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Sample records for quantitative real time

  1. 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

  2. Diagnosis of aerobic vaginitis by quantitative real-time PCR

    OpenAIRE

    Rumyantseva, T. A.; Bellen, G.; Savochkina, Y. A.; Guschin, A. E.; Donders, G.G.G.

    2016-01-01

    Abstract: Purpose To evaluate a real-time PCR-based technique to quantify bacteria associated with aerobic vaginitis (AV) as a potential test. Methods Vaginal samples from 100 women were tested by wet-mount microscopy, gram stain and quantitative real-time PCR targeting Enterobacteriacea, Staphylococcus spp., Streptococcus spp., Enterococcus spp., Escherichia coli, Streptococcus agalactiae, S. aureus; Lactobacillus spp. AV diagnosis obtained by wet-mount microscopy was used as reference. Resu...

  3. Human fecal source identification with real-time quantitative PCR

    Science.gov (United States)

    Waterborne diseases represent a significant public health risk worldwide, and can originate from contact with water contaminated with human fecal material. We describe a real-time quantitative PCR (qPCR) method that targets a Bacteroides dori human-associated genetic marker for...

  4. Quantitative analysis of real-time radiographic systems

    International Nuclear Information System (INIS)

    Barker, M.D.; Condon, P.E.; Barry, R.C.; Betz, R.A.; Klynn, L.M.

    1988-01-01

    A method was developed which yields quantitative information on the spatial resolution, contrast sensitivity, image noise, and focal spot size from real time radiographic images. The method uses simple image quality indicators and computer programs which make it possible to readily obtain quantitative performance measurements of single or multiple radiographic systems. It was used for x-ray and optical images to determine which component of the system was not operating up to standard. Focal spot size was monitored by imaging a bar pattern. This paper constitutes the second progress report on the development of the camera and radiation image quality indicators

  5. Diagnosis of aerobic vaginitis by quantitative real-time PCR.

    Science.gov (United States)

    Rumyantseva, T A; Bellen, G; Savochkina, Y A; Guschin, A E; Donders, G G G

    2016-07-01

    To evaluate a real-time PCR-based technique to quantify bacteria associated with aerobic vaginitis (AV) as a potential test. Vaginal samples from 100 women were tested by wet-mount microscopy, gram stain and quantitative real-time PCR targeting Enterobacteriacea, Staphylococcus spp., Streptococcus spp., Enterococcus spp., Escherichia coli, Streptococcus agalactiae, S. aureus; Lactobacillus spp. AV diagnosis obtained by wet-mount microscopy was used as reference. Some level of AV was diagnosed in 23 (23.7 %) cases. Various concentrations of Enterobacteriacea, Staphylococcus spp., Streptococcus spp. were detected an all patients. Enterococcus spp. were detected in 76 (78.3 %) cases. Summarized concentrations of aerobes were tenfold higher in AV-positive compared to AV-negative cases [7.30lg vs 6.06lg (p = 0.02)]. Concentrations of aerobes in severe, moderate and light AV cases did not vary significantly (p = 0.14). Concentration of lactobacilli was 1000-fold lower in AV-positive cases compared to normal cases (5.3lg vs 8.3lg, p AV-positive cases [19/22 (86.4 %) samples]. The relation of high loads of aerobes to the low numbers of Lactobacilli are a reliable marker for the presence of AV and could substitute microscopy as a test. PCR may be a good standardized substitution for AV diagnosis in settings where well-trained microscopists are lacking.

  6. 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

  7. 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

  8. Statistical aspects of quantitative real-time PCR experiment design.

    Science.gov (United States)

    Kitchen, Robert R; Kubista, Mikael; Tichopad, Ales

    2010-04-01

    Experiments using quantitative real-time PCR to test hypotheses are limited by technical and biological variability; we seek to minimise sources of confounding variability through optimum use of biological and technical replicates. The quality of an experiment design is commonly assessed by calculating its prospective power. Such calculations rely on knowledge of the expected variances of the measurements of each group of samples and the magnitude of the treatment effect; the estimation of which is often uninformed and unreliable. Here we introduce a method that exploits a small pilot study to estimate the biological and technical variances in order to improve the design of a subsequent large experiment. We measure the variance contributions at several 'levels' of the experiment design and provide a means of using this information to predict both the total variance and the prospective power of the assay. A validation of the method is provided through a variance analysis of representative genes in several bovine tissue-types. We also discuss the effect of normalisation to a reference gene in terms of the measured variance components of the gene of interest. Finally, we describe a software implementation of these methods, powerNest, that gives the user the opportunity to input data from a pilot study and interactively modify the design of the assay. The software automatically calculates expected variances, statistical power, and optimal design of the larger experiment. powerNest enables the researcher to minimise the total confounding variance and maximise prospective power for a specified maximum cost for the large study. Copyright 2010 Elsevier Inc. All rights reserved.

  9. Statistical aspects of quantitative real-time PCR experiment design

    Czech Academy of Sciences Publication Activity Database

    Kitchen, R.R.; Kubista, Mikael; Tichopád, Aleš

    2010-01-01

    Roč. 50, č. 4 (2010), s. 231-236 ISSN 1046-2023 R&D Projects: GA AV ČR IAA500520809 Institutional research plan: CEZ:AV0Z50520701 Keywords : Real-time PCR * Experiment design * Nested analysis of variance Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.527, year: 2010

  10. A Quantitative Approach to the Formal Verification of Real-Time Systems.

    Science.gov (United States)

    1996-09-01

    Computer Science A Quantitative Approach to the Formal Verification of Real - Time Systems Sergio Vale Aguiar Campos September 1996 CMU-CS-96-199...ptisiic raieaiSI v Diambimos Lboiamtad _^ A Quantitative Approach to the Formal Verification of Real - Time Systems Sergio Vale Aguiar Campos...implied, of NSF, the Semiconduc- tor Research Corporation, ARPA or the U.S. government. Keywords: real - time systems , formal verification, symbolic

  11. Quantitative Real-Time PCR Fecal Source Identification in the ...

    Science.gov (United States)

    Rivers in the Tillamook Basin play a vital role in supporting a thriving dairy and cheese-making industry, as well as providing a safe water resource for local human and wildlife populations. Historical concentrations of fecal bacteria in these waters are at times too high to allow for safe use leading to economic loss, endangerment of local wildlife, and poor conditions for recreational use. In this study, we employ host-associated qPCR methods for human (HF183/BacR287 and HumM2), ruminant (Rum2Bac), cattle (CowM2 and CowM3), canine (DG3 and DG37), and avian (GFD) fecal pollution combined with high-resolution geographic information system (GIS) land use data and general indicator bacteria measurements to elucidatewater quality spatial and temporal trends. Water samples (n=584) were collected over a 1-year period at 29 sites along the Trask, Kilchis, and Tillamook rivers and tributaries (Tillamook Basin, OR). A total of 16.6% of samples (n=97) yielded E. coli levels considered impaired based on Oregon Department of Environmental Quality bacteria criteria (406 MPN/100mL). Hostassociated genetic indicators were detected at frequencies of 39.2% (HF183/BacR287), 16.3% (HumM2), 74.6% (Rum2Bac), 13.0% (CowM2), 26.7% (CowM3), 19.8% (DG3), 3.2% (DG37), and 53.4% (GFD) across all water samples (n=584). Seasonal trends in avian, cattle, and human fecal pollution sources were evident over the study area. On a sample site basis, quantitative fecal source identification and

  12. Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma

    DEFF Research Database (Denmark)

    Gjerdrum, Lise Mette; Sorensen, Boe Sandahl; Kjeldsen, Eigil

    2004-01-01

    We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods...... tumors as being amplified. Interestingly, all these scored 2+ with the HercepTest, but were negative using FISH. We believe that real-time quantitative PCR analysis of HER-2 DNA amplification following microdissection represents a useful supplementary or perhaps even an alternative technique...

  13. Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma

    DEFF Research Database (Denmark)

    Gjerdrum, Lise Mette; Sorensen, Boe Sandahl; Kjeldsen, Eigil

    2004-01-01

    We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods...

  14. Quantitative real-time RT-PCR and chromogenic in situ hybridization

    DEFF Research Database (Denmark)

    Rosa, Fabíola E; Silveira, Sara M; Silveira, Cássia G T

    2009-01-01

    . METHODS: To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH. RESULTS: The concordance...

  15. Periodontal pathogens: a quantitative comparison of anaerobic culture and real-time PCR

    NARCIS (Netherlands)

    Boutaga, Khalil; van Winkelhoff, Arie Jan; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.

    2005-01-01

    Periodontitis is a multi-factorial chronic inflammatory and destructive disease of the tooth-supporting tissues. Quantitative anaerobic culture techniques have been used for microbial diagnosis of the different forms of the disease. The aim of this study was to compare real-time PCR with

  16. Real-time quantitative PCR of Staphylococcus aureus and application in restaurant meals.

    Science.gov (United States)

    Berrada, H; Soriano, J M; Mañes, J; Picó, Y

    2006-01-01

    Staphylococcus aureus is considered the second most common pathogen to cause outbreaks of food poisoning, exceeded only by Campylobacter. Consumption of foods containing this microorganism is often identified as the cause of illness. In this study, a rapid, reliable, and sensitive real-time quantitative PCR was developed and compared with conventional culture methods. Real-time quantitative PCR was carried out by purifying DNA extracts of S. aureus with a Staphylococcus sample preparation kit and quantifying it in the LightCycler system with hybridization probes. The assay was linear from a range of 10 to 10(6) S. aureus cells (r2 > 0.997). The PCR reaction presented an efficiency of >85%. Accuracy of the PCR-based assay, expressed as percent bias, was around 13%, and the precision, expressed as a percentage of the coefficient of variation, was 7 to 10%. Intraday and interday variability were studied at 10(2) CFU/g and was 12 and 14%, respectively. The proposed method was applied to the analysis of 77 samples of restaurant meals in Valencia (Spain). In 11.6% of samples S. aureus was detected by real-time quantitative PCR, as well as by the conventional microbiological method. An excellent correspondence between real-time quantitative PCR and microbiological numbers (CFU/g) was observed with deviations of < 28%.

  17. Quantitative density measurements from a real-time neutron radiography system

    International Nuclear Information System (INIS)

    McRae, D.D.; Jenkins, R.W. Jr.; Brenizer, J.S.; Tobin, K.W.; Hosticka, B.; Sulcoski, M.F.

    1986-01-01

    An advanced video system has been assembled from commercially available equipment to support the real-time neutron radiography facility established jointly by the University of Virginia Department of Nuclear Engineering and Engineering Physics, and the Philip Morris Research Center. A schematic diagram of the equipment used for real-time neutron radiography is presented. To obtain quantitative density measurements with this system, several modifications of both hardware and image processing software were required. After implementation of these changes, the system was capable of determining material densities by measuring the degree of neutron attenuation

  18. 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....

  19. 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....

  20. Legionellosis and Lung Abscesses: Contribution of Legionella Quantitative Real-Time PCR to an Adapted Followup

    Directory of Open Access Journals (Sweden)

    G. Descours

    2013-01-01

    Full Text Available We report a case of severe Legionnaires' disease (LD complicated by a lung abscess in an immunocompetent patient who required ECMO therapy and thoracic surgery. The results of repeated Legionella quantitative real-time PCR performed on both sera and respiratory samples correlated with the LD severity and the poor clinical outcome. Moreover, the PCR allowed for the detection of Legionella DNA in the lung abscess specimen, which was negative when cultured for Legionella. This case report provides a logical basis for further investigations to examine whether the Legionella quantitative PCR could improve the assessment of LD severity and constitute a prognostic marker.

  1. Quantitative real-time monitoring of dryer effluent using fiber optic near-infrared spectroscopy.

    Science.gov (United States)

    Harris, S C; Walker, D S

    2000-09-01

    This paper describes a method for real-time quantitation of the solvents evaporating from a dryer. The vapor stream in the vacuum line of a dryer was monitored in real time using a fiber optic-coupled acousto-optic tunable filter near-infrared (AOTF-NIR) spectrometer. A balance was placed in the dryer, and mass readings were recorded for every scan of the AOTF-NIR. A partial least-squares (PLS) calibration was subsequently built based on change in mass over change in time for solvents typically used in a chemical manufacturing plant. Controlling software for the AOTF-NIR was developed. The software collects spectra, builds the PLS calibration model, and continuously fits subsequently collected spectra to the calibration, allowing the operator to follow the mass loss of solvent from the dryer. The results indicate that solvent loss can be monitored and quantitated in real time using NIR for the optimization of drying times. These time-based mass loss values have also been used to calculate "dynamic" vapor density values for the solvents. The values calculated are in agreement with values determined from the ideal gas law and could prove valuable as tools to measure temperature or pressure indirectly.

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Real-time quantitative phase reconstruction in off-axis digital holography using multiplexing.

    Science.gov (United States)

    Girshovitz, Pinhas; Shaked, Natan T

    2014-04-15

    We present a new approach for obtaining significant speedup in the digital processing of extracting unwrapped phase profiles from off-axis digital holograms. The new technique digitally multiplexes two orthogonal off-axis holograms, where the digital reconstruction, including spatial filtering and two-dimensional phase unwrapping on a decreased number of pixels, can be performed on both holograms together, without redundant operations. Using this technique, we were able to reconstruct, for the first time to our knowledge, unwrapped phase profiles from off-axis holograms with 1 megapixel in more than 30 frames per second using a standard single-core personal computer on a MATLAB platform, without using graphic-processing-unit programming or parallel computing. This new technique is important for real-time quantitative visualization and measurements of highly dynamic samples and is applicable for a wide range of applications, including rapid biological cell imaging and real-time nondestructive testing. After comparing the speedups obtained by the new technique for holograms of various sizes, we present experimental results of real-time quantitative phase visualization of cells flowing rapidly through a microchannel.

  5. 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.

  6. Rapid quantitative detection of Lactobacillus sakei in meat and fermented sausages by real-time PCR.

    Science.gov (United States)

    Martín, Belén; Jofré, Anna; Garriga, Margarita; Pla, Maria; Aymerich, Teresa

    2006-09-01

    A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.

  7. Exploring Valid Reference Genes for Quantitative Real-Time PCR Analysis in Sesamia inferens (Lepidoptera: Noctuidae)

    OpenAIRE

    Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

    2015-01-01

    The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study...

  8. Complementary techniques: validation of gene expression data by quantitative real time PCR.

    Science.gov (United States)

    Provenzano, Maurizio; Mocellin, Simone

    2007-01-01

    Microarray technology can be considered the most powerful tool for screening gene expression profiles of biological samples. After data mining, results need to be validated with highly reliable biotechniques allowing for precise quantitation of transcriptional abundance of identified genes. Quantitative real time PCR (qrt-PCR) technology has recently reached a level of sensitivity, accuracy and practical ease that support its use as a routine bioinstrumentation for gene level measurement. Currently, qrt-PCR is considered by most experts the most appropriate method to confirm or confute microarray-generated data. The knowledge of the biochemical principles underlying qrt-PCR as well as some related technical issues must be beard in mind when using this biotechnology.

  9. Real time quantitative phase microscopy based on single-shot transport of intensity equation (ssTIE) method

    Science.gov (United States)

    Yu, Wei; Tian, Xiaolin; He, Xiaoliang; Song, Xiaojun; Xue, Liang; Liu, Cheng; Wang, Shouyu

    2016-08-01

    Microscopy based on transport of intensity equation provides quantitative phase distributions which opens another perspective for cellular observations. However, it requires multi-focal image capturing while mechanical and electrical scanning limits its real time capacity in sample detections. Here, in order to break through this restriction, real time quantitative phase microscopy based on single-shot transport of the intensity equation method is proposed. A programmed phase mask is designed to realize simultaneous multi-focal image recording without any scanning; thus, phase distributions can be quantitatively retrieved in real time. It is believed the proposed method can be potentially applied in various biological and medical applications, especially for live cell imaging.

  10. Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos

    Directory of Open Access Journals (Sweden)

    Van Zeveren Alex

    2005-12-01

    Full Text Available Abstract Background Real-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the succesful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published GAPD, ACTB or 18S rRNA have been used as a single reference gene without prior verification of their expression stability. Normalization of the data using unstable controls can result in erroneous conclusions, especially when only one reference gene is used. Results In this study the transcription levels of 8 commonly used reference genes (ACTB, GAPD, Histone H2A, TBP, HPRT1, SDHA, YWHAZ and 18S rRNA were determined at different preimplantation stages (2-cell, 8-cell, blastocyst and hatched blastocyst in order to select the most stable genes to normalize quantitative data within different preimplantation embryo stages. Conclusion Using the geNorm application YWHAZ, GAPD and SDHA were found to be the most stable genes across the examined embryonic stages, while the commonly used ACTB was shown to be highly regulated. We recommend the use of the geometric mean of those 3 reference genes as an accurate normalization factor, which allows small expression differences to be reliably measured.

  11. Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR.

    Science.gov (United States)

    Furet, Jean-Pierre; Quénée, Pascal; Tailliez, Patrick

    2004-12-15

    Real-time quantitative PCR assays were developed for the absolute quantification of lactic acid bacteria (LAB) (Streptococcus thermophilus, Lactobacillus delbrueckii, L. casei, L. paracasei, L. rhamnosus, L. acidophilus and L. johnsonii) in fermented milk products. The results of molecular quantification and classic bacterial enumeration did not differ significantly with respect to S. thermophilus and the species of the L. casei group which were detected in the six commercial fermented products tested, thus showing that DNA extraction was efficient and that genomic DNA solutions were free of PCR inhibitors. For L. delbrueckii, the results of bacterial enumeration were generally lower by a factor 10 to 100 than those of PCR quantification, suggesting a loss of viability during storage of the dairy products at 1-8 degrees C for most of the strains in this species. Real-time quantitative assays enabled identification of the species of lactic acid bacterial strains initially present in commercial fermented milk products and their accurate quantification with a detection threshold of 10(3) cells per ml of product.

  12. Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay.

    Science.gov (United States)

    Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

    2011-01-01

    Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.

  13. Quantitative detection of Campylobacter jejuni on fresh chicken carcasses by real-time PCR.

    Science.gov (United States)

    Rönner, Anna-Clara; Lindmark, Hans

    2007-06-01

    Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 microl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

  14. 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.

  15. Investigations on abundance and activity of microbial sponge symbionts using quantitative real - time PCR

    DEFF Research Database (Denmark)

    Kumala, Lars; Hentschel, Ute; Bayer, Kristina

    Marine sponges are hosts to dense and diverse microbial consortia that are likely to play a key role in the metabolic processes of the host sponge due to their enormous abundance. Common symbioses between nitrogen transforming microorganisms and sponges indicate complex nitrogen cycling within...... the host. Of particular interest is determining the community structure and function of microbial symbionts in order to gain deeper insight into host-symbiont interactions. We investigated the abundance and activity of microbial symbionts in two Mediterranean sponge species using quantitative real-time PCR....... An absolute quantification of functional genes and transcripts in archaeal and bacterial symbionts was conducted to determine their involvement in nitrification and denitrification, comparing the low microbial abundance (LMA) sponge Dysidea avara with the high microbial abundance (HMA) representative Aplysina...

  16. Comparison of high and low virulence serotypes of Actinobacillus pleuropneumoniae by quantitative real-time PCR

    DEFF Research Database (Denmark)

    Schou, Kirstine Klitgaard; Angen, Øystein; Boye, Mette

    PCR data. Preliminary results showed that in both serotype 2 and serotype 6, the toxin producing gene apxIV was the most highly expressed of the investigated genes. The major difference observed between the two serotypes was that apfA, involved in type IV vili production, was significantly upregulated...... of high virulence while serotype 6 strains are normally found to be less pathogenic. To gain an understanding of the differential virulence of serotype 2 and 6, the expression of a panel of Ap genes during infection of porcine epithelial lung cells (SJPL) were examined by quantitative real-time PCR (q...... to be important for early establishment of the bacteria in the host were examined by qPCR. The genes examined were apfA, coding for a subunit of Type IV pili, kdsB coding for a gene involved in lippopolysacceride biosynthesis, and pgaB which is involved in biofilm formation, all three believed to be important...

  17. Real-time and quantitative isotropic spatial resolution susceptibility imaging for magnetic nanoparticles

    Science.gov (United States)

    Pi, Shiqiang; Liu, Wenzhong; Jiang, Tao

    2018-03-01

    The magnetic transparency of biological tissue allows the magnetic nanoparticle (MNP) to be a promising functional sensor and contrast agent. The complex susceptibility of MNPs, strongly influenced by particle concentration, excitation magnetic field and their surrounding microenvironment, provides significant implications for biomedical applications. Therefore, magnetic susceptibility imaging of high spatial resolution will give more detailed information during the process of MNP-aided diagnosis and therapy. In this study, we present a novel spatial magnetic susceptibility extraction method for MNPs under a gradient magnetic field, a low-frequency drive magnetic field, and a weak strength high-frequency magnetic field. Based on this novel method, a magnetic particle susceptibility imaging (MPSI) of millimeter-level spatial resolution (<3 mm) was achieved using our homemade imaging system. Corroborated by the experimental results, the MPSI shows real-time (1 s per frame acquisition) and quantitative abilities, and isotropic high resolution.

  18. Quantitative analysis of the dystrophin gene by real-time PCR

    Directory of Open Access Journals (Sweden)

    Maksimovic Nela

    2012-01-01

    Full Text Available Duchenne and Becker muscular dystrophy (DMD/BMD are severe X-linked neuromuscular disorders caused by mutations in the dystrophin gene. Our aim was to optimize a quantitative real-time PCR method based on SYBR® Green I chemistry for routine diagnostics of DMD/BMD deletion carriers. Twenty female relatives of DMD/BMD patients with previously detected partial gene deletions were studied. The relative quantity of the target exons was calculated by a comparative threshold cycle method (ΔΔCt. The carrier status of all subjects was successfully determined. The gene dosage ratio for non-carriers was 1.07±0.20, and for carriers 0.56±0.11. This assay proved to be simple, rapid, reliable and cost-effective.

  19. Expression analysis of fusarium wilt resistance gene in melon by real-time quantitative pcr

    International Nuclear Information System (INIS)

    Wang, X.; Xu, B.; Zhao, L.; Gao, P.; Luan, F.

    2014-01-01

    Melon Actin gene was used as a reference gene, to explore the gene expression profiles of the Fom-2 gene in roots, stems, and leaves of melon MR-1 under induction by Fusarium oxysporum f. sp. melonis. Monitoring using real-time quantitative PCR showed similar accumulation patterns of Fom-2 in roots, stems, and leaves over the observation period of 1 to 11 days; the expression level in stems was the highest. The expression of the Fom-2 gene was strengthened by the prolongation of induction time. In stems, the expression of Fom-2 was 5.737 times higher than in the control at three days; in roots, expression of Fom-2 was 5.617 times higher than in the control at five days. Similarly, the expression of Fom-2 in leaves obviously increased. It was 4.441 times higher than in the control at 5 days. The expression of Fom-2 was non-tissue specific, up-regulated under induction by Fusarium, and related to early resistance to Fusarium wilt. (author)

  20. A quantitative method to evaluate mesenchymal stem cell lipofection using real-time PCR.

    Science.gov (United States)

    Ribeiro, S C; Mendes, R; Madeira, C; Monteiro, G A; da Silva, C L; Cabral, J M S

    2010-01-01

    Genetic modification of human mesenchymal stem cells (MSC) is a powerful tool to improve the therapeutic utility of these cells and to increase the knowledge on their regulation mechanisms. In this context, strong efforts have been made recently to develop efficient nonviral gene delivery systems. Although several studies addressed this question most of them use the end product of a reporter gene instead of the DNA uptake quantification to test the transfection efficiency. In this study, we established a method based on quantitative real-time PCR (RT-PCR) to determine the intracellular plasmid DNA copy number in human MSC after lipofection. The procedure requires neither specific cell lysis nor DNA purification. The influence of cell number on the RT-PCR sensitivity was evaluated. The method showed good reproducibility, high sensitivity, and a wide linear range of 75-2.5 x 10⁶ plasmid DNA copies per cell. RT-PCR results were then compared with the percentage of transfected cells assessed by flow cytometry analysis, which showed that flow cytometry-based results are not always proportional to plasmid cellular uptake determined by RT-PCR. This work contributed for the establishment of a rapid quantitative assay to determine intracellular plasmid DNA in stem cells, which will be extremely beneficial for the optimization of gene delivery strategies. © 2010 American Institute of Chemical Engineers

  1. Verus: A Tool for Quantitative Analysis of Finite-State Real-Time Systems.

    Science.gov (United States)

    1996-08-12

    Symbolic model checking is a technique for verifying finite-state concurrent systems that has been extended to handle real - time systems . Models with...up to 10(exp 30) states can often be verified in minutes. In this paper, we present a new tool to analyze real - time systems , based on this technique...We have designed a language, called Verus, for the description of real - time systems . Such a description is compiled into a state-transition graph and

  2. [Quantitative fluorogenic real-time PCR assay for respiratory syncytial virus detection].

    Science.gov (United States)

    Zhang, Qi-wei; You, Shang-you; Sun, Ji-min; Wu, Qi; Yu, Chun-hua; Zhang, Chu-yu

    2005-07-01

    To Establish a rapid and objective quantitative fluorogenic real-time PCR assay for early detection of human respiratory syncytial virus (hRSV). Two pairs of primers and one TaqMan Fluorogenic probe that are specific for the recognition of the most conservative N gene of hRSV for virus detection with LighCycler PCR in 93 nasopharyngeal secretion specimens collected from infants and young children. The assay was compared with virus isolation, routine PCR, nested PCR, and enzyme-linked immunosorbent assay (ELISA). This TaqMan assay had a sensitivity of 1 x 10(2) cDNA copies/microl with a dynamic range between 1 x 10(2) and 1 x 10(7) cDNA copies/microl, which was the same as that of nested PCR, but 10 times more sensitive than routine PCR. The specificity of the assay was evaluated by comparing hRSV with polivirus type 1, coxsackie virus type 2, influenza A, influenza B and adenovirus type 7. A PCR product of the expected size (195 bp) was produced and fluorescence signal detected for hRSV, but not for any of the other viruses. The results in LightCycler and Rotor-Gene instrument were consistent. Forty-four specimens (43.9%) were hRSV-positive with this assay and 4 (4/93,4.3%) were hRSV-positive with ELISA, showing rather low correlation between the two methods. No visible relation was found between the concentration of hRSV RNA and severity of the disease. This assay is rapid, sensitive, specific and quantitative, and has the potential of wide application for early diagnosis of hRSV infection and evaluation of the therapeutic effect.

  3. Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

    Science.gov (United States)

    Ogrean, Christy; Jackson, Ben; Covino, James

    2010-01-01

    The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to >98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts. Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process. Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method. PMID:20567213

  4. A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl

    Science.gov (United States)

    Smith, Matthew M.; Schmutz, Joel A.; Apelgren, Chloe; Ramey, Andy M.

    2015-01-01

    Microscopic examination of blood smears can be effective at diagnosing and quantifying hematozoa infections. However, this method requires highly trained observers, is time consuming, and may be inaccurate for detection of infections at low levels of parasitemia. To develop a molecular methodology for identifying and quantifying Leucocytozoon parasite infection in wild waterfowl (Anseriformes), we designed a real-time, quantitative PCR protocol to amplify Leucocytozoon mitochondrial DNA using TaqMan fluorogenic probes and validated our methodology using blood samples collected from waterfowl in interior Alaska during late summer and autumn (n = 105). By comparing our qPCR results to those derived from a widely used nested PCR protocol, we determined that our assay showed high levels of sensitivity (91%) and specificity (100%) in detecting Leucocytozoon DNA from host blood samples. Additionally, results of a linear regression revealed significant correlation between the raw measure of parasitemia produced by our qPCR assay (Ct values) and numbers of parasites observed on blood smears (R2 = 0.694, P = 0.003), indicating that our assay can reliably determine the relative parasitemia levels among samples. This methodology provides a powerful new tool for studies assessing effects of haemosporidian infection in wild avian species.

  5. Real-time elastography with a novel quantitative technology for assessment of liver fibrosis in chronic hepatitis B

    International Nuclear Information System (INIS)

    Wang Juan; Guo Long; Shi Xiuying; Pan Wenqian; Bai Yunfei; Ai Hong

    2012-01-01

    Background: The accurate evaluation of liver fibrosis stage is important in determining the treatment strategy. The limitations of percutaneous liver biopsy as the gold standard are obvious for invasion. Real-time elastography with conventional ultrasound probes and a new quantitative technology for diffuse histological lesion is a novel approach for staging of liver fibrosis. Purpose: This study aimed to evaluate the value of real-time tissue elastography with a new quantitative technology for the assessment of liver fibrosis stage. Materials and methods: Real-time elastography was performed in 55 patients with liver fibrosis and chronic hepatitis B and in 20 healthy volunteers. Eleven parameters for every patient in colorcode image obtained from the real-time elastography were analyzed with principal components analysis. We analyzed the correlation between elasticity index and liver fibrosis stage and the accuracy of real-time elastography for liver fibrosis staging. Additionally, aspartate transaminase-to-platelet ratio index was also included in the analysis. Results: The Spearman's correlation coefficient between the elasticity index and the histologic fibrosis stage was 0.81, which is highly significant (p 0.05), respectively. Conclusions: Real-time elastography with a new quantitative technology for diffuse histological lesion is a new and promising sonography-based noninvasive method for the assessment of liver fibrosis in patients with chronic hepatitis B.

