to test the performance of the proposed method. Numerical results show that the proposed methodology is an efficient method for comprehensive...

A distributed agent architecture for real-time knowledge-based systems: Real-time expert systems project, phase 1

Science.gov (United States)

Lee, S. Daniel

1990-01-01

We propose a distributed agent architecture (DAA) that can support a variety of paradigms based on both traditional real-time computing and artificial intelligence. DAA consists of distributed agents that are classified into two categories: reactive and cognitive. Reactive agents can be implemented directly in Ada to meet hard real-time requirements and be deployed on on-board embedded processors. A traditional real-time computing methodology under consideration is the rate monotonic theory that can guarantee schedulability based on analytical methods. AI techniques under consideration for reactive agents are approximate or anytime reasoning that can be implemented using Bayesian belief networks as in Guardian. Cognitive agents are traditional expert systems that can be implemented in ART-Ada to meet soft real-time requirements. During the initial design of cognitive agents, it is critical to consider the migration path that would allow initial deployment on ground-based workstations with eventual deployment on on-board processors. ART-Ada technology enables this migration while Lisp-based technologies make it difficult if not impossible. In addition to reactive and cognitive agents, a meta-level agent would be needed to coordinate multiple agents and to provide meta-level control.

Validation of tumor markers in central nervous system germ cell tumors by real-time reverse transcriptase polymerase chain reaction using formalin-fixed paraffin-embedded tissues.

Science.gov (United States)

Kim, Dowhan; Lee, Da Hye; Choi, Junjeong; Shim, Kyu Won; Kim, Se Hoon

2013-01-01

The therapeutic protocols for treatment of germinomas and non-germinomatous germ cell tumors (NGGCTs) are completely different, so it is important to distinguish pure germinomas from NGGCTs. As it can be difficult to diagnose by morphology alone, immunohisto-chemistry (IHC) has been widely used as an ancillary test to improve diagnostic accuracy. However, IHC has limitations due to the misinterpretation of results or the aberrant loss of immunoreactivity. However, real-time RT-PCR has certain advantages over IHC, including its quantitative nature. The aim of our study was to evaluate the usefulness of real-time RT-PCR on formalin-fixed paraffin-embedded (FFPE) tissue blocks for the diagnosis of germ cell tumors of the central nervous system. We selected eight markers of germ cell tumors using a literature search, and validated them using real-time RT-PCR. Among them, POU5F1, NANOG and TGFB2 were statistically significant (P=0.05) in multiple comparisons (MANOVA) of three groups (pure germinomas, mature teratomas and malignant germ cell tumors). Two-group (pure germinomas and NGGCTs) discriminant analysis achieved a 70.0% success rate in cross-validation. We concluded that real-time RT-PCR using FFPE tissue has adequate validating power comparable to IHC in the diagnosis of central nervous system germ cell tumors; therefore, when IHC is not available, not conclusive or not informative, RT-PCR is a potential alternative to a repeat biopsy.

Real time application of whole genome sequencing for outbreak investigation - What is an achievable turnaround time?

Science.gov (United States)

McGann, Patrick; Bunin, Jessica L; Snesrud, Erik; Singh, Seema; Maybank, Rosslyn; Ong, Ana C; Kwak, Yoon I; Seronello, Scott; Clifford, Robert J; Hinkle, Mary; Yamada, Stephen; Barnhill, Jason; Lesho, Emil

2016-07-01

Whole genome sequencing (WGS) is increasingly employed in clinical settings, though few assessments of turnaround times (TAT) have been performed in real-time. In this study, WGS was used to investigate an unfolding outbreak of vancomycin resistant Enterococcus faecium (VRE) among 3 patients in the ICU of a tertiary care hospital. Including overnight culturing, a TAT of just 48.5 h for a comprehensive report was achievable using an Illumina Miseq benchtop sequencer. WGS revealed that isolates from patient 2 and 3 differed from that of patient 1 by a single nucleotide polymorphism (SNP), indicating nosocomial transmission. However, the unparalleled resolution provided by WGS suggested that nosocomial transmission involved two separate events from patient 1 to patient 2 and 3, and not a linear transmission suspected by the time line. Rapid TAT's are achievable using WGS in the clinical setting and can provide an unprecedented level of resolution for outbreak investigations. Published by Elsevier Inc.