  6. Evaluation of Housekeeping Genes for Quantitative Real-Time PCR Analysis of Bradysia odoriphaga (Diptera: Sciaridae

    Directory of Open Access Journals (Sweden)

    Caihua Shi

    2016-07-01

    Full Text Available The soil insect Bradysia odoriphaga (Diptera: Sciaridae causes substantial damage to Chinese chive. Suitable reference genes in B. odoriphaga (Bradysia odoriphaga have yet to be identified for normalizing target gene expression among samples by quantitative real-time PCR (qRT-PCR. This study was focused on identifying the expression stability of 12 candidate housekeeping genes in B. odoriphaga under various experiment conditions. The final stability ranking of 12 housekeeping genes was obtained with RefFinder, and the most suitable number of reference genes was analyzed by GeNorm. The results revealed that the most appropriate sets of internal controls were RPS15, RPL18, and RPS18 across developmental phases; RPS15, RPL28, and GAPDH across temperatures; RPS15 and RPL18 across pesticide treatments; RSP5, RPS18, and SDHA across photoperiods; ACTb, RPS18, and RPS15 across diets; RPS13 and RPL28 across populations; and RPS15, ACTb, and RPS18 across all samples. The use of the most suitable reference genes versus an arbitrarily selected reference gene resulted in significant differences in the analysis of a target gene expression. HSP23 in B. odoriphaga was found to be up-regulated under low temperatures. These results will contribute to the standardization of qRT-PCR and will also be valuable for further research on gene function in B. odoriphaga.

  7. 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.

  8. Detection of Ophiocordyceps sinensis in soil by quantitative real-time PCR.

    Science.gov (United States)

    Peng, Qingyun; Zhong, Xin; Lei, Wei; Zhang, Guren; Liu, Xin

    2013-03-01

    Ophiocordyceps sinensis, one of the best known entomopathogenic fungi in traditional Chinese medicine, parasitizes larvae of the moth genus Thitarodes, which lives in soil tunnels. However, little is known about the spatial distribution of O. sinensis in the soil. We established a protocol for DNA extraction, purification, and quantification of O. sinensis in soil with quantitative real-time PCR targeting the internal transcribed spacer region. The method was assessed using 34 soil samples from Tibet. No inhibitory effects in purified soil DNA extracts were detected. The standard curve method for absolute DNA quantification generated crossing point values that were strongly and linearly correlated to the log10 of the initial amount of O. sinensis genomic DNA (r(2) = 0.999) over 7 orders of magnitude (4 × 10(1) to 4 × 10(7) fg). The amplification efficiency and y-intercept value of the standard curve were 1.953 and 37.70, respectively. The amount of O. sinensis genomic DNA decreased with increasing soil depth and horizontal distance from a sclerotium (P protocol is rapid, specific, sensitive, and provides a powerful tool for quantification of O. sinensis from soil.

  9. Identification of circulating miRNA biomarkers based on global quantitative real-time PCR profiling

    Directory of Open Access Journals (Sweden)

    Kang Kang

    2012-02-01

    Full Text Available Abstract MicroRNAs (miRNAs are small noncoding RNAs (18-25 nucleotides that regulate gene expression at the post-transcriptional level. Recent studies have demonstrated the presence of miRNAs in the blood circulation. Deregulation of miRNAs in serum or plasma has been associated with many diseases including cancers and cardiovascular diseases, suggesting the possible use of miRNAs as diagnostic biomarkers. However, the detection of the small amount of miRNAs found in serum or plasma requires a method with high sensitivity and accuracy. Therefore, the current study describes polymerase chain reaction (PCR-based methods for measuring circulating miRNAs. Briefly, the procedure involves four major steps: (1 sample collection and preparation; (2 global miRNAs profiling using quantitative real-time PCR (qRT-PCR; (3 data normalization and analysis; and (4 selection and validation of miRNA biomarkers. In conclusion, qRT-PCR is a promising method for profiling of circulating miRNAs as biomarkers.

  10. Selecting and validating reference genes for quantitative real-time PCR in Plutella xylostella (L.).

    Science.gov (United States)

    You, Yanchun; Xie, Miao; Vasseur, Liette; You, Minsheng

    2018-05-01

    Gene expression analysis provides important clues regarding gene functions, and quantitative real-time PCR (qRT-PCR) is a widely used method in gene expression studies. Reference genes are essential for normalizing and accurately assessing gene expression. In the present study, 16 candidate reference genes (ACTB, CyPA, EF1-α, GAPDH, HSP90, NDPk, RPL13a, RPL18, RPL19, RPL32, RPL4, RPL8, RPS13, RPS4, α-TUB, and β-TUB) from Plutella xylostella were selected to evaluate gene expression stability across different experimental conditions using five statistical algorithms (geNorm, NormFinder, Delta Ct, BestKeeper, and RefFinder). The results suggest that different reference genes or combinations of reference genes are suitable for normalization in gene expression studies of P. xylostella according to the different developmental stages, strains, tissues, and insecticide treatments. Based on the given experimental sets, the most stable reference genes were RPS4 across different developmental stages, RPL8 across different strains and tissues, and EF1-α across different insecticide treatments. A comprehensive and systematic assessment of potential reference genes for gene expression normalization is essential for post-genomic functional research in P. xylostella, a notorious pest with worldwide distribution and a high capacity to adapt and develop resistance to insecticides.

  11. Species identification of Cannabis sativa using real-time quantitative PCR (qPCR).

    Science.gov (United States)

    Johnson, Christopher E; Premasuthan, Amritha; Satkoski Trask, Jessica; Kanthaswamy, Sree

    2013-03-01

    Most narcotics-related cases in the United States involve Cannabis sativa. Material is typically identified based on the cystolithic hairs on the leaves and with chemical tests to identify of the presence of cannabinoids. Suspect seeds are germinated into a viable plant so that morphological and chemical tests can be conducted. Seed germination, however, causes undue analytical delays. DNA analyses that involve the chloroplast and nuclear genomes have been developed for identification of C. sativa materials, but they require several nanograms of template DNA. Using the trnL 3' exon-trnF intragenic spacer regions within the C. sativa chloroplast, we have developed a real-time quantitative PCR assay that is capable of identifying picogram amounts of chloroplast DNA for species determination of suspected C. sativa material. This assay provides forensic science laboratories with a quick and reliable method to identify an unknown sample as C. sativa. © 2013 American Academy of Forensic Sciences.

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

    Science.gov (United States)

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

    2018-06-01

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

  13. Exploring Valid Reference Genes for Quantitative Real-time PCR Analysis in Plutella xylostella (Lepidoptera: Plutellidae)

    Science.gov (United States)

    Fu, Wei; Xie, Wen; Zhang, Zhuo; Wang, Shaoli; Wu, Qingjun; Liu, Yong; Zhou, Xiaomao; Zhou, Xuguo; Zhang, Youjun

    2013-01-01

    Abstract: Quantitative real-time PCR (qRT-PCR), a primary tool in gene expression analysis, requires an appropriate normalization strategy to control for variation among samples. The best option is to compare the mRNA level of a target gene with that of reference gene(s) whose expression level is stable across various experimental conditions. In this study, expression profiles of eight candidate reference genes from the diamondback moth, Plutella xylostella, were evaluated under diverse experimental conditions. RefFinder, a web-based analysis tool, integrates four major computational programs including geNorm, Normfinder, BestKeeper, and the comparative ΔCt method to comprehensively rank the tested candidate genes. Elongation factor 1 (EF1) was the most suited reference gene for the biotic factors (development stage, tissue, and strain). In contrast, although appropriate reference gene(s) do exist for several abiotic factors (temperature, photoperiod, insecticide, and mechanical injury), we were not able to identify a single universal reference gene. Nevertheless, a suite of candidate reference genes were specifically recommended for selected experimental conditions. Our finding is the first step toward establishing a standardized qRT-PCR analysis of this agriculturally important insect pest. PMID:23983612

  14. 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.

  15. GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR.

    Science.gov (United States)

    Gubelmann, Carine; Gattiker, Alexandre; Massouras, Andreas; Hens, Korneel; David, Fabrice; Decouttere, Frederik; Rougemont, Jacques; Deplancke, Bart

    2011-01-01

    The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/. Database URL: http://deplanckelab.epfl.ch.

  16. A method for accurate detection of genomic microdeletions using real-time quantitative PCR

    Directory of Open Access Journals (Sweden)

    Bassett Anne S

    2005-12-01

    Full Text Available Abstract Background Quantitative Polymerase Chain Reaction (qPCR is a well-established method for quantifying levels of gene expression, but has not been routinely applied to the detection of constitutional copy number alterations of human genomic DNA. Microdeletions or microduplications of the human genome are associated with a variety of genetic disorders. Although, clinical laboratories routinely use fluorescence in situ hybridization (FISH to identify such cryptic genomic alterations, there remains a significant number of individuals in which constitutional genomic imbalance is suspected, based on clinical parameters, but cannot be readily detected using current cytogenetic techniques. Results In this study, a novel application for real-time qPCR is presented that can be used to reproducibly detect chromosomal microdeletions and microduplications. This approach was applied to DNA from a series of patient samples and controls to validate genomic copy number alteration at cytoband 22q11. The study group comprised 12 patients with clinical symptoms of chromosome 22q11 deletion syndrome (22q11DS, 1 patient trisomic for 22q11 and 4 normal controls. 6 of the patients (group 1 had known hemizygous deletions, as detected by standard diagnostic FISH, whilst the remaining 6 patients (group 2 were classified as 22q11DS negative using the clinical FISH assay. Screening of the patients and controls with a set of 10 real time qPCR primers, spanning the 22q11.2-deleted region and flanking sequence, confirmed the FISH assay results for all patients with 100% concordance. Moreover, this qPCR enabled a refinement of the region of deletion at 22q11. Analysis of DNA from chromosome 22 trisomic sample demonstrated genomic duplication within 22q11. Conclusion In this paper we present a qPCR approach for the detection of chromosomal microdeletions and microduplications. The strategic use of in silico modelling for qPCR primer design to avoid regions of repetitive

  17. Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR

    Directory of Open Access Journals (Sweden)

    López-Revilla Rubén

    2010-05-01

    Full Text Available Abstract Background We have developed an ultrasensitive method based on conventional PCR preamplification followed by nested amplification through real time PCR (qPCR in the presence of the DNA intercalating agent EvaGreen. Results Amplification mixtures calibrated with a known number of pHV101 copies carrying a 645 base pair (bp-long insert of the human papillomavirus type 16 (HPV16 E6 oncogene were used to generate the E6-1 amplicon of 645 bp by conventional PCR and then the E6-2 amplicon of 237 bp by nested qPCR. Direct and nested qPCR mixtures for E6-2 amplification corresponding to 2.5 × 102-2.5 × 106 initial pHV101 copies had threshold cycle (Ct values in the ranges of 18.7-29.0 and 10.0-25.0, respectively. The Ct of qPCR mixtures prepared with 1/50 volumes of preamplified mixtures containing 50 ng of DNA of the SiHa cell line (derived from an invasive cervical cancer with one HPV16 genome per cell was 19.9. Thermal fluorescence extinction profiles of E6-2 amplicons generated from pHV101 and SiHa DNA were identical, with a peak at 85.5°C. Conclusions Our method based on conventional preamplification for 15 cycles increased 10,750 times the sensitivity of nested qPCR for the quantitation of the E6 viral oncogene and confirmed that the SiHa cell line contains one E6-HPV16 copy per cell.

  18. Exploring valid reference genes for quantitative real - time rt - pce studies of hydrogenperoxide signaling in arabidopsis

    International Nuclear Information System (INIS)

    Zhou, H.; Han, B.; Xie, Y.; Zhang, J.; Shen, W.

    2015-01-01

    Hydrogen peroxide (H/sub 2/O/sub 2/ ) acts as a signaling molecule modulating the expression of various genes in plants. However, the reference gene(s) used for gene expression analysis of H/sub 2/O/sub 2/ signaling is still arbitrary. A reliable result obtained by quantitative real-time RT-PCR (RT-qPCR) highly depends on accurate transcript normalization using stably expressed reference genes, whereas the inaccurate normalization could easily lead to the false conclusions. In this report, by using geNorm and NormFinder algorithms, 12 candidate reference genes were evaluated and compared in root and shoot tissues of Arabidopsis upon different doses of H/sub 2/O/sub 2/. The results revealed that, in our experimental conditions, three novel reference genes (TIP41-like, UKN, and UBC21) were identified and validated as suitable reference genes for RT-qPCR normalization in both root and shoot tissues under oxidative stress. This conclusion was further confirmed by publicly available microarray data of methyl viologen and drought stress. In comparison with a single reference gene (EF-1a), the expression pattern of ZAT12 modulated by H/sub 2/O/sub 2/, when using TIP41-like, UKN, and UBC21 as multiple reference gene(s), was similar with the previous reports by using northern blotting. Thus, we proposed that these three reference genes might be good candidates for other researchers to include in their reference gene validation in gene expression studies under H/sub 2/O/sub 2/ related oxidative stress. (author)

  19. The quantification of spermatozoa by real-time quantitative PCR, spectrophotometry, and spermatophore cap size.

    Science.gov (United States)

    Doyle, Jacqueline M; McCormick, Cory R; DeWoody, J Andrew

    2011-01-01

    Many animals, such as crustaceans, insects, and salamanders, package their sperm into spermatophores, and the number of spermatozoa contained in a spermatophore is relevant to studies of sexual selection and sperm competition. We used two molecular methods, real-time quantitative polymerase chain reaction (RT-qPCR) and spectrophotometry, to estimate sperm numbers from spermatophores. First, we designed gene-specific primers that produced a single amplicon in four species of ambystomatid salamanders. A standard curve generated from cloned amplicons revealed a strong positive relationship between template DNA quantity and cycle threshold, suggesting that RT-qPCR could be used to quantify sperm in a given sample. We then extracted DNA from multiple Ambystoma maculatum spermatophores, performed RT-qPCR on each sample, and estimated template copy numbers (i.e. sperm number) using the standard curve. Second, we used spectrophotometry to determine the number of sperm per spermatophore by measuring DNA concentration relative to the genome size. We documented a significant positive relationship between the estimates of sperm number based on RT-qPCR and those based on spectrophotometry. When these molecular estimates were compared to spermatophore cap size, which in principle could predict the number of sperm contained in the spermatophore, we also found a significant positive relationship between sperm number and spermatophore cap size. This linear model allows estimates of sperm number strictly from cap size, an approach which could greatly simplify the estimation of sperm number in future studies. These methods may help explain variation in fertilization success where sperm competition is mediated by sperm quantity. © 2010 Blackwell Publishing Ltd.

  20. Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae.

    Directory of Open Access Journals (Sweden)

    Meng Sun

    Full Text Available The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA, elongation factor 1 (EF1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ribosomal protein S13 (RPS13, ribosomal protein S20 (RPS20, tubulin (TUB, and β-actin (ACTB were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1 were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands. 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults. 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C. To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83 was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

  1. Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

    2015-01-01

    The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

  2. A human fecal contamination index for ranking impaired recreational watersusing the HF183 quantitative real-time PCR method

    Science.gov (United States)

    Human fecal pollution of surface water remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for recreational water quality risk managem...

  3. Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

    Science.gov (United States)

    There is a growing interest in the application of human-associated fecal sourceidentification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data q...

  4. 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....

  5. 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...

  6. EVALUATION OF A RAPID, QUANTITATIVE REAL-TIME PCR METHOD FOR ENUMERATION OF PATHOGENIC CANDIDA CELLS IN WATER

    Science.gov (United States)

    Quantitative Real-Time PCR (QRT-PCR) technology, incorporating fluorigenic 5' nuclease (TaqMan?) chemistry, was developed for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C....

  7. Decay Of Bacterial Pathogens, Fecal Indicators, And Real-Time Quantitative PCR Genetic Markers In Manure-Amended Soils

    Science.gov (United States)

    This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria (FIB), and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manure-amended agricultural soils. Known concentrations of transformed green...

  8. Decay Of Bacterial Pathogen, Fecal Indicators, And Real-Time Quantitative PCR Genetic Markers In Manure Amended Soils

    Science.gov (United States)

    This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria, and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manre-amended agricultural soils. Known concentrations of transformed green fluore...

  9. An integrated real-time diagnostic concept using expert systems, qualitative reasoning and quantitative analysis

    International Nuclear Information System (INIS)

    Edwards, R.M.; Lee, K.Y.; Kumara, S.; Levine, S.H.

    1989-01-01

    An approach for an integrated real-time diagnostic system is being developed for inclusion as an integral part of a power plant automatic control system. In order to participate in control decisions and automatic closed loop operation, the diagnostic system must operate in real-time. Thus far, an expert system with real-time capabilities has been developed and installed on a subsystem at the Experimental Breeder Reactor (EBR-II) in Idaho, USA. Real-time simulation testing of advanced power plant concepts at the Pennsylvania State University has been developed and was used to support the expert system development and installation at EBR-II. Recently, the US National Science Foundation (NSF) and the US Department of Energy (DOE) have funded a Penn State research program to further enhance application of real-time diagnostic systems by pursuing implementation in a distributed power plant computer system including microprocessor based controllers. This paper summarizes past, current, planned, and possible future approaches to power plant diagnostic systems research at Penn State. 34 refs., 9 figs

  10. Real-time quantitative PCR for retrovirus-like particle quantification in CHO cell culture.

    Science.gov (United States)

    de Wit, C; Fautz, C; Xu, Y

    2000-09-01

    Chinese hamster ovary (CHO) cells have been widely used to manufacture recombinant proteins intended for human therapeutic uses. Retrovirus-like particles, which are apparently defective and non-infectious, have been detected in all CHO cells by electron microscopy (EM). To assure viral safety of CHO cell-derived biologicals, quantification of retrovirus-like particles in production cell culture and demonstration of sufficient elimination of such retrovirus-like particles by the down-stream purification process are required for product market registration worldwide. EM, with a detection limit of 1x10(6) particles/ml, is the standard retrovirus-like particle quantification method. The whole process, which requires a large amount of sample (3-6 litres), is labour intensive, time consuming, expensive, and subject to significant assay variability. In this paper, a novel real-time quantitative PCR assay (TaqMan assay) has been developed for the quantification of retrovirus-like particles. Each retrovirus particle contains two copies of the viral genomic particle RNA (pRNA) molecule. Therefore, quantification of retrovirus particles can be achieved by quantifying the pRNA copy number, i.e. every two copies of retroviral pRNA is equivalent to one retrovirus-like particle. The TaqMan assay takes advantage of the 5'-->3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 Sequence Detection System of PE Applied Biosystems (Foster City, CA, U.S.A.) for automated pRNA quantification through a dual-labelled fluorogenic probe. The TaqMan quantification technique is highly comparable to the EM analysis. In addition, it offers significant advantages over the EM analysis, such as a higher sensitivity of less than 600 particles/ml, greater accuracy and reliability, higher sample throughput, more flexibility and lower cost. Therefore, the TaqMan assay should be used as a substitute for EM analysis for retrovirus-like particle quantification in CHO cell

  11. On-Orbit Quantitative Real-Time Gene Expression Analysis Using the Wetlab-2 System

    Science.gov (United States)

    Parra, Macarena; Jung, Jimmy; Almeida, Eduardo; Boone, Travis; Tran, Luan; Schonfeld, Julie

    2015-01-01

    NASA Ames Research Center's WetLab-2 Project enables on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The resulting RNA is dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The project selected the Cepheid SmartCycler (TradeMark), a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, because of its advantages including rugged modular design, low power consumption, rapid thermal ramp times and four-color multiplex detection. Single tube multiplex assays can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to conduct qRT-PCR and generate results on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also eliminate the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples and provide on-orbit gene expression benchmarking prior to sample return. Finally, the system can also be used for analysis of

  12. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data

    NARCIS (Netherlands)

    Ramakers, Christian; Ruijter, Jan M.; Deprez, Ronald H. Lekanne; Moorman, Antoon F. M.

    2003-01-01

    Quantification of mRNAs using real-time polymerase chain reaction (PCR) by monitoring the product formation with the fluorescent dye SYBR Green I is being extensively used in neurosciences, developmental biology, and medical diagnostics. Most PCR data analysis procedures assume that the PCR

  13. Estimating marginal properties of quantitative real-time PCR data using nonlinear mixed models

    DEFF Research Database (Denmark)

    Gerhard, Daniel; Bremer, Melanie; Ritz, Christian

    2014-01-01

    A unified modeling framework based on a set of nonlinear mixed models is proposed for flexible modeling of gene expression in real-time PCR experiments. Focus is on estimating the marginal or population-based derived parameters: cycle thresholds and ΔΔc(t), but retaining the conditional mixed mod...

  14. Development of a real-time quantitative assay for detection of Epstein-Barr virus

    NARCIS (Netherlands)

    Niesters, H. G.; van Esser, J.; Fries, E.; Wolthers, K. C.; Cornelissen, J.; Osterhaus, A. D.

    2000-01-01

    With the use of real-time PCR, we developed and evaluated a rapid, sensitive, specific, and reproducible method for the detection of Epstein-Barr virus (EBV) DNA in plasma samples. This method allowed us to screen plasma and serum samples over a range between 100 and 10(7) copies of DNA per ml using

  15. Development of a real-time quantitative assay for detection of Epstein-Barr virus

    NARCIS (Netherlands)

    H.G.M. Niesters (Bert); E. Fries; K.C. Wolthers (Katja); A.D.M.E. Osterhaus (Albert); J.J. Cornelissen (Jan); J.W.J. van Esser (Joost)

    2000-01-01

    textabstractWith the use of real-time PCR, we developed and evaluated a rapid, sensitive, specific, and reproducible method for the detection of Epstein-Barr virus (EBV) DNA in plasma samples. This method allowed us to screen plasma and serum samples over a range between 100 and

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

    Directory of Open Access Journals (Sweden)

    Kai Wang

    2017-04-01

    Full Text Available Quantitative real-time reverse transcription PCR (RT-qPCR has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes. Amorphophallus is a perennial plant with a high content of konjac glucomannan (KGM in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes in Amorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genes Amorphophallus were cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging in A. albus and A. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated that EF1-a, EIF4A, H3 and UBQ were the best reference genes under heat stress in Amorphophallus. Furthermore, EF1-a, EIF4A, TUB, and RP were the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined that EF1-α, EIF4A, and CYP were stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock protein SHSP, which is related to heat stress in Amorphophallus. In sum, EF1-α and EIF4A were the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different

  17. Quantitative Real-time PCR detection of putrescine-producing Gram-negative bacteria

    Directory of Open Access Journals (Sweden)

    Kristýna Maršálková

    2017-01-01

    Full Text Available Biogenic amines are indispensable components of living cells; nevertheless these compounds could be toxic for human health in higher concentrations. Putrescine is supposed to be the major biogenic amine associated with microbial food spoilage. Development of reliable, fast and culture-independent molecular methods to detect bacteria producing biogenic amines deserves the attention, especially of the food industry in purpose to protect health. The objective of this study was to verify the newly designed primer sets for detection of two inducible genes adiA and speF together in Salmonella enterica and Escherichia coli genome by Real-time PCR. These forenamed genes encode enzymes in the metabolic pathway which leads to production of putrescine in Gram-negative bacteria. Moreover, relative expression of these genes was studied in E. coli CCM 3954 strain using Real-time PCR. In this study, sets of new primers for the detection two inducible genes (speF and adiA in Salmonella enterica and E. coli by Real-time PCR were designed and tested. Amplification efficiency of a Real-time PCR was calculated from the slope of the standard curves (adiA, speF, gapA. An efficiency in a range from 95 to 105 % for all tested reactions was achieved. The gene expression (R of adiA and speF genes in E. coli was varied depending on culture conditions. The highest gene expression of adiA and speF was observed at 6, 24 and 36 h (RadiA ~ 3, 5, 9; RspeF ~11, 10, 9; respectively after initiation of growth of this bacteria in nutrient broth medium enchired with amino acids. The results show that these primers could be used for relative quantification analysis of E. coli.

  18. Development of quantitative real-time PCR for detection and enumeration of Enterobacteriaceae.

    Science.gov (United States)

    Takahashi, Hajime; Saito, Rumi; Miya, Satoko; Tanaka, Yuichiro; Miyamura, Natsumi; Kuda, Takashi; Kimura, Bon

    2017-04-04

    The family Enterobacteriaceae, members of which are widely distributed in the environment, includes many important human pathogens. In this study, a rapid real-time PCR method targeting rplP, coding for L16 protein, a component of the ribosome large subunit, was developed for enumerating Enterobacteriaceae strains, and its efficiency was evaluated using naturally contaminated food products. The rplP-targeted real-time PCR amplified Enterobacteriaceae species with Ct values of 14.0-22.8, whereas the Ct values for non-Enterobacteriaceae species were >30, indicating the specificity of this method for the Enterobacteriaceae. Using a calibration curve of Ct=-3.025 (log CFU/g)+37.35, which was calculated from individual plots of the cell numbers in different concentrations of 5 Enterobacteriaceae species, the rplP-targeted real-time PCR was applied to 51 food samples. A Enterobacteriaceae species in foods rapidly and accurately, and therefore, it can be used for the microbiological risk analysis of foods. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. 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.

  20. 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...

  1. 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.

  2. Quantitative analysis of diet structure by real-time PCR, reveals different feeding patterns by two dominant grasshopper species

    Science.gov (United States)

    Huang, Xunbing; Wu, Huihui; McNeill, Mark Richard; Qin, Xinghu; Ma, Jingchuan; Tu, Xiongbing; Cao, Guangchun; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

    2016-01-01

    Studies on grasshopper diets have historically employed a range of methodologies, each with certain advantages and disadvantages. For example, some methodologies are qualitative instead of quantitative. Others require long experimental periods or examine population-level effects, only. In this study, we used real-time PCR to examine diets of individual grasshoppers. The method has the advantage of being both fast and quantitative. Using two grasshopper species, Oedaleus asiaticus and Dasyhippus barbipes, we designed ITS primer sequences for their three main host plants, Stipa krylovii, Leymus chinensis and Cleistogenes squarrosa and used real-time PCR method to test diet structure both qualitatively and quantitatively. The lowest detection efficiency of the three grass species was ~80% with a strong correlation between actual and PCR-measured food intake. We found that Oedaleus asiaticus maintained an unchanged diet structure across grasslands with different grass communities. By comparison, Dasyhippus barbipes changed its diet structure. These results revealed why O. asiaticus distribution is mainly confined to Stipa-dominated grassland, and D. barbipes is more widely distributed across Inner Mongolia. Overall, real-time PCR was shown to be a useful tool for investigating grasshopper diets, which in turn offers some insight into grasshopper distributions and improved pest management. PMID:27562455

  3. Effect of ionizing radiation on the quantitative detection of Salmonella using real-time PCR

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Sangyong; Jung, Jinwoo [Radiation Research Center for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Kim, Minjeong; Ryu, Sangryeol [Department of Food and Animal Biotechnology, School of Agricultural Biotechnology, Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921 (Korea, Republic of); Kim, Dongho [Radiation Research Center for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of)], E-mail: fungikim@kaeri.re.kr

    2008-09-15

    Food irradiation is an economically viable technology for inactivating foodborne pathogens, but irradiation can mask pathogens in unhygienically prepared food. The aim of this study was to investigate the effect of irradiation treatment on the detection of Salmonella using real-time PCR. Three commercially available kits were tested, of which the InstaGene Matrix procedure was most effective in preparing template DNA from Salmonella exposed to radiation in broth culture. The minimum level of detection by real-time PCR combined with InstaGene Matrix was 3 log units of Salmonella per milliliter. However, when pure cultures of Salmonella were irradiated at 3 and 5 kGy, the cycle threshold (C{sub T}) increased 1-1.5-fold compared to irradiation at 0 and 1 kGy. This indicated that irradiation treatment may result in an underestimation of bacterial counts due to radiation-induced DNA lesions. We also compared C{sub T} values in inoculated chicken homogenates before and after irradiation, which in this model caused a 1.3-3.3-fold underestimation of bacterial counts with respect to irradiation dose.

  4. Design and Validation of Real-Time PCR: Quantitative Diagnosis of Common Leishmania Species in Iran.

    Science.gov (United States)

    Fekri Soofi Abadi, Maryam; Dabiri, Shahriar; Fotouhi Ardakani, Reza; Fani Malaki, Lina; Amirpoor Rostami, Sahar; Ziasistani, Mahsa; Dabiri, Donya

    2016-07-01

    Design and validation of Real-time PCR on the protected gene region ITS2 to quantify the parasite load in common leishmania (L) species. Probe and primer were designed from the ITS2 region between the rRNA genes with minimum gene variation in three common leishmania species followed by a Real-time PCR using the Taq man probe method in the form of absolute quantification. A series of different concentrations of leishmania were analyzed. After the purified PCR product was successfully placed in a PTG19-T plasmid vector, specialized ITS2 region was cloned in this plasmid. In the last phase, the cloned gene was transferred to the Ecoli.Top10F bacteria. The standard plasmid was provided in 10(7) to 10(1) copies/rxn concentrations. The specification and clinical sensitivity of the data was analyzed using inter and intra scales. The probe and primer were designed using three species, including L. infantum, L. major, and L.tropica. Seven concentrations of purified parasite in culture media showed that the selected region for quantifying the parasite is suitable. Clinical and analytical specificity and sensitivity were both 100%, respectively. The Taq man method for the ITS2 region in leishmania is one the most sensitive diagnostic test for identifying the parasite load and is suggested as a tool for fast identification and quantification of species.

  5. Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus

    Directory of Open Access Journals (Sweden)

    Tandale Babasaheb V

    2008-12-01

    Full Text Available Abstract Background Chandipura virus (CHPV, a member of family Rhabdoviridae was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55–75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR using TaqMan technology was developed for rapid diagnosis. Methods Primers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [in vivo (mice in ovo (eggs, in vitro (Vero E6, PS, RD and Sand fly cell line] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard. Results Real-time one step RT-PCR was optimized using in vitro transcribed (IVT RNA. Standard curve showed linear relationship for wide range of 102-1010 (r2 = 0.99 with maximum Coefficient of variation (CV = 5.91% for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 × 100 PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 102 PFU/ml. Vero and PS cell-lines (1.2 × 103 PFU/ml were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy

  6. Intra-laboratory validation of chronic bee paralysis virus quantitation using an accredited standardised real-time quantitative RT-PCR method.

    Science.gov (United States)

    Blanchard, Philippe; Regnault, Julie; Schurr, Frank; Dubois, Eric; Ribière, Magali

    2012-03-01

    Chronic bee paralysis virus (CBPV) is responsible for chronic bee paralysis, an infectious and contagious disease in adult honey bees (Apis mellifera L.). A real-time RT-PCR assay to quantitate the CBPV load is now available. To propose this assay as a reference method, it was characterised further in an intra-laboratory study during which the reliability and the repeatability of results and the performance of the assay were confirmed. The qPCR assay alone and the whole quantitation method (from sample RNA extraction to analysis) were both assessed following the ISO/IEC 17025 standard and the recent XP U47-600 standard issued by the French Standards Institute. The performance of the qPCR assay and of the overall CBPV quantitation method were validated over a 6 log range from 10(2) to 10(8) with a detection limit of 50 and 100 CBPV RNA copies, respectively, and the protocol of the real-time RT-qPCR assay for CBPV quantitation was approved by the French Accreditation Committee. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Evaluation of methods for oligonucleotide array data via quantitative real-time PCR.

    Science.gov (United States)

    Qin, Li-Xuan; Beyer, Richard P; Hudson, Francesca N; Linford, Nancy J; Morris, Daryl E; Kerr, Kathleen F

    2006-01-17

    There are currently many different methods for processing and summarizing probe-level data from Affymetrix oligonucleotide arrays. It is of great interest to validate these methods and identify those that are most effective. There is no single best way to do this validation, and a variety of approaches is needed. Moreover, gene expression data are collected to answer a variety of scientific questions, and the same method may not be best for all questions. Only a handful of validation studies have been done so far, most of which rely on spike-in datasets and focus on the question of detecting differential expression. Here we seek methods that excel at estimating relative expression. We evaluate methods by identifying those that give the strongest linear association between expression measurements by array and the "gold-standard" assay. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is generally considered the "gold-standard" assay for measuring gene expression by biologists and is often used to confirm findings from microarray data. Here we use qRT-PCR measurements to validate methods for the components of processing oligo array data: background adjustment, normalization, mismatch adjustment, and probeset summary. An advantage of our approach over spike-in studies is that methods are validated on a real dataset that was collected to address a scientific question. We initially identify three of six popular methods that consistently produced the best agreement between oligo array and RT-PCR data for medium- and high-intensity genes. The three methods are generally known as MAS5, gcRMA, and the dChip mismatch mode. For medium- and high-intensity genes, we identified use of data from mismatch probes (as in MAS5 and dChip mismatch) and a sequence-based method of background adjustment (as in gcRMA) as the most important factors in methods' performances. However, we found poor reliability for methods using mismatch probes for low-intensity genes

  8. Evaluation of methods for oligonucleotide array data via quantitative real-time PCR

    Directory of Open Access Journals (Sweden)

    Morris Daryl E

    2006-01-01

    Full Text Available Abstract Background There are currently many different methods for processing and summarizing probe-level data from Affymetrix oligonucleotide arrays. It is of great interest to validate these methods and identify those that are most effective. There is no single best way to do this validation, and a variety of approaches is needed. Moreover, gene expression data are collected to answer a variety of scientific questions, and the same method may not be best for all questions. Only a handful of validation studies have been done so far, most of which rely on spike-in datasets and focus on the question of detecting differential expression. Here we seek methods that excel at estimating relative expression. We evaluate methods by identifying those that give the strongest linear association between expression measurements by array and the "gold-standard" assay. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR is generally considered the "gold-standard" assay for measuring gene expression by biologists and is often used to confirm findings from microarray data. Here we use qRT-PCR measurements to validate methods for the components of processing oligo array data: background adjustment, normalization, mismatch adjustment, and probeset summary. An advantage of our approach over spike-in studies is that methods are validated on a real dataset that was collected to address a scientific question. Results We initially identify three of six popular methods that consistently produced the best agreement between oligo array and RT-PCR data for medium- and high-intensity genes. The three methods are generally known as MAS5, gcRMA, and the dChip mismatch mode. For medium- and high-intensity genes, we identified use of data from mismatch probes (as in MAS5 and dChip mismatch and a sequence-based method of background adjustment (as in gcRMA as the most important factors in methods' performances. However, we found poor reliability for methods

  9. Quantitative Determination of Plasmodium vivax Gametocytes by Real-Time Quantitative Nucleic Acid Sequence-Based Amplification in Clinical Samples

    NARCIS (Netherlands)

    Beurskens, Martijn; Mens, Pètra; Schallig, Henk; Syafruddin, Din; Asih, Puji Budi Setia; Hermsen, Rob; Sauerwein, Robert

    2009-01-01

    Microscopic detection of Plasmodium vivax gametocytes, the sexual life stage of this malaria parasite, is insensitive because P vivax parasitaemia is low. To detect and quantity gametocytes a more sensitive, quantitative realtime Pvs25-QT-NASBA based oil Pvs25 mRNA was developed and tested in two

  10. Real-time PCR assays for the quantitation of rDNA from apricot and other plant species in marzipan.

    Science.gov (United States)

    Haase, Ilka; Brüning, Philipp; Matissek, Reinhard; Fischer, Markus

    2013-04-10

    Marzipan or marzipan raw paste is a typical German sweet which is consumed directly or is used as an ingredient in the bakery industry/confectionery (e.g., in stollen) and as filling for chocolate candies. Almonds (blanched and pealed) and sugar are the only ingredients for marzipan production according to German food guidelines. Especially for the confectionery industry, the use of persipan, which contains apricot or peach kernels instead of almonds, is preferred due to its stronger aroma. In most of the companies, both raw pastes are produced, in most cases on the same production line, running the risk of an unintended cross contamination. Additionally, due to high almond market values, dilutions of marzipan with cheaper seeds may occur. Especially in the case of apricot and almond, the close relationship of both species is a challenge for the analysis. DNA based methods for the qualitative detection of apricot, peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea in marzipan have recently been published. In this study, different quantitation strategies on the basis of real-time PCR have been evaluated and a relative quantitation method with a reference amplification product was shown to give the best results. As the real-time PCR is based on the high copy rDNA-cluster, even contaminations <1% can be reliably quantitated.

  11. Chemisorption of iodine-125 to gold nanoparticles allows for real-time quantitation and potential use in nanomedicine

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Adrian A, E-mail: a.walsh@nanobiosols.com [Liverpool Science Park, Nano Biosols Ltd (United Kingdom)

    2017-04-15

    Gold nanoparticles have been available for many years as a research tool in the life sciences due to their electron density and optical properties. New applications are continually being developed, particularly in nanomedicine. One drawback is the need for an easy, real-time quantitation method for gold nanoparticles so that the effects observed in in vitro cell toxicity assays and cell uptake studies can be interpreted quantitatively in terms of nanoparticle loading. One potential method of quantifying gold nanoparticles in real time is by chemisorption of iodine-125, a gamma emitter, to the nanoparticles. This paper revisits the labelling of gold nanoparticles with iodine-125, first described 30 years ago and never fully exploited since. We explore the chemical properties and usefulness in quantifying bio-functionalised gold nanoparticle binding in a quick and simple manner. The gold particles were labelled specifically and quantitatively simply by mixing the two items. The nature of the labelling is chemisorption and is robust, remaining bound over several weeks in a variety of cell culture media. Chemisorption was confirmed as potassium iodide can remove the label whereas sodium chloride and many other buffers had no effect. Particles precoated in polymers or proteins can be labelled just as efficiently allowing for post-labelling experiments in situ rather than using radioactive gold atoms in the production process. We also demonstrate that interparticle exchange of I-125 between different size particles does not appear to take place confirming the affinity of the binding.

  12. Chemisorption of iodine-125 to gold nanoparticles allows for real-time quantitation and potential use in nanomedicine

    Science.gov (United States)

    Walsh, Adrian A.

    2017-04-01

    Gold nanoparticles have been available for many years as a research tool in the life sciences due to their electron density and optical properties. New applications are continually being developed, particularly in nanomedicine. One drawback is the need for an easy, real-time quantitation method for gold nanoparticles so that the effects observed in in vitro cell toxicity assays and cell uptake studies can be interpreted quantitatively in terms of nanoparticle loading. One potential method of quantifying gold nanoparticles in real time is by chemisorption of iodine-125, a gamma emitter, to the nanoparticles. This paper revisits the labelling of gold nanoparticles with iodine-125, first described 30 years ago and never fully exploited since. We explore the chemical properties and usefulness in quantifying bio-functionalised gold nanoparticle binding in a quick and simple manner. The gold particles were labelled specifically and quantitatively simply by mixing the two items. The nature of the labelling is chemisorption and is robust, remaining bound over several weeks in a variety of cell culture media. Chemisorption was confirmed as potassium iodide can remove the label whereas sodium chloride and many other buffers had no effect. Particles precoated in polymers or proteins can be labelled just as efficiently allowing for post-labelling experiments in situ rather than using radioactive gold atoms in the production process. We also demonstrate that interparticle exchange of I-125 between different size particles does not appear to take place confirming the affinity of the binding.

  13. Chemisorption of iodine-125 to gold nanoparticles allows for real-time quantitation and potential use in nanomedicine

    International Nuclear Information System (INIS)

    Walsh, Adrian A

    2017-01-01

    Gold nanoparticles have been available for many years as a research tool in the life sciences due to their electron density and optical properties. New applications are continually being developed, particularly in nanomedicine. One drawback is the need for an easy, real-time quantitation method for gold nanoparticles so that the effects observed in in vitro cell toxicity assays and cell uptake studies can be interpreted quantitatively in terms of nanoparticle loading. One potential method of quantifying gold nanoparticles in real time is by chemisorption of iodine-125, a gamma emitter, to the nanoparticles. This paper revisits the labelling of gold nanoparticles with iodine-125, first described 30 years ago and never fully exploited since. We explore the chemical properties and usefulness in quantifying bio-functionalised gold nanoparticle binding in a quick and simple manner. The gold particles were labelled specifically and quantitatively simply by mixing the two items. The nature of the labelling is chemisorption and is robust, remaining bound over several weeks in a variety of cell culture media. Chemisorption was confirmed as potassium iodide can remove the label whereas sodium chloride and many other buffers had no effect. Particles precoated in polymers or proteins can be labelled just as efficiently allowing for post-labelling experiments in situ rather than using radioactive gold atoms in the production process. We also demonstrate that interparticle exchange of I-125 between different size particles does not appear to take place confirming the affinity of the binding.

  14. 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.

  15. Comparative evaluation of a laboratory developed real-time PCR assay and the RealStar® HHV-6 PCR Kit for quantitative detection of human herpesvirus 6.

    Science.gov (United States)

    Yip, Cyril C Y; Sridhar, Siddharth; Cheng, Andrew K W; Fung, Ami M Y; Cheng, Vincent C C; Chan, Kwok-Hung; Yuen, Kwok-Yung

    2017-08-01

    HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar ® HHV-6 PCR Kit. The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated. The diagnostic performance of the in-house HHV-6 qPCR assay was compared with the RealStar ® HHV-6 PCR Kit, using 72 clinical specimens and 17 proficiency testing samples. Linear regression analysis of the quantitative results showed a dynamic range from 2 to 10 log 10 copies/ml and a coefficient of determination (R 2 ) of 0.999 for the in-house assay. A dilution series demonstrated a limit of detection and a limit of quantification of 1.7 log 10 and 2 log 10 copies/ml, respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.27% to 4.37%. A comparison of 27 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house HHV-6 qPCR assay and the RealStar ® HHV-6 PCR Kit (R 2 =0.926; PPCR Kit. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Interlaboratory validation of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

    Science.gov (United States)

    Takabatake, Reona; Koiwa, Tomohiro; Kasahara, Masaki; Takashima, Kaori; Futo, Satoshi; Minegishi, Yasutaka; Akiyama, Hiroshi; Teshima, Reiko; Oguchi, Taichi; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

    2011-01-01

    To reduce the cost and time required to routinely perform the genetically modified organism (GMO) test, we developed a duplex quantitative real-time PCR method for a screening analysis simultaneously targeting an event-specific segment for GA21 and Cauliflower Mosaic Virus 35S promoter (P35S) segment [Oguchi et al., J. Food Hyg. Soc. Japan, 50, 117-125 (2009)]. To confirm the validity of the method, an interlaboratory collaborative study was conducted. In the collaborative study, conversion factors (Cfs), which are required to calculate the GMO amount (%), were first determined for two real-time PCR instruments, the ABI PRISM 7900HT and the ABI PRISM 7500. A blind test was then conducted. The limit of quantitation for both GA21 and P35S was estimated to be 0.5% or less. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)). The determined bias and RSD(R) were each less than 25%. We believe the developed method would be useful for the practical screening analysis of GM maize.

  17. Development and validation of a real-time quantitative PCR assay to detect Xanthomonas axonopodis pv. allii from onion seed.

    Science.gov (United States)

    Robène, Isabelle; Perret, Marion; Jouen, Emmanuel; Escalon, Aline; Maillot, Marie-Véronique; Chabirand, Aude; Moreau, Aurélie; Laurent, Annie; Chiroleu, Frédéric; Pruvost, Olivier

    2015-07-01

    Bacterial blight of onion is an emerging disease threatening world onion production. The causal agent Xanthomonas axonopodis pv. allii is seed transmitted and a reliable and sensitive tool is needed to monitor seed exchanges. A triplex quantitative real-time PCR assay was developed targeting two X. axonopodis pv. allii-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Amplification of at least one marker indicates the presence of the bacterium in seed extracts. This real-time PCR assay detected all the 79 X. axonopodis pv. allii strains tested and excluded 85.2% of the 135 non-target strains and particularly all 39 saprophytic and pathogenic bacteria associated with onion. Cross-reactions were mainly obtained for strains assigned to nine phylogenetically related X. axonopodis pathovars. The cycle cut-off was estimated statistically at 36.3 considering a risk of false positive of 1%. The limit of detection obtained in at least 95% of the time (LOD 95%) was 5×10(3) CFU/g (colony forming unit/g). The sensitivity threshold was found to be 1 infected seed in 32,790 seeds. This real-time PCR assay should be useful for preventing the long-distance spread of X. axonopodis pv. allii via contaminated seed lots and determining the epidemiology of the bacterium. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Visualisation and quantitative analysis of the rodent malaria liver stage by real time imaging.

    NARCIS (Netherlands)

    Ploemen, I.H.J.; Prudencio, M.; Douradinha, B.G.; Ramesar, J.; Fonager, J.; Gemert, G.J.A. van; Luty, A.J.F.; Hermsen, C.C.; Sauerwein, R.W.; Baptista, F.G.; Mota, M.M.; Waters, A.P.; Que, I.; Lowik, C.W.G.M.; Khan, S.M.; Janse, C.J.; Franke-Fayard, B.

    2009-01-01

    The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been

  19. TaqMan MGB probe fluorescence real-time quantitative PCR for rapid detection of Chinese Sacbrood virus.

    Directory of Open Access Journals (Sweden)

    Ma Mingxiao

    Full Text Available Sacbrood virus (SBV is a picorna-like virus that affects honey bees (Apis mellifera and results in the death of the larvae. Several procedures are available to detect Chinese SBV (CSBV in clinical samples, but not to estimate the level of CSBV infection. The aim of this study was develop an assay for rapid detection and quantification of this virus. Primers and probes were designed that were specific for CSBV structural protein genes. A TaqMan minor groove binder (MGB probe-based, fluorescence real-time quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed; specificity was high and there were no cross-reactivity with healthy larvae or other bee viruses. The assay was applied to detect CSBV in 37 clinical samples and its efficiency was compared with clinical diagnosis, electron microscopy observation, and conventional RT-PCR. The TaqMan MGB-based probe fluorescence real-time quantitative PCR for CSBV was more sensitive than other methods tested. This assay was a reliable, fast, and sensitive method that was used successfully to detect CSBV in clinical samples. The technology can provide a useful tool for rapid detection of CSBV. This study has established a useful protocol for CSBV testing, epidemiological investigation, and development of animal models.

  20. 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...

  1. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process

    Directory of Open Access Journals (Sweden)

    Borges-Pérez Andrés

    2008-12-01

    Full Text Available Abstract Background The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR. Results In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (CAC, SGN-U321250 (TIP41, SGN-U346908 ("Expressed" and SGN-U316474 (SAND genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process. Conclusion This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time

  2. A triplex quantitative real-time PCR assay for differential detection of human adenovirus serotypes 2, 3 and 7.

    Science.gov (United States)

    Qiu, Fang-Zhou; Shen, Xin-Xin; Zhao, Meng-Chuan; Zhao, Li; Duan, Su-Xia; Chen, Chen; Qi, Ju-Ju; Li, Gui-Xia; Wang, Le; Feng, Zhi-Shan; Ma, Xue-Jun

    2018-05-02

    Human adenovirus (HAdV) serotypes 2, 3 and 7 are more prevalent than other serotypes and have been associated with severe pneumonia in pediatric children. Molecular typing of HAdV is not routinely performed in clinical diagnostic laboratories as it is time-consuming and labor-intensive. In the present study, we developed a triplex quantitative real-time PCR assay (tq-PCR) in a single closed tube for differential detection and quantitative analysis of HAdV serotypes 2, 3 and 7. The sensitivity, specificity, reproducibility and clinical performance of tq-PCR were evaluated. The analytical sensitivity of the tq-PCR was 100 copies/reaction for each of HAdV serotypes 2, 3 and 7, and no cross-reaction with other common respiratory viruses or HAdV serotypes 1,4,5,6,31,55 and 57 was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.6% to 3.6%. Of 138 previously-defined HAdV-positive nasopharyngeal aspirates samples tested, the detection agreement between tq-PCR and nested PCR was 96.38% (133/138). The proposed tq-PCR assay is a sensitive, specific and reproducible method and has the potential for clinical use in the rapid and differential detection and quantitation of HAdV serotypes 2, 3 and 7.

  3. Sequential processing of quantitative phase images for the study of cell behaviour in real-time digital holographic microscopy.

    Science.gov (United States)

    Zikmund, T; Kvasnica, L; Týč, M; Křížová, A; Colláková, J; Chmelík, R

    2014-11-01

    Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The study of the cell is based on extraction of the dynamic data on cell behaviour from the time-lapse sequence of the phase images. However, the phase images are affected by the phase aberrations that make the analysis particularly difficult. This is because the phase deformation is prone to change during long-term experiments. Here, we present a novel algorithm for sequential processing of living cells phase images in a time-lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least-squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. All these procedures are performed automatically and applied immediately after obtaining every single phase image. This property of the algorithm is important for real-time cell quantitative phase imaging and instantaneous control of the course of the experiment by playback of the recorded sequence up to actual time. Such operator's intervention is a forerunner of process automation derived from image analysis. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off-axis holographic microscope. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

  4. Real-time quantitative Schlieren imaging by fast Fourier demodulation of a checkered backdrop

    Science.gov (United States)

    Wildeman, Sander

    2018-06-01

    A quantitative synthetic Schlieren imaging (SSI) method based on fast Fourier demodulation is presented. Instead of a random dot pattern (as usually employed in SSI), a 2D periodic pattern (such as a checkerboard) is used as a backdrop to the refractive object of interest. The range of validity and accuracy of this "Fast Checkerboard Demodulation" (FCD) method are assessed using both synthetic data and experimental recordings of patterns optically distorted by small waves on a water surface. It is found that the FCD method is at least as accurate as sophisticated, multi-stage, digital image correlation (DIC) or optical flow (OF) techniques used with random dot patterns, and it is significantly faster. Efficient, fully vectorized, implementations of both the FCD and DIC/OF schemes developed for this study are made available as open source Matlab scripts.

  5. Use of quantitative real-time PCR for direct detection of serratia marcescens in marine and other aquatic environments.

    Science.gov (United States)

    Joyner, Jessica; Wanless, David; Sinigalliano, Christopher D; Lipp, Erin K

    2014-03-01

    Serratia marcescens is the etiological agent of acroporid serratiosis, a distinct form of white pox disease in the threatened coral Acropora palmata. The pathogen is commonly found in untreated human waste in the Florida Keys, which may contaminate both nearshore and offshore waters. Currently there is no direct method for detection of this bacterium in the aquatic or reef environment, and culture-based techniques may underestimate its abundance in marine waters. A quantitative real-time PCR assay was developed to detect S. marcescens directly from environmental samples, including marine water, coral mucus, sponge tissue, and wastewater. The assay targeted the luxS gene and was able to distinguish S. marcescens from other Serratia species with a reliable quantitative limit of detection of 10 cell equivalents (CE) per reaction. The method could routinely discern the presence of S. marcescens for as few as 3 CE per reaction, but it could not be reliably quantified at this level. The assay detected environmental S. marcescens in complex sewage influent samples at up to 761 CE ml(-1) and in septic system-impacted residential canals in the Florida Keys at up to 4.1 CE ml(-1). This detection assay provided rapid quantitative abilities and good sensitivity and specificity, which should offer an important tool for monitoring this ubiquitous pathogen that can potentially impact both human health and coral health.

  6. 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.

  7. 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.

  8. Applying real-time quantitative PCR to diagnosis of freemartin in Holstein cattle by quantifying SRY gene: a comparison experiment

    Directory of Open Access Journals (Sweden)

    Qinghua Qiu

    2018-04-01

    Full Text Available Background Freemartinism generally occurs in female offspring of dizygotic twins in a mixed-sex pregnancy. Most bovine heterosexual twin females are freemartins. However, about 10% of bovine heterosexual twin females are fertile. Farmers mostly cull bovine fertile heterosexual twin females due to the lack of a practical diagnostic approach. Culling of such animals results in economic and genetic-material losses both for dairy and beef industry. Methods In this study, a comparative test, including qualitative detection of SRY gene by polymerase chain reaction (PCR, quantitative detection of relative content of SRY by real-time quantitative polymerase chain reaction (qPCR, and quantitative detection of H-Y antigen, was performed to establish the most accurate diagnosis for freemartin. Twelve Holstein heterosexual twin females were used in this study, while three normal Holstein bulls and three normal Holstein cows were used as a positive and negative control, respectively. Results Polymerase chain reaction results revealed that SRY gene were absent in three heterosexual twin females and only two of them were verified as fertile in later age. The qPCR results showed that relative content of SRY was more than 14.2% in freemartins and below 0.41% in fertile heterosexual twin females. The H-Y antigen test showed no significant numerical difference between freemartin and fertile heterosexual twin female. Discussion Our results show that relative content of SRY quantified by qPCR is a better detection method for diagnosis of freemartin in Holstein cattle as compare to qualitative detection of SRY gene by PCR or quantitative detection of H-Y antigen. To the authors’ knowledge, this is the first time we applied qPCR to diagnosing freemartin by quantifying SRY gene and got relative SRY content of each freemartin and fertile heterosexual twin female. We concluded that low-level of SRY would not influence fertility of bovine heterosexual twin female.

  9. Real-Time PCR-Based Quantitation Method for the Genetically Modified Soybean Line GTS 40-3-2.

    Science.gov (United States)

    Kitta, Kazumi; Takabatake, Reona; Mano, Junichi

    2016-01-01

    This chapter describes a real-time PCR-based method for quantitation of the relative amount of genetically modified (GM) soybean line GTS 40-3-2 [Roundup Ready(®) soybean (RRS)] contained in a batch. The method targets a taxon-specific soybean gene (lectin gene, Le1) and the specific DNA construct junction region between the Petunia hybrida chloroplast transit peptide sequence and the Agrobacterium 5-enolpyruvylshikimate-3-phosphate synthase gene (epsps) sequence present in GTS 40-3-2. The method employs plasmid pMulSL2 as a reference material in order to quantify the relative amount of GTS 40-3-2 in soybean samples using a conversion factor (Cf) equal to the ratio of the RRS-specific DNA to the taxon-specific DNA in representative genuine GTS 40-3-2 seeds.

  10. Species-specific detection and quantification of common barnacle larvae from the Japanese coast using quantitative real-time PCR.

    Science.gov (United States)

    Endo, Noriyuki; Sato, Kana; Matsumura, Kiyotaka; Yoshimura, Erina; Odaka, Yukiko; Nogata, Yasuyuki

    2010-11-01

    Species-specific detection and quantification methods for barnacle larvae using quantitative real-time polymerase chain reaction (qPCR) were developed. Species-specific primers for qPCR were designed for 13 barnacle species in the mitochondrial 12S ribosomal RNA gene region. Primer specificity was examined by PCR using template DNA extracted from each of the 13 barnacle species, other unidentified barnacle species, and field collected zooplankton samples. The resulting PCR products comprised single bands following agarose gel electrophoresis when the templates corresponded to primers. The amplifications were highly species-specific even for the field plankton samples. The field plankton samples were subjected to qPCR assay. The calculated DNA contents for each barnacle species were closely correlated with the number of larvae measured by microscopic examination. The method could be applied to quantify barnacle larvae in natural plankton samples.

  11. Culture-independent identification and quantification of Gallibacterium anatis (G. anatis) by real-time quantitative PCR

    DEFF Research Database (Denmark)

    Wang, Chong; Robles, Francisco; Ramirez, Saul

    2016-01-01

    Gallibacterium is a genus within the family Pasteurellaceae characterized by a high level of phenotypic and genetic diversity. No diagnostic method has yet been described, which allows species-specific identification of Gallibacterium anatis. The aim of this study was to develop a real...... published conventional PCR method and culture-based identification, respectively. The detection rates were 97%, 78% and 34% for the current qPCR, the conventional PCR and the culture-based identification method, respectively. The qPCR assay was able to detect the gene gyrB in serial dilutions of 10......-time quantitative PCR (qPCR) method allowing species-specific identification and quantification of G. anatis. A G. anatis specific DNA sequence was identified in the gyrase subunit B gene (gyrB) and used to design a TaqMan probe and corresponding primers. The specificity of the assay was tested on 52 bacterial...

  12. Evaluation of changes in periodontal bacteria in healthy dogs over 6 months using quantitative real-time PCR.

    Science.gov (United States)

    Maruyama, N; Mori, A; Shono, S; Oda, H; Sako, T

    2018-03-01

    Porphyromonas gulae, Tannerella forsythia and Campylobacter rectus are considered dominant periodontal pathogens in dogs. Recently, quantitative real-time PCR (qRT-PCR) methods have been used for absolute quantitative determination of oral bacterial counts. The purpose of the present study was to establish a standardized qRT-PCR procedure to quantify bacterial counts of the three target periodontal bacteria (P. gulae, T. forsythia and C. rectus). Copy numbers of the three target periodontal bacteria were evaluated in 26 healthy dogs. Then, changes in bacterial counts of the three target periodontal bacteria were evaluated for 24 weeks in 7 healthy dogs after periodontal scaling. Analytical evaluation of each self-designed primer indicated acceptable analytical imprecision. All 26 healthy dogs were found to be positive for P. gulae, T. forsythia and C. rectus. Median total bacterial counts (copies/ng) of each target genes were 385.612 for P. gulae, 25.109 for T. forsythia and 5.771 for C. rectus. Significant differences were observed between the copy numbers of the three target periodontal bacteria. Periodontal scaling reduced median copy numbers of the three target periodontal bacteria in 7 healthy dogs. However, after periodontal scaling, copy numbers of all three periodontal bacteria significantly increased over time (pperiodontal bacteria in dogs. Furthermore, the present study has revealed that qRT-PCR method can be considered as a new objective evaluation system for canine periodontal disease. Copyright© by the Polish Academy of Sciences.

  13. 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.

  14. 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.

  15. Comparison of real-time quantitative PCR and culture for the diagnosis of emerging Rickettsioses.

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    Emmanouil Angelakis

    Full Text Available BACKGROUND: Isolation of Rickettsia species from skin biopsies may be replaced by PCR. We evaluated culture sensitivity compared to PCR based on sampling delay and previous antibiotic treatment. METHODOLOGY/PRINCIPAL FINDINGS: Skin biopsies and ticks from patients with suspected Rickettsia infection were screened for Rickettsia spp. using qPCR, and positive results were amplified and sequenced for the gltA and ompA genes. Immunofluorescence for spotted fever group rickettsial antigens was done for 79 patients. All skin biopsies and only ticks that tested positive using qPCR were cultured in human embryonic lung (HEL fibroblasts using the centrifugation-shell vial technique. Patients and ticks were classified as definitely having rickettsioses if there was direct evidence of infection with a Rickettsia sp. using culture or molecular assays or in patients if serology was positive. Data on previous antibiotic treatments were obtained for patients with rickettsiosis. Rickettsia spp. infection was diagnosed in 47 out of 145 patients (32%, 41 by PCR and 12 by culture, whereas 3 isolates were obtained from PCR negative biopsies. For 3 of the patients serology was positive although PCR and culture were negative. Rickettsia africae was the most common detected species (n = 25, [17.2%] and isolated bacterium (n = 5, [3.4%]. The probability of isolating Rickettsia spp. was 12 times higher in untreated patients and 5.4 times higher in patients from our hometown. Rickettsia spp. was amplified in 24 out of 95 ticks (25% and we isolated 7 R. slovaca and 1 R. raoultii from Dermacentor marginatus. CONCLUSIONS/SIGNIFICANCE: We found a positive correlation between the bacteria copies and the isolation success in skin biopsies and ticks. Culture remains critical for strain analysis but is less sensitive than serology and PCR for the diagnosis of a Rickettsia infection.

  16. A two-step real-time PCR assay for quantitation and genotyping of human parvovirus 4.

    Science.gov (United States)

    Väisänen, E; Lahtinen, A; Eis-Hübinger, A M; Lappalainen, M; Hedman, K; Söderlund-Venermo, M

    2014-01-01

    Human parvovirus 4 (PARV4) of the family Parvoviridae was discovered in a plasma sample of a patient with an undiagnosed acute infection in 2005. Currently, three PARV4 genotypes have been identified, however, with an unknown clinical significance. Interestingly, these genotypes seem to differ in epidemiology. In Northern Europe, USA and Asia, genotypes 1 and 2 have been found to occur mainly in persons with a history of injecting drug use or other parenteral exposure. In contrast, genotype 3 appears to be endemic in sub-Saharan Africa, where it infects children and adults without such risk behaviour. In this study, a novel straightforward and cost-efficient molecular assay for both quantitation and genotyping of PARV4 DNA was developed. The two-step method first applies a single-probe pan-PARV4 qPCR for screening and quantitation of this relatively rare virus, and subsequently, only the positive samples undergo a real-time PCR-based multi-probe genotyping. The new qPCR-GT method is highly sensitive and specific regardless of the genotype, and thus being suitable for studying the clinical impact and occurrence of the different PARV4 genotypes. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2.