A Process For Performance Evaluation Of Real-Time Systems

Directory of Open Access Journals (Sweden)

Andrew J. Kornecki

2003-12-01

Full Text Available Real-time developers and engineers must not only meet the system functional requirements, but also the stringent timing requirements. One of the critical decisions leading to meeting these timing requirements is the selection of an operating system under which the software will be developed and run. Although there is ample documentation on real-time systems performance and evaluation, little can be found that combines such information into an efficient process for use by developers. As the software industry moves towards clearly defined processes, creation of appropriate guidelines describing a process for performance evaluation of real-time system would greatly benefit real-time developers. This technology transition research focuses on developing such a process. PROPERT (PROcess for Performance Evaluation of Real Time systems - the process described in this paper - is based upon established techniques for evaluating real-time systems. It organizes already existing real-time performance criteria and assessment techniques in a manner consistent with a well-formed process, based on the Personal Software Process concepts.

Quantitative (real-time) PCR

International Nuclear Information System (INIS)

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

2005-01-01

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

Multi-time scale energy management of wind farms based on comprehensive evaluation technology

Science.gov (United States)

Xu, Y. P.; Huang, Y. H.; Liu, Z. J.; Wang, Y. F.; Li, Z. Y.; Guo, L.

2017-11-01

A novel energy management of wind farms is proposed in this paper. Firstly, a novel comprehensive evaluation system is proposed to quantify economic properties of each wind farm to make the energy management more economical and reasonable. Then, a combination of multi time-scale schedule method is proposed to develop a novel energy management. The day-ahead schedule optimizes unit commitment of thermal power generators. The intraday schedule is established to optimize power generation plan for all thermal power generating units, hydroelectric generating sets and wind power plants. At last, the power generation plan can be timely revised in the process of on-line schedule. The paper concludes with simulations conducted on a real provincial integrated energy system in northeast China. Simulation results have validated the proposed model and corresponding solving algorithms.

Market analysis of food products for detection of allergenic walnut (Juglans regia) and pecan (Carya illinoinensis) by real-time PCR.

Science.gov (United States)

López-Calleja, Inés María; de la Cruz, Silvia; González, Isabel; García, Teresa; Martín, Rosario

2015-06-15

Two real-time polymerase chain reaction (PCR)-based assays for detection of walnut (Juglans regia) and pecan (Carya illinoinensis) traces in a wide range of processed foods are described here. The method consists on a real-time PCR assay targeting the ITS1 region, using a nuclease (TaqMan) probe labeled with FAM and BBQ. The method was positive for walnut and pecan respectively, and negative for all other heterologous plants and animals tested. Using a series of model samples with defined raw walnut in wheat flour and heat-treated walnut in wheat flour with a range of concentrations of 0.1-100,000 mg kg(-1), a practical detection limit of 0.1 mg kg(-1) of walnut content was estimated. Identical binary mixtures were done for pecan, reaching the same limit of detection of 0.1 mg kg(-1). The assay was successfully trialed on a total of 232 commercial foodstuffs. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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

2013-07-10

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

Real-time holographic endoscopy

Science.gov (United States)

Smigielski, Paul; Albe, Felix; Dischli, Bernard

1992-08-01

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

Real-time computational photon-counting LiDAR

Science.gov (United States)

Edgar, Matthew; Johnson, Steven; Phillips, David; Padgett, Miles

2018-03-01

The availability of compact, low-cost, and high-speed MEMS-based spatial light modulators has generated widespread interest in alternative sampling strategies for imaging systems utilizing single-pixel detectors. The development of compressed sensing schemes for real-time computational imaging may have promising commercial applications for high-performance detectors, where the availability of focal plane arrays is expensive or otherwise limited. We discuss the research and development of a prototype light detection and ranging (LiDAR) system via direct time of flight, which utilizes a single high-sensitivity photon-counting detector and fast-timing electronics to recover millimeter accuracy three-dimensional images in real time. The development of low-cost real time computational LiDAR systems could have importance for applications in security, defense, and autonomous vehicles.