    Science.gov (United States)

    Gatto, Francesca; Cassina, Giulia; Broccolo, Francesco; Morreale, Giuseppe; Lanino, Edoardo; Di Marco, Eddi; Vardas, Efthiya; Bernasconi, Daniela; Buttò, Stefano; Principi, Nicola; Esposito, Susanna; Scarlatti, Gabriella; Lusso, Paolo; Malnati, Mauro S

    2011-12-01

    Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Quantitative real-time PCR approaches for microbial community studies in wastewater treatment systems: applications and considerations.

    Science.gov (United States)

    Kim, Jaai; Lim, Juntaek; Lee, Changsoo

    2013-12-01

    Quantitative real-time PCR (qPCR) has been widely used in recent environmental microbial ecology studies as a tool for detecting and quantifying microorganisms of interest, which aids in better understandings of the complexity of wastewater microbial communities. Although qPCR can be used to provide more specific and accurate quantification than other molecular techniques, it does have limitations that must be considered when applying it in practice. This article reviews the principle of qPCR quantification and its applications to microbial ecology studies in various wastewater treatment environments. Here we also address several limitations of qPCR-based approaches that can affect the validity of quantification data: template nucleic acid quality, nucleic acid extraction efficiency, specificity of group-specific primers and probes, amplification of nonviable DNA, gene copy number variation, and limited number of sequences in the database. Even with such limitations, qPCR is reportedly among the best methods for quantitatively investigating environmental microbial communities. The application of qPCR is and will continue to be increasingly common in studies of wastewater treatment systems. To obtain reliable analyses, however, the limitations that have often been overlooked must be carefully considered when interpreting the results. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. [Comparison of two quantitative methods of endobronchial ultrasound real-time elastography for evaluating intrathoracic lymph nodes].

    Science.gov (United States)

    Mao, X W; Yang, J Y; Zheng, X X; Wang, L; Zhu, L; Li, Y; Xiong, H K; Sun, J Y

    2017-06-12

    Objective: To compare the clinical value of two quantitative methods in analyzing endobronchial ultrasound real-time elastography (EBUS-RTE) images for evaluating intrathoracic lymph nodes. Methods: From January 2014 to April 2014, EBUS-RTE examination was performed in patients who received EBUS-TBNA examination in Shanghai Chest Hospital. Each intrathoracic lymph node had a selected EBUS-RTE image. Stiff area ratio and mean hue value of region of interest (ROI) in each image were calculated respectively. The final diagnosis of lymph node was based on the pathologic/microbiologic results of EBUS-TBNA, pathologic/microbiologic results of other examinations and clinical following-up. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy were evaluated for distinguishing malignant and benign lesions. Results: Fifty-six patients and 68 lymph nodes were enrolled in this study, of which 35 lymph nodes were malignant and 33 lymph nodes were benign. The stiff area ratio and mean hue value of benign and malignant lesions were 0.32±0.29, 0.62±0.20 and 109.99±28.13, 141.62±17.52, respectively, and statistical differences were found in both of those two methods ( t =-5.14, P methods can be used for analyzing EBUS-RTE images quantitatively, having the value of differentiating benign and malignant intrathoracic lymph nodes, and the stiff area ratio is better than the mean hue value between the two methods.

  20. Development and application of an oligonucleotide microarray and real-time quantitative PCR for detection of wastewater bacterial pathogens

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Dae-Young [National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6 (Canada)], E-mail: daeyoung.lee@ec.gc.ca; Lauder, Heather; Cruwys, Heather; Falletta, Patricia [National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6 (Canada); Beaudette, Lee A. [Environmental Science and Technology Centre, Environment Canada, 335 River Road South, Ottawa, Ontario, K1A 0H3 (Canada)], E-mail: lee.beaudette@ec.gc.ca

    2008-07-15

    Conventional microbial water quality test methods are well known for their technical limitations, such as lack of direct pathogen detection capacity and low throughput capability. The microarray assay has recently emerged as a promising alternative for environmental pathogen monitoring. In this study, bacterial pathogens were detected in municipal wastewater using a microarray equipped with short oligonucleotide probes targeting 16S rRNA sequences. To date, 62 probes have been designed against 38 species, 4 genera, and 1 family of pathogens. The detection sensitivity of the microarray for a waterborne pathogen Aeromonas hydrophila was determined to be approximately 1.0% of the total DNA, or approximately 10{sup 3}A. hydrophila cells per sample. The efficacy of the DNA microarray was verified in a parallel study where pathogen genes and E. coli cells were enumerated using real-time quantitative PCR (qPCR) and standard membrane filter techniques, respectively. The microarray and qPCR successfully detected multiple wastewater pathogen species at different stages of the disinfection process (i.e. secondary effluents vs. disinfected final effluents) and at two treatment plants employing different disinfection methods (i.e. chlorination vs. UV irradiation). This result demonstrates the effectiveness of the DNA microarray as a semi-quantitative, high throughput pathogen monitoring tool for municipal wastewater.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Recombinant plasmid-based quantitative Real-Time PCR analysis of Salmonella enterica serotypes and its application to milk samples.

    Science.gov (United States)

    Gokduman, Kurtulus; Avsaroglu, M Dilek; Cakiris, Aris; Ustek, Duran; Gurakan, G Candan

    2016-03-01

    The aim of the current study was to develop, a new, rapid, sensitive and quantitative Salmonella detection method using a Real-Time PCR technique based on an inexpensive, easy to produce, convenient and standardized recombinant plasmid positive control. To achieve this, two recombinant plasmids were constructed as reference molecules by cloning the two most commonly used Salmonella-specific target gene regions, invA and ttrRSBC. The more rapid detection enabled by the developed method (21 h) compared to the traditional culture method (90 h) allows the quantitative evaluation of Salmonella (quantification limits of 10(1)CFU/ml and 10(0)CFU/ml for the invA target and the ttrRSBC target, respectively), as illustrated using milk samples. Three advantages illustrated by the current study demonstrate the potential of the newly developed method to be used in routine analyses in the medical, veterinary, food and water/environmental sectors: I--The method provides fast analyses including the simultaneous detection and determination of correct pathogen counts; II--The method is applicable to challenging samples, such as milk; III--The method's positive controls (recombinant plasmids) are reproducible in large quantities without the need to construct new calibration curves. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform.

    Science.gov (United States)

    Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

    2015-12-14

    Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.

  4. Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR.

    Directory of Open Access Journals (Sweden)

    Yogita Maheshwari

    Full Text Available Droplet digital polymerase chain reaction (ddPCR is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative PCR (qPCR for S. citri detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect S. citri infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of S. citri compare to qPCR.

  5. A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids

    Directory of Open Access Journals (Sweden)

    Wilson Zoe A

    2008-06-01

    Full Text Available Abstract Background Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. Results We describe a real-time PCR technique employing attached universal duplex probes (AUDP, which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP and a complementary quenching probe (QP lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. Conclusion The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.

  6. Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform

    Science.gov (United States)

    Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

    2015-11-01

    Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (r

  7. Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.

    Science.gov (United States)

    Zhang, Jin-Ju; Xu, Jing-Feng; Shen, Yong-Wei; Ma, Shi-Jiao; Zhang, Ting-Ting; Meng, Qing-Lin; Lan, Wen-Jun; Zhang, Chun; Liu, Xiao-Mei

    2017-07-01

    Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  8. Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation

    Directory of Open Access Journals (Sweden)

    Burgos Lorenzo

    2010-07-01

    Full Text Available Abstract Background The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently. Results We show here that chimeras are a very frequent phenomenon observed after regenerating transgenic plants. Spatial and temporal analyses of transformed tobacco and apricot plants with a quantitative, real-time PCR amplification of the neomycin phosphotransferase (nptII transgene as well as of an internal control (β-actin, used to normalise the amount of target DNA at each reaction, allowed detection of chimeras at unexpected rates. The amount of the nptII transgene differed greatly along with the sub-cultivation period of these plants and was dependent on the localisation of the analysed leaves; being higher in roots and basal leaves, while in the apical leaves it remained at lower levels. These data demonstrate that, unlike the use of the gus marker gene, real-time PCR is a powerful tool for detection of chimeras. Although some authors have proposed a consistent, positive Southern analysis as an alternative methodology for monitoring the dissociation of chimeras, our data show that it does not provide enough proof of uniform transformation. In this work, however, real-time PCR was applied successfully to monitor the dissociation of chimeras in tobacco plants and apricot callus. Conclusions We have developed a rapid and reliable method to detect and estimate the level of chimeras in transgenic tobacco and apricot

  9. Detection and identification of Rift Valley fever virus in mosquito vectors by quantitative real-time PCR.

    Science.gov (United States)

    Mwaengo, D; Lorenzo, G; Iglesias, J; Warigia, M; Sang, R; Bishop, R P; Brun, A

    2012-10-01

    Diagnostic methods allowing for rapid identification of pathogens are crucial for controlling and preventing dissemination after disease outbreaks as well as for use in surveillance programs. For arboviruses, detection of the presence of virus in their arthropod hosts is important for monitoring of viral activity and quantitative information is useful for modeling of transmission dynamics. In this study, molecular detection of Rift Valley fever virus (RVFV) in mosquito samples from the 2006 to 2007 East African outbreaks was performed using quantitative real-time PCR assay (qRT-PCR). Specific RVFV sequence-based primer/fluorogenic (TaqMan) probe sets were derived from the L and S RNA segments of the virus. Both primer-probe L and S segment-based combinations detected genomic RVFV sequences, with generally comparable levels of sensitivity. Viral loads from three mosquito species, Aedes mcintoshi, Aedes ochraceus and Mansonia uniformis were estimated and significant differences of between 5- and 1000-fold were detected between Ae. mcintoshi and M. uniformis using both the L and S primer-probe-based assays. The genetic relationships of the viral sequences in mosquito samples were established by partial M segment sequencing and assigned to the two previously described viral lineages defined by analysis of livestock isolates obtained during the 2006-2007 outbreak, confirming that similar viruses were present in both the vector and mammalian host. The data confirms the utility of qRT-PCR for identification and initial quantification of virus in mosquito samples during RVFV outbreaks. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Reference gene selection for real-time quantitative PCR analysis of the mouse uterus in the peri-implantation period.

    Directory of Open Access Journals (Sweden)

    Pengfei Lin

    Full Text Available The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNorm(PLUS, NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.

  11. Comparison of commercial DNA preparation kits for the detection of Brucellae in tissue using quantitative real-time PCR

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    Straube Eberhard

    2010-04-01

    Full Text Available Abstract Background The detection of Brucellae in tissue specimens using PCR assays is difficult because the amount of bacteria is usually low. Therefore, optimised DNA extraction methods are critical. The aim of this study was to assess the performance of commercial kits for the extraction of Brucella DNA. Methods Five kits were evaluated using clinical specimens: QIAamp™ DNA Mini Kit (QIAGEN, peqGold™ Tissue DNA Mini Kit (PeqLab, UltraClean™ Tissue and Cells DNA Isolation Kit (MoBio, DNA Isolation Kit for Cells and Tissues (Roche, and NucleoSpin™ Tissue (Macherey-Nagel. DNA yield was determined using a quantitative real-time PCR assay targeting IS711 that included an internal amplification control. Results Kits of QIAGEN and Roche provided the highest amount of DNA, Macherey-Nagel and Peqlab products were intermediate whereas MoBio yielded the lowest amount of DNA. Differences were significant (p Conclusions We observed differences in DNA yield as high as two orders of magnitude for some samples between the best and the worst DNA extraction kits and inhibition was observed occasionally. This indicates that DNA purification may be more relevant than expected when the amount of DNA in tissue is very low.

  12. Effectiveness of quantitative real time PCR in long-term follow-up of chronic myeloid leukemia patients

    International Nuclear Information System (INIS)

    Savasoglu, K.; Berber, B.

    2015-01-01

    To determine the use of the Quantitative Real Time PCR (RQ-PCR) assay follow-up with Chronic Myeloid Leukemia (CML) patients. Study Design: Cross-sectional observational. Place and Duration of Study: Izmir Ataturk Education and Research Hospital, Izmir, Turkey, from 2009 to 2013. Methodology: Cytogenetic, FISH, RQ-PCR test results from 177 CML patients materials selected between 2009 - 2013 years was set up for comparison analysis. Statistical analysis was performed to compare between FISH, karyotype and RQ-PCR results of the patients. Karyotyping and FISH specificity and sensitivity rates determined by ROC analysis compared with RQ-PCR results. Chi-square test was used to compare test failure rates. Results:Sensitivity and specificity values were determined for karyotyping 17.6 - 98% (p=0.118, p > 0.05) and for FISH 22.5 - 96% (p=0.064, p > 0.05) respectively. FISH sensitivity was slightly higher than karyotyping but there was calculated a strong correlation between them (p < 0.001). RQ-PCR test failure rate did not correlate with other two tests (p > 0.05); however, karyotyping and FISH test failure rate was statistically significant (p < 0.001). Conclusion: Besides, the situation needed for karyotype analysis, RQ-PCR assay can be used alone in the follow-up of CML disease. (author)

  13. Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product.

    Science.gov (United States)

    Fazelahi, Mansoureh; Kia, Vahid; Kaghazian, Hooman; Paryan, Mahdi

    2017-11-26

    Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli's and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (10 1 to 10 7 copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.

  14. Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean

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    Renata Stolf-Moreira

    2011-01-01

    Full Text Available The objective of this work was to validate, by quantitative PCR in real time (RT-qPCR, genes to be used as reference in studies of gene expression in soybean in drought-stressed trials. Four genes commonly used in soybean were evaluated: Gmβ-actin, GmGAPDH, GmLectin and GmRNAr18S. Total RNA was extracted from six samples: three from roots in a hydroponic system with different drought intensities (0, 25, 50, 75 and 100 minutes of water stress, and three from leaves of plants grown in sand with different soil moistures (15, 5 and 2.5% gravimetric humidity. The raw cycle threshold (Ct data were analyzed, and the efficiency of each primer was calculated for an overall analysis of the Ct range among the different samples. The GeNorm application was used to evaluate the best reference gene, according to its stability. The GmGAPDH was the least stable gene, with the highest mean values of expression stability (M, and the most stable genes, with the lowest M values, were the Gmβ-actin and GmRNAr18S, when both root and leaves samples were tested. These genes can be used in RT-qPCR as reference gene for expression analysis.

  15. Effectiveness of Quantitative Real Time PCR in Long-Term Follow-up of Chronic Myeloid Leukemia Patients.

    Science.gov (United States)

    Savasoglu, Kaan; Payzin, Kadriye Bahriye; Ozdemirkiran, Fusun; Berber, Belgin

    2015-08-01

    To determine the use of the Quantitative Real Time PCR (RQ-PCR) assay follow-up with Chronic Myeloid Leukemia (CML) patients. Cross-sectional observational. Izmir Ataturk Education and Research Hospital, Izmir, Turkey, from 2009 to 2013. Cytogenetic, FISH, RQ-PCR test results from 177 CMLpatients' materials selected between 2009 - 2013 years was set up for comparison analysis. Statistical analysis was performed to compare between FISH, karyotype and RQ-PCR results of the patients. Karyotyping and FISH specificity and sensitivity rates determined by ROC analysis compared with RQ-PCR results. Chi-square test was used to compare test failure rates. Sensitivity and specificity values were determined for karyotyping 17.6 - 98% (p=0.118, p > 0.05) and for FISH 22.5 - 96% (p=0.064, p > 0.05) respectively. FISH sensitivity was slightly higher than karyotyping but there was calculated a strong correlation between them (p 0.05); however, karyotyping and FISH test failure rate was statistically significant (p < 0.001). Besides, the situation needed for karyotype analysis, RQ-PCR assay can be used alone in the follow-up of CMLdisease.

  16. 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.

  17. Identification and validation of reference genes for quantitative real-time PCR in Drosophila suzukii (Diptera: Drosophilidae.

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    Yifan Zhai

    Full Text Available To accurately evaluate gene expression levels and obtain more accurate quantitative real-time RT-PCR (qRT-PCR data, normalization relative to reliable reference gene(s is required. Drosophila suzukii, is an invasive fruit pest native to East Asia, and recently invaded Europe and North America, the stability of its reference genes have not been previously investigated. In this study, ten candidate reference genes (RPL18, RPS3, AK, EF-1β, TBP, NADH, HSP22, GAPDH, Actin, α-Tubulin, were evaluated for their suitability as normalization genes under different biotic (developmental stage, tissue and population, and abiotic (photoperiod, temperature conditions. The three statistical approaches (geNorm, NormFinder and BestKeeper and one web-based comprehensive tool (RefFinder were used to normalize analysis of the ten candidate reference genes identified α-Tubulin, TBP and AK as the most stable candidates, while HSP22 and Actin showed the lowest expression stability. We used three most stable genes (α-Tubulin, TBP and AK and one unstably expressed gene to analyze the expression of P-glycoprotein in abamectin-resistant and sensitive strains, and the results were similar to reference genes α-Tubulin, TBP and AK, which show good stability, while the result of HSP22 has a certain bias. The three validated reference genes can be widely used for quantification of target gene expression with qRT-PCR technology in D.suzukii.

  18. Accurate, Fast and Cost-Effective Diagnostic Test for Monosomy 1p36 Using Real-Time Quantitative PCR

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    Pricila da Silva Cunha

    2014-01-01

    Full Text Available Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5–0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH, which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescent in situ hybridization (FISH, and/or multiplex ligation-dependent probe amplification (MLPA all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR of PRKCZ and SKI is a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.

  19. Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve.

    Science.gov (United States)

    Rueda-Martínez, Carmen; Fernández, M Carmen; Soto-Navarrete, María Teresa; Jiménez-Navarro, Manuel; Durán, Ana Carmen; Fernández, Borja

    2016-01-01

    Bicuspid aortic valve (BAV) is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40%) incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR) assays. A total of 51 adult (180-240 days old) and 56 old (300-440 days old) animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30), or to the affected strain of hamsters with TAV (n = 45) or BAV (n = 32). The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta.

  20. Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve.

    Directory of Open Access Journals (Sweden)

    Carmen Rueda-Martínez

    Full Text Available Bicuspid aortic valve (BAV is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40% incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR assays. A total of 51 adult (180-240 days old and 56 old (300-440 days old animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30, or to the affected strain of hamsters with TAV (n = 45 or BAV (n = 32. The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta.

  1. Standardisation and evaluation of a quantitative multiplex real-time PCR assay for the rapid identification of Streptococcus pneumoniae

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    Feroze Ahmed Ganaie

    2015-01-01

    Full Text Available Rapid diagnosis of Streptococcus pneumoniae can play a significant role in decreasing morbidity and mortality of infection. The accurate diagnosis of pneumococcal disease is hampered by the difficulties in growing the isolates from clinical specimens and also by misidentification. Molecular methods have gained popularity as they offer improvement in the detection of causative pathogens with speed and ease. The present study aims at validating and standardising the use of 4 oligonucleotide primer-probe sets (pneumolysin [ply], autolysin [lytA], pneumococcal surface adhesion A [psaA] and Spn9802 [DNA fragment] in a single-reaction mixture for the detection and discrimination of S. pneumoniae. Here, we validate a quantitative multiplex real-time PCR (qmPCR assay with a panel consisting of 43 S. pneumoniae and 29 non-pneumococcal isolates, 20 culture positive, 26 culture negative and 30 spiked serum samples. A standard curve was obtained using S. pneumoniae ATCC 49619 strain and glyceraldehyde 3-phosphate dehydrogenase (GAPDH gene was used as an endogenous internal control. The experiment showed high sensitivity with lower limit of detection equivalent to 4 genome copies/µl. The efficiency of the reaction was 100% for ply, lytA, Spn9802 and 97% for psaA. The test showed sensitivity and specificity of 100% with culture isolates and serum specimens. This study demonstrates that qmPCR analysis of sera using 4 oligonucleotide primers appears to be an appropriate method for the genotypic identification of S. pneumoniae infection.

  2. Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

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    Ledderose Carola

    2011-10-01

    Full Text Available Abstract Background The choice of reliable reference genes is a prerequisite for valid results when analyzing gene expression with real-time quantitative PCR (qPCR. This method is frequently applied to study gene expression patterns in immune cells, yet a thorough validation of potential reference genes is still lacking for most leukocyte subtypes and most models of their in vitro stimulation. In the current study, we evaluated the expression stability of common reference genes in two widely used cell culture models-anti-CD3/CD28 activated T cells and lipopolysaccharide stimulated neutrophils-as well as in unselected untreated leukocytes. Results The mRNA expression of 17 (T cells, 7 (neutrophils or 8 (unselected leukocytes potential reference genes was quantified by reverse transcription qPCR, and a ranking of the preselected candidate genes according to their expression stability was calculated using the programs NormFinder, geNorm and BestKeeper. IPO8, RPL13A, TBP and SDHA were identified as suitable reference genes in T cells. TBP, ACTB and SDHA were stably expressed in neutrophils. TBP and SDHA were also the most stable genes in untreated total blood leukocytes. The critical impact of reference gene selection on the estimated target gene expression is demonstrated for IL-2 and FIH expression in T cells. Conclusions The study provides a shortlist of suitable reference genes for normalization of gene expression data in unstimulated and stimulated T cells, unstimulated and stimulated neutrophils and in unselected leukocytes.

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

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    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.

  4. 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.

  5. WetLab-2: Tools for Conducting On-Orbit Quantitative Real-Time Gene Expression Analysis on ISS

    Science.gov (United States)

    Parra, Macarena; Almeida, Eduardo; Boone, Travis; Jung, Jimmy; Schonfeld, Julie

    2014-01-01

    The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR system, the Cepheid SmartCycler and will fly it in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid ramp times and the ability to detect up to four separate fluorescent channels at one time enabling multiplex assays that can be used for normalization and to study multiple genes of interest in each module. The team is currently working with Cepheid to enable the downlink of data from the ISS to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project has adapted commercial technology to design a module that can lyse cells and extract RNA of sufficient quality and quantity for use in qRT-PCR reactions while using a housekeeping gene to normalize RNA concentration and integrity. The WetLab-2 system is capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. The ability to conduct qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples. The system can be used to validate terrestrial analyses of samples returned from ISS by providing on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experiment parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Researchers will also be able to sample multigenerational changes in organisms. Finally, the system can be

  6. Application of propidium monoazide quantitative real-time PCR to quantify the viability of Lactobacillus delbrueckii ssp. bulgaricus.

    Science.gov (United States)

    Shao, Yuyu; Wang, Zhaoxia; Bao, Qiuhua; Zhang, Heping

    2016-12-01

    In this study, a combination of propidium monoazide (PMA) and quantitative real-time PCR (qPCR) was used to develop a method to determine the viability of cells of Lactobacillus delbrueckii ssp. bulgaricus ND02 (L. bulgaricus) that may have entered into a viable but nonculturable state. This can happen due to its susceptibility to cold shock during lyophilization and storage. Propidium monoazide concentration, PMA incubation time, and light exposure time were optimized to fully exploit the PMA-qPCR approach to accurately assess the total number of living L. bulgaricus ND02. Although PMA has little influence on living cells, when concentrations of PMA were higher than 30μg/mL the number of PCR-positive living bacteria decreased from 10 6 to 10 5 cfu/mL in comparison with qPCR enumeration. Mixtures of living and dead cells were used as method verification samples for enumeration by PMA-qPCR, demonstrating that this method was feasible and effective for distinguishing living cells of L. bulgaricus when mixed with a known number of dead cells. We suggest that several conditions need to be studied further before PMA-qPCR methods can be accurately used to distinguish living from dead cells for enumeration under more realistic sampling situations. However, this research provides a rapid way to enumerate living cells of L. bulgaricus and could be used to optimize selection of cryoprotectants in the lyophilization process and develop technologies for high cell density cultivation and optimal freeze-drying processes. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  7. Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites

    NARCIS (Netherlands)

    van der Meide, Wendy; Guerra, Jorge; Schoone, Gerard; Farenhorst, Marit; Coelho, Leila; Faber, William; Peekel, Inge; Schallig, Henk

    2008-01-01

    DNA or RNA amplification methods for detection of Leishmania parasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays

  8. SNPs and real-time quantitative PCR method for constitutional allelic copy number determination, the VPREB1 marker case

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    Costa Elena

    2011-05-01

    Full Text Available Abstract Background 22q11.2 microdeletion is responsible for the DiGeorge Syndrome, characterized by heart defects, psychiatric disorders, endocrine and immune alterations and a 1 in 4000 live birth prevalence. Real-time quantitative PCR (qPCR approaches for allelic copy number determination have recently been investigated in 22q11.2 microdeletions detection. The qPCR method was performed for 22q11.2 microdeletions detection as a first-level screening approach in a genetically unknown series of patients with congenital heart defects. A technical issue related to the VPREB1 qPCR marker was pointed out. Methods A set of 100 unrelated Italian patients with congenital heart defects were tested for 22q11.2 microdeletions by a qPCR method using six different markers. Fluorescence In Situ Hybridization technique (FISH was used for confirmation. Results qPCR identified six patients harbouring the 22q11.2 microdeletion, confirmed by FISH. The VPREB1 gene marker presented with a pattern consistent with hemideletion in one 3 Mb deleted patient, suggestive for a long distal deletion, and in additional five non-deleted patients. The long distal 22q11.2 deletion was not confirmed by Comparative Genomic Hybridization. Indeed, the VPREB1 gene marker generated false positive results in association with the rs1320 G/A SNP, a polymorphism localized within the VPREB1 marker reverse primer sequence. Patients heterozygous for rs1320 SNP, showed a qPCR profile consistent with the presence of a hemideletion. Conclusions Though the qPCR technique showed advantages as a screening approach in terms of cost and time, the VPREB1 marker case revealed that single nucleotide polymorphisms can interfere with qPCR data generating erroneous allelic copy number interpretations.

  9. Validation of reference genes for quantitative real-time PCR in Périgord black truffle (Tuber melanosporum) developmental stages.

    Science.gov (United States)

    Zarivi, Osvaldo; Cesare, Patrizia; Ragnelli, Anna Maria; Aimola, Pierpaolo; Leonardi, Marco; Bonfigli, Antonella; Colafarina, Sabrina; Poma, Anna Maria; Miranda, Michele; Pacioni, Giovanni

    2015-08-01

    The symbiotic fungus Tuber melanosporum Vittad. (Périgord black truffle) belongs to the Ascomycota and forms mutualistic symbiosis with tree and shrub roots. This truffle has a high value in a global market and is cultivated in many countries of both hemispheres. The publication of the T. melanosporum genome has given researchers unique opportunities to learn more about the biology of the fungus. Real-time quantitative PCR (qRT-PCR) is a definitive technique for quantitating differences in transcriptional gene expression levels between samples. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes must show stable expression under given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in T. melanosporum development. In this study, we present a morphological and microscopical classification of the developmental stages of T. melanosporum fruit body, and investigate the expression levels of 12 candidate reference genes (18S rRNA; 5.8S rRNA; Elongation factor 1-alpha; Elongation factor 1-beta; α-tubulin; 60S ribosomal protein L29; β-tubulin; 40S ribosomal protein S1; 40S ribosomal protein S3; Glucose-6-phosphate dehydrogenase; β-actin; Ubiquitin-conjugating enzyme). To evaluate the suitability of these genes as endogenous controls, five software-based approaches and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. We demonstrate here that the 18S rRNA gene shows the most stable expression during T. melanosporum development and that a set of three genes, 18S rRNA, Elongation factor 1-alpha and 40S ribosomal protein S3, is the most suitable to normalize qRT-PCR data from all the analyzed developmental stages; conversely, 18S rRNA, Glucose-6-phosphate dehydrogenase and Elongation factor 1-alpha are the most suitable

  10. Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer

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    Miller Nicola

    2007-11-01

    Full Text Available Abstract Background Real-time quantitative PCR (RQ-PCR forms the basis of many breast cancer biomarker studies and novel prognostic assays, paving the way towards personalised cancer treatments. Normalisation of relative RQ-PCR data is required to control for non-biological variation introduced during sample preparation. Endogenous control (EC genes, used in this context, should ideally be expressed constitutively and uniformly across treatments in all test samples. Despite widespread recognition that the accuracy of the normalised data is largely dependent on the reliability of the EC, there are no reports of the systematic validation of genes commonly used for this purpose in the analysis of gene expression by RQ-PCR in primary breast cancer tissues. The aim of this study was to identify the most suitable endogenous control genes for RQ-PCR analysis of primary breast tissue from a panel of eleven candidates in current use. Oestrogen receptor alpha (ESR1 was used a target gene to compare the effect of choice of EC on the estimate of gene quantity. Results The expression and validity of candidate ECs (GAPDH, TFRC, ABL, PPIA, HPRT1, RPLP0, B2M, GUSB, MRPL19, PUM1 and PSMC4 was determined in 6 benign and 21 malignant primary breast cancer tissues. Gene expression data was analysed using two different statistical models. MRPL19 and PPIA were identified as the most stable and reliable EC genes, while GUSB, RPLP0 and ABL were least stable. There was a highly significant difference in variance between ECs. ESR1 expression was appreciably higher in malignant compared to benign tissues and there was a significant effect of EC on the magnitude of the error associated with the relative quantity of ESR1. Conclusion We have validated two endogenous control genes, MRPL19 and PPIA, for RQ-PCR analysis of gene expression in primary breast tissue. Of the genes in current use in this field, the above combination offers increased accuracy and resolution in the

  11. Ex vivo screening for immunodominant viral epitopes by quantitative real time polymerase chain reaction (qRT-PCR

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    Nagorsen Dirk

    2003-12-01

    Full Text Available Abstract The identification and characterization of viral epitopes across the Human Leukocyte Antigen (HLA polymorphism is critical for the development of actives-specific or adoptive immunotherapy of virally-mediated diseases. This work investigates whether cytokine mRNA transcripts could be used to identify epitope-specific HLA-restricted memory T lymphocytes reactivity directly in fresh peripheral blood mononuclear cells (PBMCs from viral-seropositive individuals in response to ex vivo antigen recall. PBMCs from HLA-A*0201 healthy donors, seropositive for Cytomegalovirus (CMV and Influenza (Flu, were exposed for different periods and at different cell concentrations to the HLA-A*0201-restricted viral FluM158–66 and CMVpp65495–503 peptides. Quantitative real time PCR (qRT-PCR was employed to evaluate memory T lymphocyte immune reactivation by measuring the production of mRNA encoding four cytokines: Interferon-γ (IFN-γ, Interleukin-2 (IL-2, Interleukin-4 (IL-4, and Interleukin-10 (IL-10. We could characterize cytokine expression kinetics that illustrated how cytokine mRNA levels could be used as ex vivo indicators of T cell reactivity. Particularly, IFN-γ mRNA transcripts could be consistently detected within 3 to 12 hours of short-term stimulation in levels sufficient to screen for HLA-restricted viral immune responses in seropositive subjects. This strategy will enhance the efficiency of the identification of viral epitopes independently of the individual HLA phenotype and could be used to follow the intensity of immune responses during disease progression or in response to in vivo antigen-specific immunization.

  12. Ex vivo screening for immunodominant viral epitopes by quantitative real time polymerase chain reaction (qRT-PCR)

    Science.gov (United States)

    Provenzano, Maurizio; Mocellin, Simone; Bonginelli, Paola; Nagorsen, Dirk; Kwon, Seog-Woon; Stroncek, David

    2003-01-01

    The identification and characterization of viral epitopes across the Human Leukocyte Antigen (HLA) polymorphism is critical for the development of actives-specific or adoptive immunotherapy of virally-mediated diseases. This work investigates whether cytokine mRNA transcripts could be used to identify epitope-specific HLA-restricted memory T lymphocytes reactivity directly in fresh peripheral blood mononuclear cells (PBMCs) from viral-seropositive individuals in response to ex vivo antigen recall. PBMCs from HLA-A*0201 healthy donors, seropositive for Cytomegalovirus (CMV) and Influenza (Flu), were exposed for different periods and at different cell concentrations to the HLA-A*0201-restricted viral FluM158–66 and CMVpp65495–503 peptides. Quantitative real time PCR (qRT-PCR) was employed to evaluate memory T lymphocyte immune reactivation by measuring the production of mRNA encoding four cytokines: Interferon-γ (IFN-γ), Interleukin-2 (IL-2), Interleukin-4 (IL-4), and Interleukin-10 (IL-10). We could characterize cytokine expression kinetics that illustrated how cytokine mRNA levels could be used as ex vivo indicators of T cell reactivity. Particularly, IFN-γ mRNA transcripts could be consistently detected within 3 to 12 hours of short-term stimulation in levels sufficient to screen for HLA-restricted viral immune responses in seropositive subjects. This strategy will enhance the efficiency of the identification of viral epitopes independently of the individual HLA phenotype and could be used to follow the intensity of immune responses during disease progression or in response to in vivo antigen-specific immunization. PMID:14675481

  13. Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR.

    Directory of Open Access Journals (Sweden)

    Ruby Chandna

    Full Text Available The real time quantitative reverse transcription PCR (qRT-PCR is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes, ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes, in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.

  14. Selection and validation of appropriate reference genes for quantitative real-time PCR analysis in Salvia hispanica.

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    Rahul Gopalam

    Full Text Available Quantitative real-time polymerase chain reaction (qRT-PCR has become the most popular choice for gene expression studies. For accurate expression analysis, it is pertinent to select a stable reference gene to normalize the data. It is now known that the expression of internal reference genes varies considerably during developmental stages and under different experimental conditions. For Salvia hispanica, an economically important oilseed crop, there are no reports of stable reference genes till date. In this study, we chose 13 candidate reference genes viz. Actin11 (ACT, Elongation factor 1-alpha (EF1-α, Eukaryotic translation initiation factor 3E (ETIF3E, alpha tubulin (α-TUB, beta tubulin (β-TUB, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Cyclophilin (CYP, Clathrin adaptor complex (CAC, Serine/threonine-protein phosphatase 2A (PP2A, FtsH protease (FtsH, 18S ribosomal RNA (18S rRNA, S-adenosyl methionine decarboxylase (SAMDC and Rubisco activase (RCA and the expression levels of these genes were assessed in a diverse set of tissue samples representing vegetative stages, reproductive stages and various abiotic stress treatments. Two of the widely used softwares, geNorm and Normfinder were used to evaluate the expression stabilities of these 13 candidate reference genes under different conditions. Results showed that GAPDH and CYP expression remain stable throughout in the different abiotic stress treatments, CAC and PP2A expression were relatively stable under reproductive stages and α-TUB, PP2A and ETIF3E were found to be stably expressed in vegetative stages. Further, the expression levels of Diacylglycerol acyltransferase (DGAT1, a key enzyme in triacylglycerol synthesis was analyzed to confirm the validity of reference genes identified in the study. This is the first systematic study of selection of reference genes in S. hispanica, and will benefit future expression studies in this crop.

  15. Validation of reference genes for quantitative expression analysis by real-time rt-PCR in four lepidopteran insects.

    Science.gov (United States)

    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), Chilo suppressalis Walker (Crambidae), and Spodoptera exigua Hübner (Noctuidae) to study their expression stability. The algorithms of geNorm, NormFinder, stability index, and ΔCt analysis were used to evaluate these HKGs. Across different developmental stages, actin A1 was the most stable in P. xylostella and C. suppressalis, but it was the least stable in B. mori and S. exigua. Rp49 and GAPDH were the most stable in B. mori and S. exigua, respectively. In different tissues, GAPDH, E2F, and Rp49 were the most stable in B. mori, S. exigua, and C. suppressalis, respectively. The relative abundances of Siwi genes estimated by 2(-ΔΔCt) method were tested with different HKGs as the reference gene, proving the importance of internal controls in qPCR data analysis. The results not only presented a list of suitable reference genes in four lepidopteran insects, but also proved that the expression stabilities of HKGs were different among evolutionarily close species. There was no single universal reference gene that could be used in all situations. It is indispensable to validate the expression of HKGs before using them as the internal control in qPCR.

  16. 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

  17. Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification ▿

    Science.gov (United States)

    Yankson, Kweku K.; Steck, Todd R.

    2009-01-01

    We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil. PMID:19633108

  18. MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments

    NARCIS (Netherlands)

    Bustin, S.A.; Beaulieu, J.F.; Huggett, J.; Jaggi, R.; Kibenge, F.S.; Olsvik, P.A.; Penning, L.C.; Toegel, S.

    2010-01-01

    MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments Stephen A Bustin1 , Jean-François Beaulieu2 , Jim Huggett3 , Rolf Jaggi4 , Frederick SB Kibenge5 , Pål A Olsvik6 , Louis C Penning7 and Stefan Toegel8 1 Centre for

  19. 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...

  20. A Human Fecal Contamination Score for Ranking Recreational Sites using the HF183/BacR287 Quantitative Real-Time PCR Method

    Science.gov (United States)

    Human fecal pollution of recreational waters remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality research and manag...

  1. QUANTITATION OF INTRACELLULAR NAD(P)H IN LIVING CELLS CAN MONITOR AN IMBALANCE OF DNA SINGLE STRAND BREAK REPAIR IN REAL TIME

    Science.gov (United States)

    Quantitation of intracellular NAD(P)H in living cells can monitor an imbalance of DNA single strand break repair in real time.ABSTRACTDNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or du...

  2. Quantitative Real Time PCR approach to study gene expression profile during prenatal growth of skeletal muscle in pig of Duroc and Pietrain breeds

    Directory of Open Access Journals (Sweden)

    M. Cagnazzo

    2010-01-01

    Full Text Available The quantitative real time-PCR (QRT-PCR is a very sensitive method used to quantify mRNA level in gene expression analysis. Combining amplification, detection and quantification in a single step, allows a more accurate measurement compared to the traditional PCR end point analysis (Pfaffl, 2001; Bustin, 2002.

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

    Science.gov (United States)

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

    2014-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Gavin J. Nixon

    2014-12-01

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

  5. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    International Nuclear Information System (INIS)

    Huang, S-H; Tsai, M-H; Lin, C-W; Yang, T-C; Chuang, P-H; Tsai, I-S; Lu, H-C; Wan Lei; Lin, Y-J; Lai, C-H

    2008-01-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples

  6. 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

  7. 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

  8. Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses

    DEFF Research Database (Denmark)

    Stilwell, Natalie K.; Whittington, Richard J.; Hick, Paul M.

    2018-01-01

    Ranaviruses are globally emerging pathogens negatively impacting wild and cultured fish, amphibians, and reptiles. Although conventional and diagnostic real-time PCR (qPCR) assays have been developed to detect ranaviruses, these assays often have not been tested against the known diversity of ran...

  9. Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

    Directory of Open Access Journals (Sweden)

    Peng Xia

    2009-01-01

    Full Text Available Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf mitochondrial (mtDNA and nuclear (nDNA DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

  10. User satisfaction with a real-time automated feedback system for general practitioners: a quantitative and qualitative study

    NARCIS (Netherlands)

    Bindels, Rianne; Hasman, Arie; Derickx, Mieke; van Wersch, Jan W. J.; Winkens, Ron A. G.

    2003-01-01

    OBJECTIVE: The GRIF automated feedback system produces real-time comments on the appropriateness of diagnostic tests ordered by general practitioners (GPs) based on recommendations from accepted national and regional practice guidelines. We investigated the experiences of GPs with this system and,

  11. Quantitative analysis of the improvement in high zoom maritime tracking due to real-time image enhancement

    CSIR Research Space (South Africa)

    Bachoo, AK

    2011-04-01

    Full Text Available This work aims to evaluate the improvement in the performance of tracking small maritime targets due to real-time enhancement of the video streams from high zoom cameras on pan-tilt pedestal. Due to atmospheric conditions these images can frequently...

  12. Two quantitative real-time PCR assays for the detection of penaeid shrimp and blue crab, crustacean shellfish allergens.

    Science.gov (United States)

    Eischeid, Anne C; Kim, Bang-hyun; Kasko, Sasha M

    2013-06-19

    Food allergen detection methods must be able to specifically detect minute quantities of an allergenic food in a complex food matrix. One technique that can be used is real-time PCR. For the work described here, real-time PCR assays were developed to detect penaeid shrimp and blue crab, crustacean shellfish allergens. The method was tested using shrimp meat and crab meat spiked into several types of foods, including canned soups, deli foods, meat, seafood, and prepared seafood products. Foods were spiked with either shrimp or crab at levels ranging from 0.1 to 10⁶ parts per million (ppm) and analyzed either raw or cooked by a variety of methods. Real-time PCR data were used to generate linear standard curves, and assays were evaluated with respect to linear range and reaction efficiency. Results indicate that both assays performed well in a variety of food types. High reaction efficiencies were achieved across a linear range of 6-8 orders of magnitude. Limits of detection were generally between 0.1 and 1 ppm. Cooking methods used to simulate thermal processing of foods had little effect on assay performance. This work demonstrates that real-time PCR can be a valuable tool in the detection of crustacean shellfish.

  13. 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.

  14. Comparison of lung cancer cell lines representing four histopathological subtypes with gene expression profiling using quantitative real-time PCR

    Directory of Open Access Journals (Sweden)

    Kawaguchi Makoto

    2010-01-01

    Full Text Available Abstract Background Lung cancers are the most common type of human malignancy and are intractable. Lung cancers are generally classified into four histopathological subtypes: adenocarcinoma (AD, squamous cell carcinoma (SQ, large cell carcinoma (LC, and small cell carcinoma (SC. Molecular biological characterization of these subtypes has been performed mainly using DNA microarrays. In this study, we compared the gene expression profiles of these four subtypes using twelve human lung cancer cell lines and the more reliable quantitative real-time PCR (qPCR. Results We selected 100 genes from public DNA microarray data and examined them by DNA microarray analysis in eight test cell lines (A549, ABC-1, EBC-1, LK-2, LU65, LU99, STC 1, RERF-LC-MA and a normal control lung cell line (MRC-9. From this, we extracted 19 candidate genes. We quantified the expression of the 19 genes and a housekeeping gene, GAPDH, with qPCR, using the same eight cell lines plus four additional validation lung cancer cell lines (RERF-LC-MS, LC-1/sq, 86-2, and MS-1-L. Finally, we characterized the four subtypes of lung cancer cell lines using principal component analysis (PCA of gene expression profiling for 12 of the 19 genes (AMY2A, CDH1, FOXG1, IGSF3, ISL1, MALL, PLAU, RAB25, S100P, SLCO4A1, STMN1, and TGM2. The combined PCA and gene pathway analyses suggested that these genes were related to cell adhesion, growth, and invasion. S100P in AD cells and CDH1 in AD and SQ cells were identified as candidate markers of these lung cancer subtypes based on their upregulation and the results of PCA analysis. Immunohistochemistry for S100P and RAB25 was closely correlated to gene expression. Conclusions These results show that the four subtypes, represented by 12 lung cancer cell lines, were well characterized using qPCR and PCA for the 12 genes examined. Certain genes, in particular S100P and CDH1, may be especially important for distinguishing the different subtypes. Our results

  15. O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis.

    Science.gov (United States)

    Mekonnen, Solomon A; Beissner, Marcus; Saar, Malkin; Ali, Solomon; Zeynudin, Ahmed; Tesfaye, Kassahun; Adbaru, Mulatu G; Battke, Florian; Poppert, Sven; Hoelscher, Michael; Löscher, Thomas; Bretzel, Gisela; Herbinger, Karl-Heinz

    2017-10-02

    Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis.

  16. Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

    Directory of Open Access Journals (Sweden)

    Pek-Lan Chan

    Full Text Available BACKGROUND: The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR. With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. RESULTS: In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569 outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN. PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. CONCLUSIONS: Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection

  17. Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment

    Directory of Open Access Journals (Sweden)

    Li Qingdi

    2012-06-01

    Full Text Available Abstract Background The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata. Results The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25 remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1α, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4 were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-ΔΔCT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-ΔΔCT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13 as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such

  18. Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

    Science.gov (United States)

    Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

    2014-01-01

    Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate

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

    Directory of Open Access Journals (Sweden)

    Qiusheng Kong

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

  20. Quantitative Real-Time Fluoroscopy Analysis on Measurement of the Hepatic Arterial Flow During Transcatheter Arterial Chemoembolization of Hepatocellular Carcinoma: Comparison with Quantitative Digital Subtraction Angiography Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yi-Yang; Lee, Rheun-Chuan, E-mail: rclee@vghtpe.gov.tw; Guo, Wan-Yuo, E-mail: wyguo@vghtpe.gov.tw; Chu, Wei-Fa [Taipei Veterans General Hospital, Department of Radiology (China); Wu, Frank Chun-Hsien [Siemens Healthcare Ltd. (China); Gehrisch, Sonja [Siemens Healthcare GmbH (Germany)

    2016-11-15

    PurposeTo quantify the arterial flow change during transcatheter arterial chemoembolization (TACE) for hepatocellular carcinoma (HCC) using digital subtraction angiography, quantitative color-coding analysis (d-QCA), and real-time subtraction fluoroscopy QCA (f-QCA).Materials and MethodsThis prospective study enrolled 20 consecutive patients with HCC who had undergone TACE via a subsegmental approach between February 2014 and April 2015. The TACE endpoint was a sluggish antegrade tumor-feeding arterial flow. d-QCA and f-QCA were used for determining the relative maximal density time (rT{sub max}) of the selected arteries. The rT{sub max} of the selected arteries was analyzed in d-QCA and f-QCA before and after TACE, and its correlation in both analyses was evaluated.ResultsThe pre- and post-TACE rT{sub max} of the embolized segmental artery in d-QCA and f-QCA were 1.59 ± 0.81 and 2.97 ± 1.80 s (P < 0.001) and 1.44 ± 0.52 and 2.28 ± 1.02 s (P < 0.01), respectively. The rT{sub max} of the proximal hepatic artery did not significantly change during TACE in d-QCA and f-QCA. The Spearman correlation coefficients of the pre- and post-TACE rT{sub max} of the embolized segmental artery between d-QCA and f-QCA were 0.46 (P < 0.05) and 0.80 (P < 0.001). Radiation doses in one series of d-QCA and f-QCA were 140.7 ± 51.5 milligray (mGy) and 2.5 ± 0.7 mGy, respectively.Conclusionsf-QCA can quantify arterial flow changes with a higher temporal resolution and lower radiation dose. Flow quantification of the embolized segmental artery using f-QCA and d-QCA is highly correlated.

  1. Quantitative Real-Time Fluoroscopy Analysis on Measurement of the Hepatic Arterial Flow During Transcatheter Arterial Chemoembolization of Hepatocellular Carcinoma: Comparison with Quantitative Digital Subtraction Angiography Analysis

    International Nuclear Information System (INIS)

    Lin, Yi-Yang; Lee, Rheun-Chuan; Guo, Wan-Yuo; Chu, Wei-Fa; Wu, Frank Chun-Hsien; Gehrisch, Sonja

    2016-01-01

    PurposeTo quantify the arterial flow change during transcatheter arterial chemoembolization (TACE) for hepatocellular carcinoma (HCC) using digital subtraction angiography, quantitative color-coding analysis (d-QCA), and real-time subtraction fluoroscopy QCA (f-QCA).Materials and MethodsThis prospective study enrolled 20 consecutive patients with HCC who had undergone TACE via a subsegmental approach between February 2014 and April 2015. The TACE endpoint was a sluggish antegrade tumor-feeding arterial flow. d-QCA and f-QCA were used for determining the relative maximal density time (rT_m_a_x) of the selected arteries. The rT_m_a_x of the selected arteries was analyzed in d-QCA and f-QCA before and after TACE, and its correlation in both analyses was evaluated.ResultsThe pre- and post-TACE rT_m_a_x of the embolized segmental artery in d-QCA and f-QCA were 1.59 ± 0.81 and 2.97 ± 1.80 s (P < 0.001) and 1.44 ± 0.52 and 2.28 ± 1.02 s (P < 0.01), respectively. The rT_m_a_x of the proximal hepatic artery did not significantly change during TACE in d-QCA and f-QCA. The Spearman correlation coefficients of the pre- and post-TACE rT_m_a_x of the embolized segmental artery between d-QCA and f-QCA were 0.46 (P < 0.05) and 0.80 (P < 0.001). Radiation doses in one series of d-QCA and f-QCA were 140.7 ± 51.5 milligray (mGy) and 2.5 ± 0.7 mGy, respectively.Conclusionsf-QCA can quantify arterial flow changes with a higher temporal resolution and lower radiation dose. Flow quantification of the embolized segmental artery using f-QCA and d-QCA is highly correlated.

  2. Application of quantitative real-time PCR compared to filtration methods for the enumeration of Escherichia coli in surface waters within Vietnam.

    Science.gov (United States)

    Vital, Pierangeli G; Van Ha, Nguyen Thi; Tuyet, Le Thi Hong; Widmer, Kenneth W

    2017-02-01

    Surface water samples in Vietnam were collected from the Saigon River, rural and suburban canals, and urban runoff canals in Ho Chi Minh City, Vietnam, and were processed to enumerate Escherichia coli. Quantification was done through membrane filtration and quantitative real-time polymerase chain reaction (PCR). Mean log colony-forming unit (CFU)/100 ml E. coli counts in the dry season for river/suburban canals and urban canals were log 2.8 and 3.7, respectively, using a membrane filtration method, while using Taqman quantitative real-time PCR they were log 2.4 and 2.8 for river/suburban canals and urban canals, respectively. For the wet season, data determined by the membrane filtration method in river/suburban canals and urban canals samples had mean counts of log 3.7 and 4.1, respectively. While mean log CFU/100 ml counts in the wet season using quantitative PCR were log 3 and 2, respectively. Additionally, the urban canal samples were significantly lower than those determined by conventional culture methods for the wet season. These results show that while quantitative real-time PCR can be used to determine levels of fecal indicator bacteria in surface waters, there are some limitations to its application and it may be impacted by sources of runoff based on surveyed samples.

  3. Quantitative real-time monitoring of multi-elements in airborne particulates by direct introduction into an inductively coupled plasma mass spectrometer

    International Nuclear Information System (INIS)

    Suzuki, Yoshinari; Sato, Hikaru; Hiyoshi, Katsuhiro; Furuta, Naoki

    2012-01-01

    A new calibration system for real-time determination of trace elements in airborne particulates was developed. Airborne particulates were directly introduced into an inductively coupled plasma mass spectrometer, and the concentrations of 15 trace elements were determined by means of an external calibration method. External standard solutions were nebulized by an ultrasonic nebulizer (USN) coupled with a desolvation system, and the resulting aerosol was introduced into the plasma. The efficiency of sample introduction via the USN was calculated by two methods: (1) the introduction of a Cr standard solution via the USN was compared with introduction of a Cr(CO) 6 standard gas via a standard gas generator and (2) the aerosol generated by the USN was trapped on filters and then analyzed. The Cr introduction efficiencies obtained by the two methods were the same, and the introduction efficiencies of the other elements were equal to the introduction efficiency of Cr. Our results indicated that our calibration method for introduction efficiency worked well for the 15 elements (Ti, V, Cr, Mn, Co, Ni, Cu, Zn, As, Mo, Sn, Sb, Ba, Tl and Pb). The real-time data and the filter-collection data agreed well for elements with low-melting oxides (V, Co, As, Mo, Sb, Tl, and Pb). In contrast, the real-time data were smaller than the filter-collection data for elements with high-melting oxides (Ti, Cr, Mn, Ni, Cu, Zn, Sn, and Ba). This result implies that the oxides of these 8 elements were not completely fused, vaporized, atomized, and ionized in the initial radiation zone of the inductively coupled plasma. However, quantitative real-time monitoring can be realized after correction for the element recoveries which can be calculated from the ratio of real-time data/filter-collection data. - Highlights: ► APs were directly introduced into ICP-MS and real-time analysis was performed. ► The real-time data were calibrated by a multi-element standard solution from USN. ► During real-time

  4. Response of a diuron-degrading community to diuron exposure assessed by real-time quantitative PCR monitoring of phenylurea hydrolase A and B encoding genes

    OpenAIRE

    Pesce , S.; Beguet , J.; Rouard , N.; Devers Lamrani , M.; Martin Laurent , F.

    2013-01-01

    A real-time quantitative PCR method was developed to detect and quantify phenlylurea hydrolase genes' (puhA and puhB) sequences from environmental DNA samples to assess diuron-degrading genetic potential in some soil and sediment microbial communities. In the soil communities, mineralization rates (determined with [ring-14C]-labeled diuron) were linked to diuron-degrading genetic potentials estimated from puhB number copies, which increased following repeated diuron treatments. In the sedimen...

  5. 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...

  6. 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....

  7. Comparative Evaluation of Four Real-Time PCR Methods for the Quantitative Detection of Epstein-Barr Virus from Whole Blood Specimens.

    Science.gov (United States)

    Buelow, Daelynn; Sun, Yilun; Tang, Li; Gu, Zhengming; Pounds, Stanley; Hayden, Randall

    2016-07-01

    Monitoring of Epstein-Barr virus (EBV) load in immunocompromised patients has become integral to their care. An increasing number of reagents are available for quantitative detection of EBV; however, there are little published comparative data. Four real-time PCR systems (one using laboratory-developed reagents and three using analyte-specific reagents) were compared with one another for detection of EBV from whole blood. Whole blood specimens seeded with EBV were used to determine quantitative linearity, analytical measurement range, lower limit of detection, and CV for each assay. Retrospective testing of 198 clinical samples was performed in parallel with all methods; results were compared to determine relative quantitative and qualitative performance. All assays showed similar performance. No significant difference was found in limit of detection (3.12-3.49 log10 copies/mL; P = 0.37). A strong qualitative correlation was seen with all assays that used clinical samples (positive detection rates of 89.5%-95.8%). Quantitative correlation of clinical samples across assays was also seen in pairwise regression analysis, with R(2) ranging from 0.83 to 0.95. Normalizing clinical sample results to IU/mL did not alter the quantitative correlation between assays. Quantitative EBV detection by real-time PCR can be performed over a wide linear dynamic range, using three different commercially available reagents and laboratory-developed methods. EBV was detected with comparable sensitivity and quantitative correlation for all assays. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  8. Selection of internal control genes for real-time quantitative PCR in ovary and uterus of sows across pregnancy.

    Directory of Open Access Journals (Sweden)

    María Martínez-Giner

    Full Text Available BACKGROUND: Reproductive traits play a key role in pig production in order to reduce costs and increase economic returns. Among others, gene expression analyses represent a useful approach to study genetic mechanisms underlying reproductive traits in pigs. The application of reverse-transcription quantitative PCR requires the selection of appropriate reference genes, whose expression levels should not be affected by the experimental conditions, especially when comparing gene expression across different physiological stages. RESULTS: The gene expression stability of ten potential reference genes was studied by three different methods (geNorm, NormFinder and BestKeeper in ovary and uterus collected at five different physiological time points (heat, and 15, 30, 45 and 60 days of pregnancy. Although final ranking differed, the three algorithms gave very similar results. Thus, the most stable genes across time were TBP and UBC in uterus and TBP and HPRT1 in ovary, while HMBS and ACTB showed the less stable expression in uterus and ovary, respectively. When studied as a systematic effect, the reproductive stage did not significantly affect the expression of the candidate reference genes except at 30d and 60d of pregnancy, when a general drop in expression was observed in ovary. CONCLUSIONS: Based in our results, we propose the use of TBP, UBC and SDHA in uterus and TBP, GNB2L1 and HPRT1 in ovary for normalization of longitudinal expression studies using quantitative PCR in sows.

  9. Soil Baiting, Rapid PCR Assay and Quantitative Real Time PCR to Diagnose Late Blight of Potato in Quarantine Programs

    Directory of Open Access Journals (Sweden)

    Touseef Hussain

    2018-05-01

    Full Text Available Phytophthora infestans (mont de Bary is a pathogen of great concern across the globe, and accurate detection is an important component in responding to the outbreaks of potential disease. Although the molecular diagnostic protocol used in regulatory programs has been evaluated but till date methods implying direct comparison has rarely used. In this study, a known area soil samples from potato fields where light blight appear every year (both A1 and A2 mating type was assayed by soil bait method, PCR assay detection and quantification of the inoculums. Suspected disease symptoms appeared on bait tubers were further confirmed by rapid PCR, inoculums were quantified through Real Time PCR, which confirms presence of P. infestans. These diagnostic methods can be highly correlated with one another. Potato tuber baiting increased the sensitivity of the assay compared with direct extraction of DNA from tuber and soil samples. Our study determines diagnostic sensitivity and specificity of the assays to determine the performance of each method. Overall, molecular techniques based on different types of PCR amplification and Real-time PCR can lead to high throughput, faster and more accurate detection method which can be used in quarantine programmes in potato industry and diagnostic laboratory.

  10. 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.

  11. Quantitation of hepatitis B virus DNA in plasma using a sensitive cost-effective "in-house" real-time PCR assay

    Directory of Open Access Journals (Sweden)

    Daniel Hubert Darius

    2009-01-01

    Full Text Available Background: Sensitive nucleic acid testing for the detection and accurate quantitation of hepatitis B virus (HBV is necessary to reduce transmission through blood and blood products and for monitoring patients on antiviral therapy. The aim of this study is to standardize an "in-house" real-time HBV polymerase chain reaction (PCR for accurate quantitation and screening of HBV. Materials and Methods: The "in-house" real-time assay was compared with a commercial assay using 30 chronically infected individuals and 70 blood donors who are negative for hepatitis B surface antigen, hepatitis C virus (HCV antibody and human immunodeficiency virus (HIV antibody. Further, 30 HBV-genotyped samples were tested to evaluate the "in-house" assay′s capacity to detect genotypes prevalent among individuals attending this tertiary care hospital. Results: The lower limit of detection of this "in-house" HBV real-time PCR was assessed against the WHO international standard and found to be 50 IU/mL. The interassay and intra-assay coefficient of variation (CV of this "in-house" assay ranged from 1.4% to 9.4% and 0.0% to 2.3%, respectively. Virus loads as estimated with this "in-house" HBV real-time assay correlated well with the commercial artus HBV RG PCR assay ( r = 0.95, P < 0.0001. Conclusion: This assay can be used for the detection and accurate quantitation of HBV viral loads in plasma samples. This assay can be employed for the screening of blood donations and can potentially be adapted to a multiplex format for simultaneous detection of HBV, HIV and HCV to reduce the cost of testing in blood banks.

  12. 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

  13. Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods.

    Science.gov (United States)

    Edagawa, Akiko; Kimura, Akio; Kawabuchi-Kurata, Takako; Adachi, Shinichi; Furuhata, Katsunori; Miyamoto, Hiroshi

    2015-10-19

    We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR), and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%). Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%). In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8%) compared with real-time qPCR alone (46/68, 67.6%). Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1%) compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%). Legionella was not detected in the remaining six samples (6/68, 8.8%), irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.

  14. Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods

    Directory of Open Access Journals (Sweden)

    Akiko Edagawa

    2015-10-01

    Full Text Available We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR, and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%. Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%. In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8% compared with real-time qPCR alone (46/68, 67.6%. Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1% compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%. Legionella was not detected in the remaining six samples (6/68, 8.8%, irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.

  15. Isothermal microcalorimetry - A quantitative method to monitor Trypanosoma congolense growth and growth inhibition by trypanocidal drugs in real time.

    Science.gov (United States)

    Gysin, M; Braissant, O; Gillingwater, K; Brun, R; Mäser, P; Wenzler, T

    2018-03-16

    Trypanosoma congolense is a protozoan parasite that is transmitted by tsetse flies, causing African Animal Trypanosomiasis, also known as Nagana, in sub-Saharan Africa. Nagana is a fatal disease of livestock that causes severe economic losses. Two drugs are available, diminazene and isometamidium, yet successful treatment is jeopardized by drug resistant T. congolense. Isothermal microcalorimetry is a highly sensitive tool that can be used to study growth of the extracellular T. congolense parasites or to study parasite growth inhibition after the addition of antitrypanosomal drugs. Time of drug action and time to kill can be quantified in a simple way by real time heat flow measurements. We established a robust protocol for the microcalorimetric studies of T. congolense and developed mathematical computations in R to calculate different parameters related to growth and the kinetics of drug action. We demonstrate the feasibility and benefit of the method exemplary with the two standard drugs, diminazene aceturate and isometamidium chloride. The method and the mathematical approach can be translated to study other pathogenic or non-pathogenic cells if they are metabolically active and grow under axenic conditions. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. 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.)