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

Directory of Open Access Journals (Sweden)

Anita Chakravarti

2016-01-01

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

Real time magnetic resonance guided endomyocardial local delivery

Science.gov (United States)

Corti, R; Badimon, J; Mizsei, G; Macaluso, F; Lee, M; Licato, P; Viles-Gonzalez, J F; Fuster, V; Sherman, W

2005-01-01

Objective: To investigate the feasibility of targeting various areas of left ventricle myocardium under real time magnetic resonance (MR) imaging with a customised injection catheter equipped with a miniaturised coil. Design: A needle injection catheter with a mounted resonant solenoid circuit (coil) at its tip was designed and constructed. A 1.5 T MR scanner with customised real time sequence combined with in-room scan running capabilities was used. With this system, various myocardial areas within the left ventricle were targeted and injected with a gadolinium-diethylenetriaminepentaacetic acid (DTPA) and Indian ink mixture. Results: Real time sequencing at 10 frames/s allowed clear visualisation of the moving catheter and its transit through the aorta into the ventricle, as well as targeting of all ventricle wall segments without further image enhancement techniques. All injections were visualised by real time MR imaging and verified by gross pathology. Conclusion: The tracking device allowed real time in vivo visualisation of catheters in the aorta and left ventricle as well as precise targeting of myocardial areas. The use of this real time catheter tracking may enable precise and adequate delivery of agents for tissue regeneration. PMID:15710717

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

DEFF Research Database (Denmark)

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

2013-01-01

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

Prototyping Real-Time Control in the SPS

CERN Document Server

Andersson, J; Jensen, L; Jones, R; Lamont, M; Wenninger, J; Wijnands, Thijs; CERN. Geneva. AB Department

2003-01-01

Real-time control of beam related parameters will be required in the LHC. In order to gain experience of the issues involved in implementing distributed real-time control over large distances, a prototype local orbit feedback system is being developed in the SPS. This will use 6 pickups, each equipped with the full LHC acquisition electronics chain and linked to a real-time communication and feedback system. This reports summarises the .rst tests performed with this system in October 2002, where the data from four pickups was successfully acquired and displayed at 10 Hz in the control room.

Formal methods for dependable real-time systems

Science.gov (United States)

Rushby, John

1993-01-01

The motivation for using formal methods to specify and reason about real time properties is outlined and approaches that were proposed and used are sketched. The formal verifications of clock synchronization algorithms are concluded as showing that mechanically supported reasoning about complex real time behavior is feasible. However, there was significant increase in the effectiveness of verification systems since those verifications were performed, at it is to be expected that verifications of comparable difficulty will become fairly routine. The current challenge lies in developing perspicuous and economical approaches to the formalization and specification of real time properties.

Real time detecting system for turning force

Energy Technology Data Exchange (ETDEWEB)

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

2001-07-01

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

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.

Real-time Avatar Animation from a Single Image.

Science.gov (United States)

Saragih, Jason M; Lucey, Simon; Cohn, Jeffrey F

2011-01-01

A real time facial puppetry system is presented. Compared with existing systems, the proposed method requires no special hardware, runs in real time (23 frames-per-second), and requires only a single image of the avatar and user. The user's facial expression is captured through a real-time 3D non-rigid tracking system. Expression transfer is achieved by combining a generic expression model with synthetically generated examples that better capture person specific characteristics. Performance of the system is evaluated on avatars of real people as well as masks and cartoon characters.

An Analysis of Input/Output Paradigms for Real-Time Systems