  17. 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...

  18. 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)

  19. Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing.

    Science.gov (United States)

    Huertas, César S; Carrascosa, L G; Bonnal, S; Valcárcel, J; Lechuga, L M

    2016-04-15

    Alternative splicing of mRNA precursors enables cells to generate different protein outputs from the same gene depending on their developmental or homeostatic status. Its deregulation is strongly linked to disease onset and progression. Current methodologies for monitoring alternative splicing demand elaborate procedures and often present difficulties in discerning between closely related isoforms, e.g. due to cross-hybridization during their detection. Herein, we report a general methodology using a Surface Plasmon Resonance (SPR) biosensor for label-free monitoring of alternative splicing events in real-time, without any cDNA synthesis or PCR amplification requirements. We applied this methodology to RNA isolated from HeLa cells for the quantification of alternatively spliced isoforms of the Fas gene, involved in cancer progression through regulation of programmed cell death. We demonstrate that our methodology is isoform-specific, with virtually no cross-hybridization, achieving limits of detection (LODs) in the picoMolar (pM) range. Similar results were obtained for the detection of the BCL-X gene mRNA isoforms. The results were independently validated by RT-qPCR, with excellent concordance in the determination of isoform ratios. The simplicity and robustness of this biosensor technology can greatly facilitate the exploration of alternative splicing biomarkers in disease diagnosis and therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. A two-stage method of quantitative flood risk analysis for reservoir real-time operation using ensemble-based hydrologic forecasts

    Science.gov (United States)

    Liu, P.

    2013-12-01

    Quantitative analysis of the risk for reservoir real-time operation is a hard task owing to the difficulty of accurate description of inflow uncertainties. The ensemble-based hydrologic forecasts directly depict the inflows not only the marginal distributions but also their persistence via scenarios. This motivates us to analyze the reservoir real-time operating risk with ensemble-based hydrologic forecasts as inputs. A method is developed by using the forecast horizon point to divide the future time into two stages, the forecast lead-time and the unpredicted time. The risk within the forecast lead-time is computed based on counting the failure number of forecast scenarios, and the risk in the unpredicted time is estimated using reservoir routing with the design floods and the reservoir water levels of forecast horizon point. As a result, a two-stage risk analysis method is set up to quantify the entire flood risks by defining the ratio of the number of scenarios that excessive the critical value to the total number of scenarios. The China's Three Gorges Reservoir (TGR) is selected as a case study, where the parameter and precipitation uncertainties are implemented to produce ensemble-based hydrologic forecasts. The Bayesian inference, Markov Chain Monte Carlo, is used to account for the parameter uncertainty. Two reservoir operation schemes, the real operated and scenario optimization, are evaluated for the flood risks and hydropower profits analysis. With the 2010 flood, it is found that the improvement of the hydrologic forecast accuracy is unnecessary to decrease the reservoir real-time operation risk, and most risks are from the forecast lead-time. It is therefore valuable to decrease the avarice of ensemble-based hydrologic forecasts with less bias for a reservoir operational purpose.

  1. 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...

  2. Visually estimated ejection fraction by two dimensional and triplane echocardiography is closely correlated with quantitative ejection fraction by real-time three dimensional echocardiography

    Directory of Open Access Journals (Sweden)

    Manouras Aristomenis

    2009-08-01

    Full Text Available Abstract Background Visual assessment of left ventricular ejection fraction (LVEF is often used in clinical routine despite general recommendations to use quantitative biplane Simpsons (BPS measurements. Even thou quantitative methods are well validated and from many reasons preferable, the feasibility of visual assessment (eyeballing is superior. There is to date only sparse data comparing visual EF assessment in comparison to quantitative methods available. The aim of this study was to compare visual EF assessment by two-dimensional echocardiography (2DE and triplane echocardiography (TPE using quantitative real-time three-dimensional echocardiography (RT3DE as the reference method. Methods Thirty patients were enrolled in the study. Eyeballing EF was assessed using apical 4-and 2 chamber views and TP mode by two experienced readers blinded to all clinical data. The measurements were compared to quantitative RT3DE. Results There were an excellent correlation between eyeballing EF by 2D and TP vs 3DE (r = 0.91 and 0.95 respectively without any significant bias (-0.5 ± 3.7% and -0.2 ± 2.9% respectively. Intraobserver variability was 3.8% for eyeballing 2DE, 3.2% for eyeballing TP and 2.3% for quantitative 3D-EF. Interobserver variability was 7.5% for eyeballing 2D and 8.4% for eyeballing TP. Conclusion Visual estimation of LVEF both using 2D and TP by an experienced reader correlates well with quantitative EF determined by RT3DE. There is an apparent trend towards a smaller variability using TP in comparison to 2D, this was however not statistically significant.

  3. Visually estimated ejection fraction by two dimensional and triplane echocardiography is closely correlated with quantitative ejection fraction by real-time three dimensional echocardiography.

    Science.gov (United States)

    Shahgaldi, Kambiz; Gudmundsson, Petri; Manouras, Aristomenis; Brodin, Lars-Ake; Winter, Reidar

    2009-08-25

    Visual assessment of left ventricular ejection fraction (LVEF) is often used in clinical routine despite general recommendations to use quantitative biplane Simpsons (BPS) measurements. Even thou quantitative methods are well validated and from many reasons preferable, the feasibility of visual assessment (eyeballing) is superior. There is to date only sparse data comparing visual EF assessment in comparison to quantitative methods available. The aim of this study was to compare visual EF assessment by two-dimensional echocardiography (2DE) and triplane echocardiography (TPE) using quantitative real-time three-dimensional echocardiography (RT3DE) as the reference method. Thirty patients were enrolled in the study. Eyeballing EF was assessed using apical 4-and 2 chamber views and TP mode by two experienced readers blinded to all clinical data. The measurements were compared to quantitative RT3DE. There were an excellent correlation between eyeballing EF by 2D and TP vs 3DE (r = 0.91 and 0.95 respectively) without any significant bias (-0.5 +/- 3.7% and -0.2 +/- 2.9% respectively). Intraobserver variability was 3.8% for eyeballing 2DE, 3.2% for eyeballing TP and 2.3% for quantitative 3D-EF. Interobserver variability was 7.5% for eyeballing 2D and 8.4% for eyeballing TP. Visual estimation of LVEF both using 2D and TP by an experienced reader correlates well with quantitative EF determined by RT3DE. There is an apparent trend towards a smaller variability using TP in comparison to 2D, this was however not statistically significant.

  4. Development of real-time and quantitative monitoring of thrombus formation in an extracorporeal centrifugal blood pump

    Science.gov (United States)

    Sakota, Daisuke; Fujiwara, Tatsuki; Ohuchi, Katsuhiro; Kuwana, Katsuyuki; Yamazaki, Hiroyuki; Kosaka, Ryo; Maruyama, Osamu

    2018-02-01

    We developed an optical detector of thrombus formed on the pivot bearing of an extracorporeal centrifugal blood pump (MERA HCF-MP23; Senko Medical Instrument Mfg. Co., Ltd., Tokyo, Japan) which is frequently used for long-term extracorporeal circulation support to bridge to an implantable artificial heart, which in turn is used for bridge to heart transplantation in Japan. In this study, we investigated the quantitative performance of the thrombus formation in acute animal experiments. A total of three experiments of extracorporeal left ventricular assist using Japanese specific pathogen-free pigs were conducted. The optical fibers were set in the pump driver unit. The incident light at nearinfrared wavelength aiming at the pivot bearing and the resulting scattered light were guided to respective fibers. The detected signal was analyzed to obtain thrombus formation level (TFL) calculated by a specially developed software. When the increase in TFL was confirmed, the pump was exchanged and the extracorporeal circulation was restarted. The number of pump exchanges were four times at each experiment so a total of twelve pumps were evaluated. 3-dimentional data surrounding the pivot bearing and the adhered thrombus was captured by a 3-dimantional surface measurement system to calculate the thrombus surface area (TSA) formed on the pivot bearing. As a result, the correlation coefficient between TFL and TSA was 0.878. The accuracy of TSA estimated by the optical detector was 3.6+/-2.3 mm2. This was small enough to not have the pump exchanged in clinical judgement. The developed detector would be useful for optimal anti-coagulation management.

  5. Quantitative real-time PCR identifies a critical region of deletion on 22q13 related to prognosis in oral cancer

    DEFF Research Database (Denmark)

    Reis, Patricia P; Rogatto, Silvia R; Kowalski, Luiz P

    2002-01-01

    Quantitative real time PCR was performed on genomic DNA from 40 primary oral carcinomas and the normal adjacent tissues. The target genes ECGFB, DIA1, BIK, and PDGFB and the microsatellite markers D22S274 and D22S277, mapped on 22q13, were selected according to our previous loss of heterozygosity...... findings in head and neck tumors. Quantitative PCR relies on the comparison of the amount of product generated from a target gene and that generated from a disomic reference gene (GAPDH-housekeeping gene). Reactions have been performed with normal control in triplicates, using the 7700 Sequence Detection.......0018) for patients with DIA1 gene loss. Relative copy number losses detected in these sequences may be related to disease progression and a worse prognosis in patients with oral cancer....

  6. 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

  7. Detection, quantitation and identification of enteroviruses from surface waters and sponge tissue from the Florida Keys using real-time RT-PCR

    Science.gov (United States)

    Donaldson, K.A.; Griffin, Dale W.; Paul, J.H.

    2002-01-01

    A method was developed for the quantitative detection of pathogenic human enteroviruses from surface waters in the Florida Keys using Taqman (R) one-step Reverse transcription (RT)-PCR with the Model 7700 ABI Prism (R) Sequence Detection System. Viruses were directly extracted from unconcentrated grab samples of seawater, from seawater concentrated by vortex flow filtration using a 100kD filter and from sponge tissue. Total RNA was extracted from the samples, purified and concentrated using spin-column chromatography. A 192-196 base pair portion of the 5??? untranscribed region was amplified from these extracts. Enterovirus concentrations were estimated using real-time RT-PCR technology. Nine of 15 sample sites or 60% were positive for the presence of pathogenic human enteroviruses. Considering only near-shore sites, 69% were positive with viral concentrations ranging from 9.3viruses/ml to 83viruses/g of sponge tissue (uncorrected for extraction efficiency). Certain amplicons were selected for cloning and sequencing for identification. Three strains of waterborne enteroviruses were identified as Coxsackievirus A9, Coxsackievirus A16, and Poliovirus Sabin type 1. Time and cost efficiency of this one-step real-time RT-PCR methodology makes this an ideal technique to detect, quantitate and identify pathogenic enteroviruses in recreational waters. Copyright ?? 2002 Elsevier Science Ltd.

  8. Identification of pyrG Used as an Endogenous Reference Gene in Qualitative and Real-Time Quantitative PCR Detection of Pleurotus ostreatus.

    Science.gov (United States)

    Zheng, Shi; Shan, Luying; Zhuang, Yongliang; Shang, Ying

    2018-03-01

    As a well-known edible fungus rich in nutrients, Pleurotus ostreatus has been used as an alternative to expensive wild edible fungi. Specifically, the fact that using P. ostreatus instead of other expensive wild edible fungi has damaged the rights and interests of consumers. Among the existing methods for detection of food adulteration, the amplification of endogenous reference gene is the most accurate method. However, an ideal endogenous reference gene for P. ostreatus has yet to be developed. In this study, a DNA extraction method for P. ostreatus was optimized, and pyrG was selected as a species-specific gene through sequence alignment. This gene was subsequently subjected to qualitative and quantitative Polymerase Chain Reaction (PCR) assays with 3 different P. ostreatus varieties and 7 other species. A low detection limit of 5 pg/μL was obtained by TaqMan quantitative PCR, and no pyrG amplification product was observed in the 7 other species. No allelic variation was detected in P. ostreatus varieties. These experiments confirmed that pyrG was an ideal endogenous reference gene for the qualitative and real-time quantitative PCR detection of P. ostreatus. This method was also suitable for the examination of processed P. ostreatus samples and determination of adulteration in wild mushrooms. The pyrG gene was chosen as an ideal endogenous reference gene for the qualitative and real-time quantitative PCR detection of P. ostreatus, and the detection limit was 5 pg/μL for the quantification. This method is used not only for raw materials but also for processed P. ostreatus products and other processed mushroom foods. © 2018 Institute of Food Technologists®.

  9. Alchemy: A web 2.0 real-time quality assurance platform for human immunodeficiency Virus, hepatitis C Virus, and BK Virus quantitation assays

    Directory of Open Access Journals (Sweden)

    Emmanuel Agosto-Arroyo

    2017-01-01

    Full Text Available Background: The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT. Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs contain all the data elements necessary to do accurate quality assurance (QA reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Methods: Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV quantitation, hepatitis C virus (HCV quantitation, and BK virus (BKV quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Results: Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45–60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Conclusions: Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician

  10. Alchemy: A Web 2.0 Real-time Quality Assurance Platform for Human Immunodeficiency Virus, Hepatitis C Virus, and BK Virus Quantitation Assays.

    Science.gov (United States)

    Agosto-Arroyo, Emmanuel; Coshatt, Gina M; Winokur, Thomas S; Harada, Shuko; Park, Seung L

    2017-01-01

    The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT). Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs) contain all the data elements necessary to do accurate quality assurance (QA) reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV) quantitation, hepatitis C virus (HCV) quantitation, and BK virus (BKV) quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45-60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician-supervised resident/fellow programming projects as learning

  11. Selecting a set of housekeeping genes for quantitative real-time PCR in normal and tetraploid haemocytes of soft-shell clams, Mya arenaria.

    Science.gov (United States)

    Siah, A; Dohoo, C; McKenna, P; Delaporte, M; Berthe, F C J

    2008-09-01

    The transcripts involved in the molecular mechanisms of haemic neoplasia in relation to the haemocyte ploidy status of the soft-shell clam, Mya arenaria, have yet to be identified. For this purpose, real-time quantitative RT-PCR constitutes a sensitive and efficient technique, which can help determine the gene expression involved in haemocyte tetraploid status in clams affected by haemic neoplasia. One of the critical steps in comparing transcription profiles is the stability of selected housekeeping genes, as well as an accurate normalization. In this study, we selected five reference genes, S18, L37, EF1, EF2 and actin, generally used as single control genes. Their expression was analyzed by real-time quantitative RT-PCR at different levels of haemocyte ploidy status in order to select the most stable genes. Using the geNorm software, our results showed that L37, EF1 and S18 represent the most stable gene expressions related to various ploidy status ranging from 0 to 78% of tetraploid haemocytes in clams sampled in North River (Prince Edward Island, Canada). However, actin gene expression appeared to be highly regulated. Hence, using it as a housekeeping gene in tetraploid haemocytes can result in inaccurate data. To compare gene expression levels related to haemocyte ploidy status in Mya arenaria, using L37, EF1 and S18 as housekeeping genes for accurate normalization is therefore recommended.

  12. Identification of three novel OA1 gene mutations identified in three families misdiagnosed with congenital nystagmus and carrier status determination by real-time quantitative PCR assay

    Directory of Open Access Journals (Sweden)

    Hamel Christian

    2003-01-01

    Full Text Available Abstract Background X-linked ocular albinism type 1 (OA1 is caused by mutations in OA1 gene, which encodes a membrane glycoprotein localised to melanosomes. OA1 mainly affects pigment production in the eye, resulting in optic changes associated with albinism including hypopigmentation of the retina, nystagmus, strabismus, foveal hypoplasia, abnormal crossing of the optic fibers and reduced visual acuity. Affected Caucasian males usually appear to have normal skin and hair pigment. Results We identified three previously undescribed mutations consisting of two intragenic deletions (one encompassing exon 6, the other encompassing exons 7–8, and a point mutation (310delG in exon 2. We report the development of a new method for diagnosis of heterozygous deletions in OA1 gene based on measurement of gene copy number using real-time quantitative PCR from genomic DNA. Conclusion The identification of OA1 mutations in families earlier reported as families with hereditary nystagmus indicate that ocular albinism type 1 is probably underdiagnosed. Our method of real-time quantitative PCR of OA1 exons with DMD exon as external standard performed on the LightCycler™ allows quick and accurate carrier-status assessment for at-risk females.

  13. [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.

  14. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

    2002-01-01

    A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD......) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect ... 6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro...

  15. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

    2002-01-01

    6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro...... axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer......A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD...

  16. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

    2002-01-01

    ) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect r = 0.99) over...... 6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro....... In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical...

  17. A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR

    Directory of Open Access Journals (Sweden)

    Stewart Don

    2008-05-01

    Full Text Available Abstract Background Based upon defining a common reference point, current real-time quantitative PCR technologies compare relative differences in amplification profile position. As such, absolute quantification requires construction of target-specific standard curves that are highly resource intensive and prone to introducing quantitative errors. Sigmoidal modeling using nonlinear regression has previously demonstrated that absolute quantification can be accomplished without standard curves; however, quantitative errors caused by distortions within the plateau phase have impeded effective implementation of this alternative approach. Results Recognition that amplification rate is linearly correlated to amplicon quantity led to the derivation of two sigmoid functions that allow target quantification via linear regression analysis. In addition to circumventing quantitative errors produced by plateau distortions, this approach allows the amplification efficiency within individual amplification reactions to be determined. Absolute quantification is accomplished by first converting individual fluorescence readings into target quantity expressed in fluorescence units, followed by conversion into the number of target molecules via optical calibration. Founded upon expressing reaction fluorescence in relation to amplicon DNA mass, a seminal element of this study was to implement optical calibration using lambda gDNA as a universal quantitative standard. Not only does this eliminate the need to prepare target-specific quantitative standards, it relegates establishment of quantitative scale to a single, highly defined entity. The quantitative competency of this approach was assessed by exploiting "limiting dilution assay" for absolute quantification, which provided an independent gold standard from which to verify quantitative accuracy. This yielded substantive corroborating evidence that absolute accuracies of ± 25% can be routinely achieved. Comparison

  18. Identification of internal control genes for quantitative expression analysis by real-time PCR in bovine peripheral lymphocytes.

    Science.gov (United States)

    Spalenza, Veronica; Girolami, Flavia; Bevilacqua, Claudia; Riondato, Fulvio; Rasero, Roberto; Nebbia, Carlo; Sacchi, Paola; Martin, Patrice

    2011-09-01

    Gene expression studies in blood cells, particularly lymphocytes, are useful for monitoring potential exposure to toxicants or environmental pollutants in humans and livestock species. Quantitative PCR is the method of choice for obtaining accurate quantification of mRNA transcripts although variations in the amount of starting material, enzymatic efficiency, and the presence of inhibitors can lead to evaluation errors. As a result, normalization of data is of crucial importance. The most common approach is the use of endogenous reference genes as an internal control, whose expression should ideally not vary among individuals and under different experimental conditions. The accurate selection of reference genes is therefore an important step in interpreting quantitative PCR studies. Since no systematic investigation in bovine lymphocytes has been performed, the aim of the present study was to assess the expression stability of seven candidate reference genes in circulating lymphocytes collected from 15 dairy cows. Following the characterization by flow cytometric analysis of the cell populations obtained from blood through a density gradient procedure, three popular softwares were used to evaluate the gene expression data. The results showed that two genes are sufficient for normalization of quantitative PCR studies in cattle lymphocytes and that YWAHZ, S24 and PPIA are the most stable genes. Copyright © 2010 Elsevier Ltd. All rights reserved.

  19. Detection and semi-quantification of Strongylus vulgaris DNA in equine faeces by real-time quantitative PCR.

    Science.gov (United States)

    Nielsen, Martin K; Peterson, David S; Monrad, Jesper; Thamsborg, Stig M; Olsen, Susanne N; Kaplan, Ray M

    2008-03-01

    Strongylus vulgaris is an important strongyle nematode with high pathogenic potential infecting horses world-wide. Several decades of intensive anthelmintic use has virtually eliminated clinical disease caused by S. vulgaris, but has also caused high levels of anthelmintic resistance in equine small strongyle (cyathostomin) nematodes. Recommendations aimed at limiting the development of anthelmintic resistance by reducing treatment intensity raises a simultaneous demand for reliable and accurate diagnostic tools for detecting important parasitic pathogens. Presently, the only means available to differentiate among strongyle species in a faecal sample is by identifying individual L3 larvae following a two week coproculture procedure. The aim of the present study is to overcome this diagnostic obstacle by developing a fluorescence-based quantitative PCR assay capable of identifying S. vulgaris eggs in faecal samples from horses. Species-specific primers and a TaqMan probe were designed by alignment of published ribosomal DNA sequences of the second internal transcribed spacer of cyathostomin and Strongylus spp. nematodes. The assay was tested for specificity and optimized using genomic DNA extracted from identified male worms of Strongylus and cyathostomin species. In addition, eggs were collected from adult female worms and used to evaluate the quantitative potential of the assay. Statistically significant linear relationships were found between egg numbers and cycle of threshold (Ct) values. PCR results were unaffected by the presence of cyathostomin DNA in the sample and there was no indication of PCR inhibition by faecal sources. A field evaluation on faecal samples obtained from four Danish horse farms revealed a good agreement with the traditional larval culture (kappa-value=0.78), but with a significantly higher performance of the PCR assay. An association between Ct values and S. vulgaris larval counts was statistically significant. The present assay can

  20. A real-time fluorescent sensor specific to Mg2+: crystallographic evidence, DFT calculation and its use for quantitative determination of magnesium in drinking water.

    Science.gov (United States)

    Men, Guangwen; Chen, Chunrong; Zhang, Shitong; Liang, Chunshuang; Wang, Ying; Deng, Mengyu; Shang, Hongxing; Yang, Bing; Jiang, Shimei

    2015-02-14

    An "off-the-shelf" fluorescence "turn-on" Mg(2+) chemosensor 3,5-dichlorosalicylaldehyde (BCSA) was rationally designed and developed. This proposed sensor works based on Mg(2+)-induced formation of the 2 : 1 BCSA-Mg(2+) complex. The coordination of BSCA to Mg(2+) increases its structural rigidity generating a chelation-enhanced fluorescence (CHEF) effect which was confirmed by single crystal XRD studies of the BSCA-Mg(2+) complex and TD/DFT calculations. This sensor exhibits high sensitivity and selectivity for the quantitative monitoring of Mg(2+) with a wide detection range (0-40 μM), a low detection limit (2.89 × 10(-7) mol L(-1)) and a short response time (sensor can be utilized to monitor Mg(2+) in real time within actual samples from drinking water.

  1. 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...

  2. Combining real-time PCR and next-generation DNA sequencing to provide quantitative comparisons of fungal aerosol populations

    Science.gov (United States)

    Dannemiller, Karen C.; Lang-Yona, Naama; Yamamoto, Naomichi; Rudich, Yinon; Peccia, Jordan

    2014-02-01

    We examined fungal communities associated with the PM10 mass of Rehovot, Israel outdoor air samples collected in the spring and fall seasons. Fungal communities were described by 454 pyrosequencing of the internal transcribed spacer (ITS) region of the fungal ribosomal RNA encoding gene. To allow for a more quantitative comparison of fungal exposure in humans, the relative abundance values of specific taxa were transformed to absolute concentrations through multiplying these values by the sample's total fungal spore concentration (derived from universal fungal qPCR). Next, the sequencing-based absolute concentrations for Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, and Penicillium/Aspergillus spp. were compared to taxon-specific qPCR concentrations for A. alternata, C. cladosporioides, E. nigrum, and Penicillium/Aspergillus spp. derived from the same spring and fall aerosol samples. Results of these comparisons showed that the absolute concentration values generated from pyrosequencing were strongly associated with the concentration values derived from taxon-specific qPCR (for all four species, p 0.70). The correlation coefficients were greater for species present in higher concentrations. Our microbial aerosol population analyses demonstrated that fungal diversity (number of fungal operational taxonomic units) was higher in the spring compared to the fall (p = 0.02), and principal coordinate analysis showed distinct seasonal differences in taxa distribution (ANOSIM p = 0.004). Among genera containing allergenic and/or pathogenic species, the absolute concentrations of Alternaria, Aspergillus, Fusarium, and Cladosporium were greater in the fall, while Cryptococcus, Penicillium, and Ulocladium concentrations were greater in the spring. The transformation of pyrosequencing fungal population relative abundance data to absolute concentrations can improve next-generation DNA sequencing-based quantitative aerosol exposure assessment.

  3. Route around real time

    International Nuclear Information System (INIS)

    Terrier, Francois

    1996-01-01

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

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

    Science.gov (United States)

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

    2007-02-01

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

  5. Real time falling leaves

    OpenAIRE

    Vázquez Alcocer, Pere Pau; Balsa, Marcos

    2007-01-01

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

  6. Real Time Strategy Language

    OpenAIRE

    Hayes, Roy; Beling, Peter; Scherer, William

    2014-01-01

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

  7. Real Time Processing

    CERN Multimedia

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

    2015-01-01

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

  8. Identification of appropriate reference genes for human mesenchymal stem cell analysis by quantitative real-time PCR.

    Science.gov (United States)

    Li, Xiuying; Yang, Qiwei; Bai, Jinping; Xuan, Yali; Wang, Yimin

    2015-01-01

    Normalization to a reference gene is the method of choice for quantitative reverse transcription-PCR (RT-qPCR) analysis. The stability of reference genes is critical for accurate experimental results and conclusions. We have evaluated the expression stability of eight commonly used reference genes found in four different human mesenchymal stem cells (MSC). Using geNorm, NormFinder and BestKeeper algorithms, we show that beta-2-microglobulin and peptidyl-prolylisomerase A were the optimal reference genes for normalizing RT-qPCR data obtained from MSC, whereas the TATA box binding protein was not suitable due to its extensive variability in expression. Our findings emphasize the significance of validating reference genes for qPCR analyses. We offer a short list of reference genes to use for normalization and recommend some commercially-available software programs as a rapid approach to validate reference genes. We also demonstrate that the two reference genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase, are frequently used are not always successful in many cases.

  9. Selection of reference genes for quantitative real time RT-PCR during dimorphism in the zygomycete Mucor circinelloides.

    Science.gov (United States)

    Valle-Maldonado, Marco I; Jácome-Galarza, Irvin E; Gutiérrez-Corona, Félix; Ramírez-Díaz, Martha I; Campos-García, Jesús; Meza-Carmen, Víctor

    2015-03-01

    Mucor circinelloides is a dimorphic fungal model for studying several biological processes including cell differentiation (yeast-mold transitions) as well as biodiesel and carotene production. The recent release of the first draft sequence of the M. circinelloides genome, combined with the availability of analytical methods to determine patterns of gene expression, such as quantitative Reverse transcription-Polymerase chain reaction (qRT-PCR), and the development of molecular genetic tools for the manipulation of the fungus, may help identify M. circinelloides gene products and analyze their relevance in different biological processes. However, no information is available on M. circinelloides genes of stable expression that could serve as internal references in qRT-PCR analyses. One approach to solve this problem consists in the use of housekeeping genes as internal references. However, validation of the usability of these reference genes is a fundamental step prior to initiating qRT-PCR assays. This work evaluates expression of several constitutive genes by qRT-PCR throughout the morphological differentiation stages of M. circinelloides; our results indicate that tfc-1 and ef-1 are the most stable genes for qRT-PCR assays during differentiation studies and they are proposed as reference genes to carry out gene expression studies in this fungus.

  10. Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment

    Science.gov (United States)

    Ghosh, Prakash; Khan, Md. Anik Ashfaq; Duthie, Malcolm S.; Vallur, Aarthy C.; Picone, Alessandro; Howard, Randall F.; Reed, Steven G.

    2017-01-01

    Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19–100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16–94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55–89.16%) for VL samples, 100% (95%CI, 69.15–100%) for RVL samples, and 52.5% (95% CI, 36.13–68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98–100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L

  11. Real-Time Monitoring of Chemical Changes in Three Kinds of Fermented Milk Products during Fermentation Using Quantitative Difference Nuclear Magnetic Resonance Spectroscopy.

    Science.gov (United States)

    Lu, Yi; Ishikawa, Hiroto; Kwon, Yeondae; Hu, Fangyu; Miyakawa, Takuya; Tanokura, Masaru

    2018-02-14

    Fermented milk products are rising in popularity throughout the world as a result of their health benefits, including improving digestion, normalizing the function of the immune system, and aiding in weight management. This study applies an in situ quantitative nuclear magnetic resonance method to monitor chemical changes in three kinds of fermented milk products, Bulgarian yogurt, Caspian Sea yogurt, and kefir, during fermentation. As a result, the concentration changes in nine organic compounds, α/β-lactose, α/β-galactose, lactic acid, citrate, ethanol, lecithin, and creatine, were monitored in real time. This revealed three distinct metabolic processes in the three fermented milk products. Moreover, pH changes were also determined by variations in the chemical shift of citric acid during the fermentation processes. These results can be applied to estimate microbial metabolism in various flora and help guide the fermentation and storage of various fermented milk products to improve their quality, which may directly influence human health.

  12. Real-Time Quantitative PCR Measurement of Ileal Lactobacillus salivarius Populations from Broiler Chickens To Determine the Influence of Farming Practices▿ †

    Science.gov (United States)

    Harrow, Sally A.; Ravindran, Velmurugu; Butler, Ruth C.; Marshall, John W.; Tannock, Gerald W.

    2007-01-01

    A real-time quantitative PCR assay targeting a 16S-23S intergenic spacer region sequence was devised to measure the sizes of populations of Lactobacillus salivarius present in ileal digesta collected from broiler chickens. This species has been associated with deconjugation of bile salts in the small bowel and reduced broiler productivity. The assay was tested as a means of monitoring the sizes of L. salivarius populations from broilers fed diets with different compositions, maintained at different stocking densities, or given the antimicrobial drugs bacitracin and monensin in the feed. Stocking densities did not influence the numbers of L. salivarius cells in the ileum. A diet containing meat and bone meal reduced the size of the L. salivarius population relative to that of chickens given the control diet, as did administration of bacitracin and monensin in the feed. These changes in the target bacterial population were associated with improved broiler weight gain. PMID:17890342

  13. Cytoglobin expression is upregulated in all tissues upon hypoxia: an in vitro and in vivo study by quantitative real-time PCR

    International Nuclear Information System (INIS)

    Fordel, E.; Geuens, E.; Dewilde, S.; Rottiers, P.; Carmeliet, P.; Grooten, J.; Moens, L.

    2004-01-01

    The vertebrate globin family has been extended with two members: neuroglobin and cytoglobin. We here investigate the changes of expression levels upon hypoxia of cytoglobin in parallel with neuroglobin, in vivo and in vitro, by using real-time quantitative PCR. Our data prove that cytoglobin is upregulated upon hypoxia in all tissues. The mechanism of induction of cytoglobin is regulated by the hypoxia-inducible factor 1, a posttranscriptionally regulated transcription factor controlling several hypoxia-inducible genes. The latter is argumented by: (1) cytoglobin is significantly upregulated upon hypoxia and this is dependent on the tissue and severity of hypoxia; (2) the regulation of cytoglobin expression in HIF-1 (+/-) knockout mice is affected; (3) the variations of the expression regulation are in the same manner as seen in the expression of our control gene VEGF, that is proven to be regulated by the HIF-1-pathway; and (4) cytoglobin promoter region contains HRE sites

  14. Four-hour quantitative real-time polymerase chain reaction-based comprehensive chromosome screening and accumulating evidence of accuracy, safety, predictive value, and clinical efficacy.

    Science.gov (United States)

    Treff, Nathan R; Scott, Richard T

    2013-03-15

    Embryonic comprehensive chromosomal euploidy may represent a powerful biomarker to improve the success of IVF. However, there are a number of aneuploidy screening strategies to consider, including different technologic platforms with which to interrogate the embryonic DNA, and different embryonic developmental stages from which DNA can be analyzed. Although there are advantages and disadvantages associated with each strategy, a series of experiments producing evidence of accuracy, safety, clinical predictive value, and clinical efficacy indicate that trophectoderm biopsy and quantitative real-time polymerase chain reaction (qPCR)-based comprehensive chromosome screening (CCS) may represent a useful strategy to improve the success of IVF. This Biomarkers in Reproductive Medicine special issue review summarizes the accumulated experience with the development and clinical application of a 4-hour blastocyst qPCR-based CCS technology. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  15. 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.

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

    DEFF Research Database (Denmark)

    Nielsen, K. K.; Boye, Mette

    2005-01-01

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

  17. Galactomannan enzyme immunoassay and quantitative Real Time PCR as tools to evaluate the exposure and response in a rat model of aspergillosis after posaconazole prophylaxis.

    Science.gov (United States)

    Cendejas-Bueno, Emilio; Forastiero, Agustina; Ruiz, Isabel; Mellado, Emilia; Buitrago, María José; Gavaldà, Joan; Gomez-Lopez, Alicia

    2016-11-01

    A steroid-immunosuppressed rat model of invasive pulmonary aspergillosis was use to examine the usefulness of galactomannan enzyme immunoassay (GM) and quantitative real time PCR (RT-PCR) in evaluating the association between response and exposure after a high dose of prophylactic posaconazole. Two different strains of Aspergillus fumigatus with different in vitro posaconazole susceptibility were used. Serum concentrations demonstrated similar posaconazole exposure for all treated animals. However, response to posaconazole relied on the in vitro susceptibility of the infecting strain. After prophylaxis, galactomannan index and fungal burden only decreased in those animals infected with the most susceptible strain. This study demonstrated that both biomarkers may be useful tools for predicting efficacy of antifungal compounds in prophylaxis. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  18. The numbers game: quantitative analysis of Neorickettsia sp. propagation through complex life cycle of its digenean host using real-time qPCR.

    Science.gov (United States)

    Greiman, Stephen E; Tkach, Vasyl V

    2016-07-01

    Bacteria of the genus Neorickettsia are obligate intracellular endosymbionts of parasitic flukes (Digenea) and are passed through the entire complex life cycle of the parasite by vertical transmission. Several species of Neorickettsia are known to cause diseases in domestic animals, wildlife, and humans. Quantitative data on the transmission of the bacteria through the digenean life cycle is almost completely lacking. This study quantified for the first time the abundance of Neorickettsia within multiple stages of the life cycle of the digenean Plagiorchis elegans. Snails Lymnaea stagnalis collected from a pond in North Dakota were screened for the presence of digenean cercariae, which were subsequently tested for the presence of Neorickettsia. Three L. stagnalis were found shedding P. elegans cercariae infected with Neorickettsia. These snails were used to initiate three separate laboratory life cycles and obtain all life cycle stages for bacterial quantification. A quantitative real-time PCR assay targeting the GroEL gene was developed to enumerate Neorickettsia sp. within different stages of the digenean life cycle. The number of bacteria significantly increased throughout all stages, from eggs to adults. The two largest increases in number of bacteria occurred during the period from eggs to cercariae and from 6-day metacercariae to 48-h juvenile worms. These two periods seem to be the most important for Neorickettsia propagation through the complex digenean life cycle and maturation in the definitive host.

  19. Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting the groEL Gene

    Science.gov (United States)

    Junick, Jana

    2012-01-01

    Quantitative real-time PCR assays targeting the groEL gene for the specific enumeration of 12 human fecal Bifidobacterium species were developed. The housekeeping gene groEL (HSP60 in eukaryotes) was used as a discriminative marker for the differentiation of Bifidobacterium adolescentis, B. angulatum, B. animalis, B. bifidum, B. breve, B. catenulatum, B. dentium, B. gallicum, B. longum, B. pseudocatenulatum, B. pseudolongum, and B. thermophilum. The bifidobacterial chromosome contains a single copy of the groEL gene, allowing the determination of the cell number by quantification of the groEL copy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a given Bifidobacterium species. Independent of the Bifidobacterium species tested, the proportion of groEL copies recovered from fecal samples spiked with 5 to 9 log10 cells/g feces was approximately 50%. The quantification limit was 5 to 6 log10 groEL copies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3 Bifidobacterium species and B. longum frequently detected. The predominant species in infant and adult fecal samples were B. breve and B. adolescentis, respectively. It was possible to distinguish B. catenulatum and B. pseudocatenulatum. We conclude that the groEL gene is a suitable molecular marker for the specific and accurate quantification of human fecal Bifidobacterium species by real-time PCR. PMID:22307308

  20. Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application

    Science.gov (United States)

    Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

    2011-01-01

    Background Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. Methods The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. Conclusions The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. Significance The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food. PMID:21283808

  1. High-level expression of podoplanin in benign and malignant soft tissue tumors: immunohistochemical and quantitative real-time RT-PCR analysis.

    Science.gov (United States)

    Xu, Yongjun; Ogose, Akira; Kawashima, Hiroyuki; Hotta, Tetsuo; Ariizumi, Takashi; Li, Guidong; Umezu, Hajime; Endo, Naoto

    2011-03-01

    Podoplanin is a 38 kDa mucin-type transmembrane glycoprotein that was first identified in rat glomerular epithelial cells (podocytes). It is expressed in normal lymphatic endothelium, but is absent from vascular endothelial cells. D2-40 is a commercially available mouse monoclonal antibody which binds to an epitope on human podoplanin. D2-40 immunoreactivity is therefore highly sensitive and specific for lymphatic endothelium. Recent investigations have shown widespread applications of immunohistochemical staining with D2-40 in evaluating podoplanin expression as an immunohistochemical marker for diagnosis and prognosis in various tumors. To determine whether the podoplanin (D2-40) antibody may be useful for the diagnosis of soft tissue tumors, 125 cases, including 4 kinds of benign tumors, 15 kinds of malignant tumors and 3 kinds of tumor-like lesions were immunostained using the D2-40 antibody. Total RNA was extracted from frozen tumor tissue obtained from 41 corresponding soft tissue tumor patients and 12 kinds of soft tissue tumor cell lines. Quantitative real-time PCR reactions were performed. Immunohistochemical and quantitative real-time RT-PCR analyses demonstrated the expression of the podoplanin protein and mRNA in the majority of benign and malignant soft tissue tumors and tumor-like lesions examined, with the exception of alveolar soft part sarcoma, embryonal and alveolar rhabdomyosarcoma, extraskeletal Ewing's sarcoma/peripheral primitive neuro-ectodermal tumor and lipoma, which were completely negative for podoplanin. Since it is widely and highly expressed in nearly all kinds of soft tissue tumors, especially in spindle cell sarcoma, myxoid type soft tissue tumors and soft tissue tumors of the nervous system, podoplanin is considered to have little value in the differential diagnosis of soft tissue tumors.

  2. DEVELOPMENT OF REAL-TIME MULTIPLEX PCR FOR THE QUANTITATIVE DETERMINATION OF TREC'S AND KREC'S IN WHOLE BLOOD AND IN DRIED BLOOD SPOTS

    Directory of Open Access Journals (Sweden)

    M. A. Gordukova

    2015-01-01

    Full Text Available Primary immunodeficiencies (PID such as severe combined immunodeficiency (SCID and X-linked agammaglobulinemia are characterized by the lack of functional Tand B-cells, respectively. Without early diagnosis and prompt treatment children with PID suffer from severe infectious diseases, leading to their death or disability. Our purpose was developing of simple, inexpensive, high throughput technique based on the quantitative determination of TREC and KREC molecules by real-time PCR, and its validation in a group of children with a verified diagnosis of SCID and X-linked agammaglobulinemia.In this study, we developed and validated multiplex real-time PCR for the TREC’s and KREC’s quantitative analysis. We have shown that linear range of Ct changes depending on the concentrations of targets with a correlation coefficient R2 not worse than 0.98 was observed at concentrations from 109 to 5 × 104 copies per ml. The lowest amount of targets reliably detected in a reaction volume was 10 TREC’s copies, 5 KREC ‘s copies and 5 copies of internal control (IL17RA. We determined the age-depended reference values of TRECs and KRECs in whole blood in 29 boys and 27 girls with normal immunological parameters. The normal cut-offs for TRECs and KRECs were defined in dry blood spots depending on the method of extraction.The proposed method showed 100% diagnostic sensitivity and specificity in the studied group. The method can be proposed as a screening tool for the diagnosis of SCID and X-linked agammaglobulinemia both in whole blood and in the dry blood spots. The further investigation is required with larger number of samples. 

  3. Development of a real-time RT-PCR assay for the simultaneous identification, quantitation and differentiation of avian metapneumovirus subtypes A and B.

    Science.gov (United States)

    Cecchinato, Mattia; Lupini, Caterina; Munoz Pogoreltseva, Olga Svetlana; Listorti, Valeria; Mondin, Alessandra; Drigo, Michele; Catelli, Elena

    2013-01-01

    In recent years, special attention has been paid to real-time polymerase chain reaction (PCR) for avian metapneumovirus (AMPV) diagnosis, due to its numerous advantages over classical PCR. A new multiplex quantitative real-time reverse transcription-PCR (qRT-PCR) with molecular beacon probe assay, designed to target the SH gene, was developed. The test was evaluated in terms of specificity, sensitivity and repeatability, and compared with conventional RT nested-PCR based on the G gene. All of the AMPV subtype A and B strains tested were amplified and specifically detected while no amplification occurred with other non-target bird respiratory pathogens. The detection limit of the assay was 10(-0.41) median infectious dose/ml and 10(1.15) median infectious dose/ml when the AMPV-B strain IT/Ty/B/Vr240/87 and the AMPV-A strain IT/Ty/A/259-01/03 were used, respectively, as templates. In all cases, the amplification efficiency was approximately 2 and the error values were 0.9375) between crossing point values and virus quantities, making the assay herein designed reliable for quantification. When the newly developed qRT-PCR was compared with a conventional RT nested-PCR, it showed greater sensitivity with RNA extracted from both positive controls and from experimentally infected birds. This assay can be effectively used for the detection, identification, differentiation and quantitation of AMPV subtype A or subtype B to assist in disease diagnosis and to carry out rapid surveillance with high levels of sensitivity and specificity.

  4. Feasibility of iFlow color-coding technique in quantitative real-time measurement of hemodynamic changes after transarterial chemoembolization for hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    WANG Yuzhe

    2018-01-01

    Full Text Available Objective To investigate the value of iFlow color-coding technique in quantitative real-time analysis of hemodynamic changes after transarterial chemoembolization (TACE for hepatocellular carcinoma (HCC. Methods A total of 31 patients who were diagnosed with HCC in Shanghai Fifth People′s Hospital from December 2015 to January 2017 were enrolled. No patient underwent surgical operation or ablation. All patients underwent TACE with the same contrast agent, high-pressure injector parameters, and place of angiographic catheter. The iFlow technique was used to generate two-dimensional color-coded images and time-density curve (TDC before and after surgery and measure the opening of the angiographic catheter and the time to peak (TTP of the starting and ending points of the major tumor feeding arteries, as well as the ratio of the areas under the curve (AUC of TDC of tumor tissue and the opening of the angiographic catheter. The paired t-test was used for comparison of continuous data between groups. Results TTP of the major tumor feeding arteries was 4.64±0.49 s before TACE and 5.97±0.84 s after TACE (t=11.57, P<0.01, and there was a significant difference in AUC between the tumor tissue and the opening of the angiographic catheter (0.53±0.15 vs 0.16±0.12, t=25.85, P<0.01. There was no significant difference in TTP between the opening of the angiographic catheter and the major tumor feeding arteries before and after TACE (P>0.05. Before TACE, the TDC of tumor feeding arteries had a shape of “rapid increase-rapid reduction” with relatively high slope and peak value, while after TACE, the TDC had a shape of “increase-flat-reduction” with reductions in slope and peak value. Conclusion The iFlow technique can perform real-time measurement of TTP and TDC of the region of interest and helps with quantitative evaluation of hemodynamic changes in HCC. Therefore, it can provide objective quantitative indices for evaluating the degree of

  5. Real Time Text Analysis

    Science.gov (United States)

    Senthilkumar, K.; Ruchika Mehra Vijayan, E.

    2017-11-01

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

  6. Real time Faraday spectrometer

    Science.gov (United States)

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

    1991-01-01

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

  7. Real time Faraday spectrometer

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  8. A quantitative method to track protein translocation between intracellular compartments in real-time in live cells using weighted local variance image analysis.

    Directory of Open Access Journals (Sweden)

    Guillaume Calmettes

    Full Text Available The genetic expression of cloned fluorescent proteins coupled to time-lapse fluorescence microscopy has opened the door to the direct visualization of a wide range of molecular interactions in living cells. In particular, the dynamic translocation of proteins can now be explored in real time at the single-cell level. Here we propose a reliable, easy-to-implement, quantitative image processing method to assess protein translocation in living cells based on the computation of spatial variance maps of time-lapse images. The method is first illustrated and validated on simulated images of a fluorescently-labeled protein translocating from mitochondria to cytoplasm, and then applied to experimental data obtained with fluorescently-labeled hexokinase 2 in different cell types imaged by regular or confocal microscopy. The method was found to be robust with respect to cell morphology changes and mitochondrial dynamics (fusion, fission, movement during the time-lapse imaging. Its ease of implementation should facilitate its application to a broad spectrum of time-lapse imaging studies.

  9. 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

  10. 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.

  11. Quantitative monitoring of HCMV DNAlactia in human milk by real time PCR assay: Implementation of internal control contributes to standardization and quality control.

    Science.gov (United States)

    Hartleif, Steffen; Göhring, Katharina; Goelz, Rangmar; Jahn, Gerhard; Hamprecht, Klaus

    2016-11-01

    For cytomegalovirus screening of breastfeeding mothers of preterm infants under risk, we present a rapid, quantitative real-time PCR protocol using the hybridization format of the viral gB target region. For quantification, we used an external gB fragment cloned into a vector system. For standardization, we created an internal control-plasmid by site-directed mutagenesis with an exchange of 9 nucleotides. Spiked with internal control, patient wildtype amplicons could be discriminated from internal controls by hybridization probes using two-channel fluorescence detection. Potential bias of formerly reported false nucleotide sequence data of gB-hybridization probes was excluded. Using this approach, we could demonstrate excellent analytical performance and high reproducibility of HCMV detection during lactation. This assay shows very good correlation with a commercial quantitative HCMV DNA PCR and may help to identify rapidly HCMV shedding mothers of very low birth weight preterm infants to prevent HCMV transmission. On the other hand, negative DNA amplification results allow feeding of milk samples of seropositive mothers to their preterm infants under risk (<30 weeks of gestational age, <1000g birth weight) during the onset and late stage of HCMV shedding during lactation. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Development of a real-time and quantitative thrombus sensor for an extracorporeal centrifugal blood pump by near-infrared light.

    Science.gov (United States)

    Sakota, Daisuke; Fujiwara, Tatsuki; Ohuchi, Katsuhiro; Kuwana, Katsuyuki; Yamazaki, Hiroyuki; Kosaka, Ryo; Nishida, Masahiro; Mizuno, Tomohiro; Arai, Hirokuni; Maruyama, Osamu

    2018-01-01

    We developed an optical thrombus sensor for a monopivot extracorporeal centrifugal blood pump. In this study, we investigated its quantitative performance for thrombus detection in acute animal experiments of left ventricular assist using the pump on pathogen-free pigs. Optical fibers were set in the driver unit of the pump. The incident light at the near-infrared wavelength of 810 nm was aimed at the pivot bearing, and the resulting scattered light was guided to the optical fibers. The detected signal was analyzed to obtain the thrombus formation level. As a result, real-time and quantitative monitoring of the thrombus surface area on the pivot bearing was achieved with an accuracy of 3.6 ± 2.3 mm 2 . In addition, the sensing method using the near-infrared light was not influenced by changes in the oxygen saturation and the hematocrit. It is expected that the developed sensor will be useful for optimal anticoagulation management for long-term extracorporeal circulation therapies.

  13. Real time production optimization

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-07-01

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

  14. Real time urbanism

    Directory of Open Access Journals (Sweden)

    Ana Ruiz Varona

    2012-12-01

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

  15. [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.

  16. Development Status of the WetLab-2 Project: New Tools for On-orbit Real-time Quantitative Gene Expression.

    Science.gov (United States)

    Jung, Jimmy; Parra, Macarena P.; Almeida, Eduardo; Boone, Travis; Chinn, Tori; Ricco, Antonio; Souza, Kenneth; Hyde, Liz; Rukhsana, Yousuf; Richey, C. Scott

    2013-01-01

    The primary objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform to facilitate gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens grown or cultured on orbit. The WetLab-2 equipment will be capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. In addition to the logistical benefits of in-situ sample processing and analysis, conducting qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms. The system can also validate terrestrial analyses of samples returned from ISS by providing quantitative on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experimental parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Finally, WetLab-2 can be used for analysis of air, surface, water, and clinical samples to monitor environmental contaminants and crew health. The verification flight of the instrument is scheduled to launch on SpaceX-5 in Aug. 2014.Progress to date: The WetLab-2 project completed a thorough study of commercially available qRT-PCR systems and performed a downselect based on both scientific and engineering requirements. The selected instrument, the Cepheid SmartCycler, has advantages including modular design (16 independent PCR modules), low power consumption, and rapid ramp times. The SmartCycler has multiplex capabilities, assaying up to four genes of interest in each of the 16 modules. The WetLab-2 team is currently working with Cepheid to modify the unit for housing within an EXPRESS rack locker on the ISS. This will enable the downlink of data to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project is

  17. Real time spectrum analysis

    International Nuclear Information System (INIS)

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

    1983-01-01

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

  18. Repeated cycles of chemical and physical disinfection and their influence on Mycobacterium avium subsp. paratuberculosis viability measured by propidium monoazide F57 quantitative real time PCR.

    Science.gov (United States)

    Kralik, Petr; Babak, Vladimir; Dziedzinska, Radka

    2014-09-01

    Mycobacterium avium subsp. paratuberculosis (MAP) has a high degree of resistance to chemical and physical procedures frequently used for the elimination of other bacteria. Recently, a method for the determination of viability by exposure of MAP to propidium monoazide (PMA) and subsequent real time quantitative PCR (qPCR) was established and found to be comparable with culture. The aim of this study was to apply the PMA qPCR method to determine the impact of increasing concentration or time and repeated cycles of the application of selected disinfectants on MAP viability. Different MAP isolates responded to the same type of stress in different ways. The laboratory strain CAPM 6381 had the highest tolerance, while the 8819 low-passage field isolate was the most sensitive. Ultraviolet exposure caused only a partial reduction in MAP viability; all MAP isolates were relatively resistant to chlorine. Only the application of peracetic acid led to the total elimination of MAP. Repeated application of the treatments resulted in more significant decreases in MAP viability compared to single increases in the concentration or time of exposure to the disinfectant. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. MATtrack: A MATLAB-Based Quantitative Image Analysis Platform for Investigating Real-Time Photo-Converted Fluorescent Signals in Live Cells.

    Science.gov (United States)

    Courtney, Jane; Woods, Elena; Scholz, Dimitri; Hall, William W; Gautier, Virginie W

    2015-01-01

    We introduce here MATtrack, an open source MATLAB-based computational platform developed to process multi-Tiff files produced by a photo-conversion time lapse protocol for live cell fluorescent microscopy. MATtrack automatically performs a series of steps required for image processing, including extraction and import of numerical values from Multi-Tiff files, red/green image classification using gating parameters, noise filtering, background extraction, contrast stretching and temporal smoothing. MATtrack also integrates a series of algorithms for quantitative image analysis enabling the construction of mean and standard deviation images, clustering and classification of subcellular regions and injection point approximation. In addition, MATtrack features a simple user interface, which enables monitoring of Fluorescent Signal Intensity in multiple Regions of Interest, over time. The latter encapsulates a region growing method to automatically delineate the contours of Regions of Interest selected by the user, and performs background and regional Average Fluorescence Tracking, and automatic plotting. Finally, MATtrack computes convenient visualization and exploration tools including a migration map, which provides an overview of the protein intracellular trajectories and accumulation areas. In conclusion, MATtrack is an open source MATLAB-based software package tailored to facilitate the analysis and visualization of large data files derived from real-time live cell fluorescent microscopy using photoconvertible proteins. It is flexible, user friendly, compatible with Windows, Mac, and Linux, and a wide range of data acquisition software. MATtrack is freely available for download at eleceng.dit.ie/courtney/MATtrack.zip.

  20. Coexistence of Epstein-Barr virus and Parvovirus B19 in tonsillar tissue samples: quantitative measurement by real-time PCR.

    Science.gov (United States)

    Sahiner, Fatih; Gümral, Ramazan; Yildizoğlu, Üzeyir; Babayiğit, Mustafa Alparslan; Durmaz, Abdullah; Yiğit, Nuri; Saraçli, Mehmet Ali; Kubar, Ayhan

    2014-08-01

    In this study, we aimed to investigate the presence and copy number of six different viruses in tonsillar tissue samples removed surgically because of chronic recurrent tonsillitis or chronic obstructive tonsillar hypertrophy. In total, 56 tissue samples (tonsillar core) collected from 44 children and 12 adults were included in this study. The presence of viruses was investigated using a new TaqMan-based quantitative real-time PCR assay. Of the 56 tissue samples, 67.9% (38/56) were positive for at least one of the six viruses. Epstein-Barr virus was the most frequently detected virus, being found in 53.6% (30/56), followed by human Parvovirus B19 21.4% (12/56), human adenovirus 12.5% (7/56), human Cytomegalovirus 5.4% (3/56), BK polyomavirus 1.8% (1/56), and Herpes simplex virus 1.8% (1/56). Precancerous or cancerous changes were not detected in the tonsillar tissue samples by pathologic examination, whereas lymphoid hyperplasia was observed in 24 patients. In contrast to other viruses, B19 virus was present in high copy number in tonsillar tissues. The rates of EBV and B19 virus with high copy number (>500.000 copies/ml) were higher in children than in adults, and a positive relationship was also found between the presence of EBV and the presence of B19 virus with high copy number (P=0.037). It is previously reported that some viral agents are associated with different chronic tonsillar pathologies. In the present study, the presence of B19 virus in tonsillar core samples was investigated quantitatively for the first time, and our data suggests that EBV infections could be associated with B19 virus infections or could facilitate B19 virus replication. However, further detailed studies are needed to clarify this observation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  1. 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.

  2. Comparison of serum pools and oral fluid samples for detection of porcine circovirus type 2 by quantitative real-time PCR in finisher pigs

    DEFF Research Database (Denmark)

    Nielsen, Gitte Blach; Nielsen, Jens Peter; Haugegaard, John

    2018-01-01

    Porcine circovirus type 2 (PCV2) diagnostics in live pigs often involves pooled serum and/or oral fluid samples for group-level determination of viral load by quantitative real-time polymerase chain reaction (qPCR). The purpose of the study was to compare the PCV2 viral load determined by q......PCR of paired samples at the pen level of pools of sera (SP) from 4 to 5 pigs and the collective oral fluid (OF) from around 30 pigs corresponding to one rope put in the same pen. Pigs in pens of 2 finishing herds were sampled by cross-sectional (Herd 1) and cross-sectional with follow-up (Herd 2) study designs....... In Herd 1, 50 sample pairs consisting of SP from 4 to 5 pigs and OF from around 23 pigs were collected. In Herd 2, 65 sample pairs consisting of 4 (SP) and around 30 (OF) pigs were collected 4 times at 3-week intervals. A higher proportion of PCV2-positive pens (86% vs. 80% and 100% vs. 91%) and higher...

  3. Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station.

    Directory of Open Access Journals (Sweden)

    Macarena Parra

    Full Text Available The International Space Station (ISS National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen Escherichia coli and mouse liver tissue. Cycle threshold (Ct values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for

  4. [Detecting HB-1 Expression Level in Bone Marrow of Acute Leukemia Patients by Real-Time Fluorescence Quantitative RT-PCR].

    Science.gov (United States)

    Wang, Qing-Yun; Li, Yuan; Ji, Li; Liang, Ze-Yin; Liu, Wei; Ren, Han-Yun; Qiu, Zhi-Xiang

    2018-02-01

    To investigate the expression level of HB-1 gene in patients with acute lymphoblastic leukemia (ALL) and the significance of HB-1 gene in monitoring of minimal residual disease (MRD). The method of real-time fluorescence quantitative RT-PCR (Taqman probe) was established to detect the expression levels of HB-1 gene; then the sensitivity, specificity and repeatability of this assay were evaluated and verified. The HB-1 gene expression levels in bone marrow of 183 cases of ALL, 70 cases of acute myeloid leukemias (AML), 52 cases of non-malignant hematologic diseases and 24 healthy hematopoietic stem cell donors were detected. The correlation of HB-1 level with diagnosis and relapse was analyzed by detecting bone marrow samples of 33 B-ALL. The sensitivity of this assay reached the 10 -4 level. The coefficient of variation for inter-batch and inter-tube of HB-1 were 6.79% and 4.80%, respectively. It was found that HB-1 gene specifically expressed in acute B lymphoblastic leukemia. The median expression levels of HB-1 gene in newly diagnosed and relapsed B-ALL patients were statistically significantly higher than those in ALL in complete remission(CR), newly diagnosed T-ALL, newly diagnosed AML, non-malignant hematologic diseases, and healthy hematopoietic stem cell donors(33.0% vs 0.68%, 0.07%, 0.02%, 0.58% and 0, respectively) (P0.05). The expression level of HB-1 gene declined sharply when B-ALL patients reached complete remission (0-7.99%, with median level 0.68%), but increased when relapsed (7.69%, 8.08% and 484.0% in 3 relapsed samples), which was in accordance with results of flow cytometry. HB-1 gene specifically expressed in acute B lymphoblastic leukemia cells. The established real-time fluorescence quantitative RT-PCR assay shows good sensitivity, specificity and repeatability, thus, can be used as a biological marker in the clinical detection, monitoring MRD and predicting of early relapse for B-ALL patients.

  5. Development of Quantitative Competitive PCR and Absolute Based Real-Time PCR Assays for Quantification of The Butyrate Producing Bacterium: Butyrivibrio fibrisolvens

    Directory of Open Access Journals (Sweden)

    Mojtaba Tahmoorespur

    2016-04-01

    Full Text Available Introduction Butyrivibrio fibrisolvens strains are presently recognized as the major butyrate-producing bacteria found in the rumen and digestive track of many animals and also in the human gut. In this study we reported the development of two DNA based techniques, quantitative competitive (QC PCR and absolute based Real-Time PCR, for enumerating Butyrivibrio fibrisolvens strains. Despite the recent introduction of real-time PCR method for the rapid quantification of the target DNA sequences, use of quantitative competitive PCR (QC-PCR technique continues to play an important role in nucleic acid quantification since it is more cost effective. The procedure relies on the co-amplification of the sequence of interest with a serially diluted synthetic DNA fragment of the known concentration (competitor, using the single set primers. A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR. It monitors the amplification of a targeted DNA molecule during the PCR. Materials and Methods At first reported species-specific primers targeting the 16S rDNA region of the bacterium Butyrivibrio fibrisolvens were used for amplifying a 213 bp fragment. A DNA competitor differing by 50 bp in length from the 213 bp fragment was constructed and cloned into pTZ57R/T vector. The competitor was quantified by NanoDrop spectrophotometer and serially diluted and co-amplified by PCR with total extracted DNA from rumen fluid samples. PCR products were quantified by photographing agarose gels and analyzed with Image J software and the amount of amplified target DNA was log plotted against the amount of amplified competitor. Coefficient of determination (R2 was used as a criterion of methodology precision. For developing the Real-time PCR technique, the 213 bp fragment was amplified and cloned into pTZ57R/T was used to draw a standard curve. Results and Discussion The specific primers of Butyrivibrio

  6. 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...

  7. Prospective Qualitative and Quantitative Analysis of Real-Time Peer Review Quality Assurance Rounds Incorporating Direct Physical Examination for Head and Neck Cancer Radiation Therapy.

    Science.gov (United States)

    Cardenas, Carlos E; Mohamed, Abdallah S R; Tao, Randa; Wong, Andrew J R; Awan, Mussadiq J; Kuruvila, Shirly; Aristophanous, Michalis; Gunn, G Brandon; Phan, Jack; Beadle, Beth M; Frank, Steven J; Garden, Adam S; Morrison, William H; Fuller, Clifton D; Rosenthal, David I

    2017-07-01

    Our department has a long-established comprehensive quality assurance (QA) planning clinic for patients undergoing radiation therapy (RT) for head and neck cancer. Our aim is to assess the impact of a real-time peer review QA process on the quantitative and qualitative radiation therapy plan changes in the era of intensity modulated RT (IMRT). Prospective data for 85 patients undergoing head and neck IMRT who presented at a biweekly QA clinic after simulation and contouring were collected. A standard data collection form was used to document alterations made during this process. The original pre-QA clinical target volumes (CTVs) approved by the treating-attending physicians were saved before QA and compared with post-QA consensus CTVs. Qualitative assessment was done according to predefined criteria. Dice similarity coefficients (DSC) and other volume overlap metrics were calculated for each CTV level and were used for quantitative comparison. Changes are categorized as major, minor, and trivial according to the degree of overlap. Patterns of failure were analyzed and correlated to plan changes. All 85 patients were examined by at least 1 head and neck subspecialist radiation oncologist who was not the treating-attending physician; 80 (94%) were examined by ≥3 faculty members. New clinical findings on physical examination were found in 12 patients (14%) leading to major plan changes. Quantitative DSC analysis revealed significantly better agreement in CTV1 (0.94 ± 0.10) contours than in CTV2 (0.82 ± 0.25) and CTV3 (0.86 ± 0.2) contours (P=.0002 and P=.03, respectively; matched-pair Wilcoxon test). The experience of the treating-attending radiation oncologist significantly affected DSC values when all CTV levels were considered (P=.012; matched-pair Wilcoxon text). After a median follow-up time of 38 months, only 10 patients (12%) had local recurrence, regional recurrence, or both, mostly in central high-dose areas. Comprehensive peer review planning

  8. 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

  9. Quantitative studies of the distribution pattern for Salmonella Enteritidis in the internal organs of chicken after oral challenge by a real-time PCR.

    Science.gov (United States)

    He, G Z; Tian, W Y; Qian, N; Cheng, A C; Deng, S X

    2010-12-01

    This research was undertaken to identify and understand the regular distribution pattern for Salmonella Enteritidis (S. enteritidis) in the internal organs of chicken after oral challenge over a 3 wk period. We used a real-time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) to detect genomic DNA of S. enteritidis in the blood and the internal organs, including heart, liver, spleen, kidney, pancreas, and gallbladder, from chicken after oral challenge at different time points. The results showed that the spleen was positive at 12 h post inoculation (PI), and the blood was at 14 h PI. The organism was detected in the liver and heart at 16 h PI, pancrea was positive at 20 h PI, and the final organ to show a positive results were the kidney and gallbladder at 22 h PI. The copy number of S. enteritidis DNA in each tissue reached a peak at 24 h-36 h PI, with the liver and spleen containing high concentrations of S. enteritidis, whereas the blood, heart, kidney, pancreas, and gallbladder had low concentrations. S. enteritidis populations began to decrease and were not detectable at 3 d PI, but were still present up to 12 d PI in the gallbladder, 2 wk for the liver, and 3 wk for the spleen without causing apparent symptoms. The results showed that the liver and spleen may be the primary sites for S. enteritidis setting itself up as a commensa over a long time after oral challenge. Interestingly, it may be the first time reported that the gallbladder is a site of carriage for S. enteritidis over a 12 d period. This study will help to understand the mechanisms of action of S. enteritidis infection in vivo.

  10. 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.

  11. A human fecal contamination score for ranking recreational sites using the HF183/BacR287 quantitative real-time PCR method.

    Science.gov (United States)

    Cao, Yiping; Sivaganesan, Mano; Kelty, Catherine A; Wang, Dan; Boehm, Alexandria B; Griffith, John F; Weisberg, Stephen B; Shanks, Orin C

    2018-01-01

    Human fecal pollution of recreational waters remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality research and management. However, there are currently no standardized approaches for field implementation and interpretation of qPCR data. In this study, a standardized HF183/BacR287 qPCR method was combined with a water sampling strategy and a novel Bayesian weighted average approach to establish a human fecal contamination score (HFS) that can be used to prioritize sampling sites for remediation based on measured human waste levels. The HFS was then used to investigate 975 study design scenarios utilizing different combinations of sites with varying sampling intensities (daily to once per week) and number of qPCR replicates per sample (2-14 replicates). Findings demonstrate that site prioritization with HFS is feasible and that both sampling intensity and number of qPCR replicates influence reliability of HFS estimates. The novel data analysis strategy presented here provides a prescribed approach for the implementation and interpretation of human-associated HF183/BacR287 qPCR data with the goal of site prioritization based on human fecal pollution levels. In addition, information is provided for future users to customize study designs for optimal HFS performance. Published by Elsevier Ltd.

  12. High abundance of JS-1- and Chloroflexi-related Bacteria in deeply buried marine sediments revealed by quantitative, real-time PCR.

    Science.gov (United States)

    Blazejak, Anna; Schippers, Axel

    2010-05-01

    Sequences of members of the bacterial candidate division JS-1 and the classes Anaerolineae and Caldilineae of the phylum Chloroflexi are frequently found in 16S rRNA gene clone libraries obtained from marine sediments. Using a newly designed quantitative, real-time PCR assay, these bacterial groups were jointly quantified in samples from near-surface and deeply buried marine sediments from the Peru margin, the Black Sea, and a forearc basin off the island of Sumatra. In near-surface sediments, sequences of the JS-1 as well as Anaerolineae- and Caldilineae-related Bacteria were quantified with significantly lower 16S rRNA gene copy numbers than the sequences of total Bacteria. In contrast, in deeply buried sediments below approximately 1 m depth, similar quantities of the 16S rRNA gene copies of these specific groups and Bacteria were found. This finding indicates that JS-1 and Anaerolineae- and Caldilineae-related Bacteria might dominate the bacterial community in deeply buried marine sediments and thus seem to play an important ecological role in the deep biosphere.

  13. Response of a diuron-degrading community to diuron exposure assessed by real-time quantitative PCR monitoring of phenylurea hydrolase A and B encoding genes.

    Science.gov (United States)

    Pesce, Stéphane; Beguet, Jérémie; Rouard, Nadine; Devers-Lamrani, Marion; Martin-Laurent, Fabrice

    2013-02-01

    A real-time quantitative PCR method was developed to detect and quantify phenlylurea hydrolase genes' (puhA and puhB) sequences from environmental DNA samples to assess diuron-degrading genetic potential in some soil and sediment microbial communities. In the soil communities, mineralization rates (determined with [ring-¹⁴C]-labeled diuron) were linked to diuron-degrading genetic potentials estimated from puhB number copies, which increased following repeated diuron treatments. In the sediment communities, mineralization potential did not depend solely on the quantity of puhB copies, underlining the need to assess gene expression. In the sediment samples, both puhB copy numbers and mineralization capacities were highly conditioned by whether or not diuron-treated soil was added. This points to transfers of degradative potential from soils to sediments. No puhA gene was detected in soil and sediment DNA extracts. Moreover, some sediments exhibited high diuron mineralization potential even though puhB genes were not detected, suggesting the existence of alternative diuron degradation pathways.

  14. 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.

  15. Differentially expressed genes of Tetrahymena thermophila in response to tributyltin (TBT) identified by suppression subtractive hybridization and real time quantitative PCR.

    Science.gov (United States)

    Feng, Lifang; Miao, Wei; Wu, Yuxuan

    2007-02-15

    Tributyltin (TBT) is widely used as antifouling paints, agriculture biocides, and plastic stabilizers around the world, resulting in great pollution problem in aquatic environments. However, it has been short of the biomonitor to detect TBT in freshwater. We constructed the suppression subtractive hybridization library of Tetrahymena thermophila exposed to TBT, and screened out 101 Expressed Sequence Tags whose expressions were significantly up- or down-regulated with TBT treatment. From this, a series of genes related to the TBT toxicity were discovered, such as glutathione-S-transferase gene (down-regulated), plasma membrane Ca2+ ATPase isoforms 3 gene (up-regulated) and NgoA (up-regulated). Furthermore, their expressions under different concentrations of TBT treatment (0.5-40 ppb) were detected by real time fluorescent quantitative PCR. The differentially expressed genes of T. thermophila in response to TBT were identified, which provide the basic to make Tetrahymena as a sensitive, rapid and convenient TBT biomonitor in freshwater based on rDNA inducible expression system.

  16. 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.

  17. Yeast Monitoring of Wine Mixed or Sequential Fermentations Made by Native Strains from D.O. “Vinos de Madrid” Using Real-Time Quantitative PCR

    Science.gov (United States)

    García, Margarita; Esteve-Zarzoso, Braulio; Crespo, Julia; Cabellos, Juan M.; Arroyo, Teresa

    2017-01-01

    There is an increasing trend toward understanding the impact of non-Saccharomyces yeasts on the winemaking process. Although Saccharomyces cerevisiae is the predominant species at the end of fermentation, it has been recognized that the presence of non-Saccharomyces species during alcoholic fermentation can produce an improvement in the quality and complexity of the final wines. A previous work was developed for selecting the best combinations between S. cerevisiae and five non-Saccharomyces (Torulaspora delbrueckii, Schizosaccharomyces pombe, Candida stellata, Metschnikowia pulcherrima, and Lachancea thermotolorans) native yeast strains from D.O. “Vinos de Madrid” at the laboratory scale. The best inoculation strategies between S. cerevisiae and non-Saccharomyces strains were chosen to analyze, by real-time quantitative PCR (qPCR) combined with the use of specific primers, the dynamics of inoculated populations throughout the fermentation process at the pilot scale using the Malvar white grape variety. The efficiency of the qPCR system was verified independently of the samples matrix, founding the inoculated yeast species throughout alcoholic fermentation. Finally, we can validate the positive effect of selected co-cultures in the Malvar wine quality, highlighting the sequential cultures of T. delbrueckii CLI 918/S. cerevisiae CLI 889 and C. stellata CLI 920/S. cerevisiae CLI 889 and, mixed and sequential cultures of L. thermotolerans 9-6C combined with S. cerevisiae CLI 889. PMID:29326669

  18. Yeast Monitoring of Wine Mixed or Sequential Fermentations Made by Native Strains from D.O. “Vinos de Madrid” Using Real-Time Quantitative PCR

    Directory of Open Access Journals (Sweden)

    Margarita García

    2017-12-01

    Full Text Available There is an increasing trend toward understanding the impact of non-Saccharomyces yeasts on the winemaking process. Although Saccharomyces cerevisiae is the predominant species at the end of fermentation, it has been recognized that the presence of non-Saccharomyces species during alcoholic fermentation can produce an improvement in the quality and complexity of the final wines. A previous work was developed for selecting the best combinations between S. cerevisiae and five non-Saccharomyces (Torulaspora delbrueckii, Schizosaccharomyces pombe, Candida stellata, Metschnikowia pulcherrima, and Lachancea thermotolorans native yeast strains from D.O. “Vinos de Madrid” at the laboratory scale. The best inoculation strategies between S. cerevisiae and non-Saccharomyces strains were chosen to analyze, by real-time quantitative PCR (qPCR combined with the use of specific primers, the dynamics of inoculated populations throughout the fermentation process at the pilot scale using the Malvar white grape variety. The efficiency of the qPCR system was verified independently of the samples matrix, founding the inoculated yeast species throughout alcoholic fermentation. Finally, we can validate the positive effect of selected co-cultures in the Malvar wine quality, highlighting the sequential cultures of T. delbrueckii CLI 918/S. cerevisiae CLI 889 and C. stellata CLI 920/S. cerevisiae CLI 889 and, mixed and sequential cultures of L. thermotolerans 9-6C combined with S. cerevisiae CLI 889.

  19. Monitoring the response of patients with cutaneous leishmaniasis to treatment with pentamidine isethionate by quantitative real-time PCR, and identification of Leishmania parasites not responding to therapy.

    Science.gov (United States)

    Mans, D R A; Kent, A D; Hu, R V; Lai A Fat, E J; Schoone, G J; Adams, E R; Rood, E J; Alba, S; Sabajo, L O A; Lai A Fat, R F; de Vries, H J C; Schallig, H D F H

    2016-08-01

    Leishmania (Viannia) guyanensis is believed to be the principal cause of cutaneous leishmaniasis (CL) in Suriname. This disease is treated with pentamidine isethionate (PI), but treatment failure has increasingly been reported. To evaluate PI for its clinical efficacy, to compare parasite load, and to assess the possibility of treatment failure due to other infecting Leishmania species. Parasite load of patients with CL was determined in skin biopsies using real-time quantitative PCR before treatment and 6 and 12 weeks after treatment. Clinical responses were evaluated at week 12 and compared with parasite load. In parallel, molecular species differentiation was performed. L. (V.) guyanensis was the main infecting species in 129 of 143 patients (about 90%). PI treatment led to a significant decrease (P Leishmania (Viannia) braziliensis, Leishmania (Leishmania) amazonensis and L. (V.) guyanensis (1/92, 1/92 and 22/92 evaluable cases, respectively). There was substantial agreement beyond chance between the parasite load at week 6 and the clinical outcome at week 12, as indicated by the κ value of 0.61. L. (V.) guyanensis is the main infecting species of CL in Suriname, followed by L. (V.) braziliensis and L. (L.) amazonensis. Furthermore, patient response to PI can be better anticipated based on the parasite load 6 weeks after the treatment rather than on parasite load before treatment. © 2015 The Authors Clinical and Experimental Dermatology published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists, North American Clinical Dermatologic Society and St Johns Dermatological Society.

  20. Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR.

    Science.gov (United States)

    Fang, Peng; Lu, Rongfei; Sun, Feng; Lan, Ying; Shen, Wenbiao; Du, Linlin; Zhou, Yijun; Zhou, Tong

    2015-10-24

    Stably expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), particularly for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia. The expression of fourteen common used reference genes of Oryza sativa L. were evaluated by RT-qPCR in RSV and RBSDV infected rice plants. Suitable normalization reference gene(s) were identified by geNorm and NormFinder algorithms. UBQ 10 + GAPDH and UBC + Actin1 were identified as suitable reference genes for RT-qPCR normalization under RSV and RBSDV infection, respectively. When using multiple reference genes, the expression patterns of OsPRIb and OsWRKY, two virus resistance genes, were approximately similar with that reported previously. Comparatively, by using single reference gene (TIP41-Like), a weaker inducible response was observed. We proposed that the combination of two reference genes could obtain more accurate and reliable normalization of RT-qPCR results in RSV- and RBSDV-infected plants. This work therefore sheds light on establishing a standardized RT-qPCR procedure in RSV- and RBSDV-infected rice plants, and might serve as an important point for discovering complex regulatory networks and identifying genes relevant to biological processes or implicated in virus.

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

    Directory of Open Access Journals (Sweden)

    Benshui Shu

    2018-04-01

    Full Text Available 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 present study, the fragments of eight candidate reference genes were cloned and identified from the pest Spodoptera litura. In addition, the expression stability of these genes in different samples from larvae of control and azadirachtin treatments were evaluated by the computational methods of NormFinder, BestKeeper, Delta CT, geNorm, and RefFinder. According to our results, two of the reference genes should be the optimal number for RT-qPCR analysis. Furthermore, the best reference genes for different samples were showed as followed: EF-1α and EF2 for cuticle, β-Tubulin and RPL7A for fat body, EF2 and Actin for midgut, EF2 and RPL13A for larva and RPL13A and RPL7A for all the samples. Our results established a reliable normalization for RT-qPCR experiments in S. litura and ensure the data more accurate for the mechanism analysis of azadirachtin.

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

    Science.gov (United States)

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

    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 present study, the fragments of eight candidate reference genes were cloned and identified from the pest Spodoptera litura . In addition, the expression stability of these genes in different samples from larvae of control and azadirachtin treatments were evaluated by the computational methods of NormFinder, BestKeeper, Delta CT, geNorm, and RefFinder. According to our results, two of the reference genes should be the optimal number for RT-qPCR analysis. Furthermore, the best reference genes for different samples were showed as followed: EF-1α and EF2 for cuticle, β-Tubulin and RPL7A for fat body, EF2 and Actin for midgut, EF2 and RPL13A for larva and RPL13A and RPL7A for all the samples. Our results established a reliable normalization for RT-qPCR experiments in S. litura and ensure the data more accurate for the mechanism analysis of azadirachtin.

  3. Gene expression profile and immunological evaluation of unique hypothetical unknown proteins of Mycobacterium leprae by using quantitative real-time PCR.

    Science.gov (United States)

    Kim, Hee Jin; Prithiviraj, Kalyani; Groathouse, Nathan; Brennan, Patrick J; Spencer, John S

    2013-02-01

    The cell-mediated immunity (CMI)-based in vitro gamma interferon release assay (IGRA) of Mycobacterium leprae-specific antigens has potential as a promising diagnostic means to detect those individuals in the early stages of M. leprae infection. Diagnosis of leprosy is a major obstacle toward ultimate disease control and has been compromised in the past by the lack of specific markers. Comparative bioinformatic analysis among mycobacterial genomes identified potential M. leprae-specific proteins called "hypothetical unknowns." Due to massive gene decay and the prevalence of pseudogenes, it is unclear whether any of these proteins are expressed or are immunologically relevant. In this study, we performed cDNA-based quantitative real-time PCR to investigate the expression status of 131 putative open reading frames (ORFs) encoding hypothetical unknowns. Twenty-six of the M. leprae-specific antigen candidates showed significant levels of gene expression compared to that of ESAT-6 (ML0049), which is an important T cell antigen of low abundance in M. leprae. Fifteen of 26 selected antigen candidates were expressed and purified in Escherichia coli. The seroreactivity to these proteins of pooled sera from lepromatous leprosy patients and cavitary tuberculosis patients revealed that 9 of 15 recombinant hypothetical unknowns elicited M. leprae-specific immune responses. These nine proteins may be good diagnostic reagents to improve both the sensitivity and specificity of detection of individuals with asymptomatic leprosy.

  4. Comparative validation using quantitative real-time PCR (qPCR and conventional PCR of bovine semen centrifuged in continuous density gradient

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    M.V. Resende

    2011-06-01

    Full Text Available The objective of the present study was to determine the sperm enrichment with X-bearing spermatozoa, after one centrifugation in a Percoll or OptiPrep continuous density gradient, using quantitative real-time polymerase chain reaction (qPCR of sperm DNA and resultant in vitro-produced bovine embryos by PCR. Frozen/thawed sperm was layered on density gradients and the tubes were centrifuged. Supernatants were gently aspirated and the sperm recovered from the bottom of the tubes. Cleavage and blastocyst rates were determined through in vitro production of embryos and PCR was performed to identify the embryos' genetic sex. A difference in blastocyst rate was found in the Percoll treatment compared to OptiPrep (P<0.05. The percentage of female embryos in the Percoll and OptiPrep groups was 62.0% and 47.1%, respectively. These results were confirmed by qPCR of spermatozoa DNA and underestimation was seen only in the Percoll group. It was possible to sexing sperm using simple approach.

  5. Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes in Jurkat T-cells treated with HIV-1 Tat protein.

    Science.gov (United States)

    Liao, Wenting; Tan, Guangguo; Zhu, Zhenyu; Chen, Qiuli; Lou, Ziyang; Dong, Xin; Zhang, Wei; Pan, Wei; Chai, Yifeng

    2012-11-02

    HIV-1 Tat protein is released by infected cells and can affect bystander uninfected T cells and induce numerous biological responses which contribute to its pathogenesis. To elucidate the complex pathogenic mechanism, we conducted a comprehensive investigation on Tat protein-related extracellular and intracellular metabolic changes in Jurkat T-cells using combined gas chromatography-mass spectrometry (GC-MS), reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and a hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS)-based metabonomics approach. Quantitative real-time PCR (qRT-PCR) analyses were further employed to measure expressions of several relevant enzymes together with perturbed metabolic pathways. Combined metabonomic and qRT-PCR analyses revealed that HIV-1 Tat caused significant and comprehensive metabolic changes, as represented by significant changes of 37 metabolites and 10 relevant enzymes in HIV-1 Tat-treated cells. Using MetaboAnalyst 2.0, it was found that 11 pathways (Impact-value >0.10) among the regulated pathways were acutely perturbed, including sphingolipid metabolism, glycine, serine and threonine metabolism, pyruvate metabolism, inositol phosphate metabolism, arginine and proline metabolism, citrate cycle, phenylalanine metabolism, tryptophan metabolism, pentose phosphate pathway, glycerophospholipid metabolism, glycolysis or gluconeogenesis. These results provide metabolic evidence of the complex pathogenic mechanism of HIV-1 Tat protein as a "viral toxin", and would help obligate Tat protein as "an important target" for therapeutic intervention and vaccine development.

  6. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan PCR Assay

    Directory of Open Access Journals (Sweden)

    Hua-Ying Fu

    2016-01-01

    Full Text Available Ratoon stunting disease (RSD of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx. A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR and a fluorogenic probe (Pat1-QP targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7% of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174 were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174 were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

  7. Selection of reference genes for quantitative real-time RT-PCR studies in tomato fruit of the genotype MT-Rg1

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    Karla L. González-Aguilera

    2016-09-01

    Full Text Available Quantitative real-time RT-PCR (qRT-PCR has become one of the most widely used methods for accurate quantification of gene expression. Since there are no universal reference genes for normalization, the optimal strategy to normalize raw qRT-PCR data is to perform an initial comparison of a set of independent reference genes to assess the most stable ones in each biological model. Normalization of a qRT-PCR experiment helps to ensure that the results are both statistically significant and biologically meaningful. Tomato is the model of choice to study fleshy fruit development. The miniature tomato (Solanum lycopersicum L. cultivar Micro-Tom (MT is considered a model system for tomato genetics and functional genomics. A new genotype, containing the Rg1 allele, improves tomato in vitro regeneration. In this work, we evaluated the expression stability of four tomato reference genes, namely CAC, SAND, Expressed and ACTIN2. We showed that the genes CAC and Exp are the best reference genes of the four we tested during fruit development in the MT-Rg1 genotype. Furthermore, we validated the reference genes by showing that the expression profiles of the transcription factors FRUITFULL1 (FUL1 and APETALA2c (AP2c during fruit development are comparable to previous reports using other tomato cultivars.

  8. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

    Science.gov (United States)

    Purcell, Maureen K.; Pearman-Gillman, Schuyler; Thompson, Rachel L.; Gregg, Jacob L.; Hart, Lucas M.; Winton, James R.; Emmenegger, Eveline J.; Hershberger, Paul K.

    2016-01-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  9. Quantitation of O6-methylguanine-DNA methyltransferase gene messenger RNA in gliomas by means of real-time RT-PCR and clinical response to nitrosoureas.

    Science.gov (United States)

    Tanaka, Satoshi; Oka, Hidehiro; Fujii, Kiyotaka; Watanabe, Kaoru; Nagao, Kumi; Kakimoto, Atsushi

    2005-09-01

    1. O6-methylguanine-DNA methyltransferase (MGMT) mRNA was measured in 50 malignant gliomas that had received 1-(4-amino-2-methyl-5-pyrimidynyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) after the resection of the tumor by real-time reverse transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. 2. The mean absolute value of MGMTmRNA normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for 50 tumors was 1.29 x 10(4)+/- 1.28 x 10(4) copy/microg RNA (mean +/- SD). The amount of MGMTmRNA less than 6 x 10(3) copy/microg RNA was the most significant factor in predicting the initial effect of treatment with ACNU by multi-variant regression analysis (p = 0.0157). 3. These results suggest that quantitation of MGMTmRNA is the excellent method for predicting for the effect of ACNU in glioma therapy.

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

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    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.

  11. 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.

  12. 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.

  13. Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

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    Soares Fernando A

    2009-03-01

    Full Text Available Abstract Background HER-2 gene testing has become an integral part of breast cancer patient diagnosis. The most commonly used assay in the clinical setting for evaluating HER-2 status is immunohistochemistry (IHC and fluorescence in situ hybridization (FISH. These procedures permit correlation between HER-2 expression and morphological features. However, FISH signals are labile and fade over time, making post-revision of the tumor difficult. CISH (chromogenic in situ hybridization is an alternative procedure, with certain advantages, although still limited as a diagnostic tool in breast carcinomas. Methods To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH. Results The concordance rate between IHC and qRT-PCR results was 78.9%, and 94.6% for qRT-PCR and CISH. Intratumoral heterogeneity of HER-2 status was identified in three cases by CISH. The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1+ immunostaining cases, which showed high-level amplification (15.4% and HER-2 transcript overexpression (20%. Moreover, 2+ immunostaining cases presented nonamplified status (50% by CISH and HER-2 downexpression (38.5% by qRT-PCR. In general, concordance occurred between qRT-PCR and CISH results. A high concordance was observed between CISH/qRT-PCR and FISH. Comparisons with clinicopathological data revealed a significant association between HER-2 downexpression and the involvement of less than four lymph nodes (P = 0.0350. Conclusion Based on these findings, qRT-PCR was more precise and reproducible than IHC. Furthermore, CISH was revealed as an alternative and useful procedure for investigating amplifications involving the HER-2 gene.

  14. Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

    International Nuclear Information System (INIS)

    Rosa, Fabíola E; Silveira, Sara M; Silveira, Cássia GT; Bérgamo, Nádia A; Neto, Francisco A Moraes; Domingues, Maria AC; Soares, Fernando A; Caldeira, José RF; Rogatto, Silvia R

    2009-01-01

    HER-2 gene testing has become an integral part of breast cancer patient diagnosis. The most commonly used assay in the clinical setting for evaluating HER-2 status is immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). These procedures permit correlation between HER-2 expression and morphological features. However, FISH signals are labile and fade over time, making post-revision of the tumor difficult. CISH (chromogenic in situ hybridization) is an alternative procedure, with certain advantages, although still limited as a diagnostic tool in breast carcinomas. To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH. The concordance rate between IHC and qRT-PCR results was 78.9%, and 94.6% for qRT-PCR and CISH. Intratumoral heterogeneity of HER-2 status was identified in three cases by CISH. The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1+ immunostaining cases, which showed high-level amplification (15.4%) and HER-2 transcript overexpression (20%). Moreover, 2+ immunostaining cases presented nonamplified status (50%) by CISH and HER-2 downexpression (38.5%) by qRT-PCR. In general, concordance occurred between qRT-PCR and CISH results. A high concordance was observed between CISH/qRT-PCR and FISH. Comparisons with clinicopathological data revealed a significant association between HER-2 downexpression and the involvement of less than four lymph nodes (P = 0.0350). Based on these findings, qRT-PCR was more precise and reproducible than IHC. Furthermore, CISH was revealed as an alternative and useful procedure for investigating amplifications involving the HER-2 gene

  15. 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.

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

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    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.

  17. Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea in soil and fecal samples

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    Durant Jean-Francois

    2012-12-01

    Full Text Available Abstract Background Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis and/or Toxocara cati (T. cati, two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount. Methods A duplex quantitative real-time PCR (2qPCR targeting the ribosomal RNA gene internal transcribed spacer (ITS2 has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum and Parascaris equorum (P. equorum. The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination. Results 2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces. Conclusion The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.

  18. Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea) in soil and fecal samples.

    Science.gov (United States)

    Durant, Jean-Francois; Irenge, Leonid M; Fogt-Wyrwas, Renata; Dumont, Catherine; Doucet, Jean-Pierre; Mignon, Bernard; Losson, Bertrand; Gala, Jean-Luc

    2012-12-07

    Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis) and/or Toxocara cati (T. cati), two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount. A duplex quantitative real-time PCR (2qPCR) targeting the ribosomal RNA gene internal transcribed spacer (ITS2) has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum) and Parascaris equorum (P. equorum). The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination. 2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD) in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces. The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.

  19. Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization Under Ethanol Stress Conditions in Oenococcus oeni SD-2a

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    Shuai Peng

    2018-05-01

    Full Text Available The powerful Quantitative real-time PCR (RT-qPCR was widely used to assess gene expression levels, which requires the optimal reference genes used for normalization. Oenococcus oeni (O. oeni, as the one of most important microorganisms in wine industry and the most resistant lactic acid bacteria (LAB species to ethanol, has not been investigated regarding the selection of stable reference genes for RT-qPCR normalization under ethanol stress conditions. In this study, nine candidate reference genes (proC, dnaG, rpoA, ldhD, ddlA, rrs, gyrA, gyrB, and dpoIII were analyzed to determine the most stable reference genes for RT-qPCR in O. oeni SD-2a under different ethanol stress conditions (8, 12, and 16% (v/v ethanol. The transcript stabilities of these genes were evaluated using the algorithms geNorm, NormFinder, and BestKeeper. The results showed that dnaG and dpoIII were selected as the best reference genes across all experimental ethanol conditions. Considering single stress experimental modes, dpoIII and dnaG would be suitable to normalize expression level for 8% ethanol shock treatment, while the combination of gyrA, gyrB, and rrs would be suitable for 12% ethanol shock treatment. proC and gyrB revealed the most stable expression in 16% ethanol shock treatment. This study selected and validated for the first time the reference genes for RT-qPCR normalization in O. oeni SD-2a under ethanol stress conditions.

  20. 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.

  1. Quantification of viable bacterial starter cultures of Virgibacillus sp. and Tetragenococcus halophilus in fish sauce fermentation by real-time quantitative PCR.

    Science.gov (United States)

    Udomsil, Natteewan; Chen, Shu; Rodtong, Sureelak; Yongsawatdigul, Jirawat

    2016-08-01

    Real-time quantitative polymerase chain reaction (qPCR) methods were developed for the quantification of Virgibacillus sp. SK37 and Tetragenococcus halophilus MS33, which were added as starter cultures in fish sauce fermentation. The PCR assays were coupled with propidium monoazide (PMA) treatment of samples to selectively quantify viable cells and integrated with exogenous recombinant Escherichia coli cells to control variabilities in analysis procedures. The qPCR methods showed species-specificity for both Virgibacillus halodenitrificans and T. halophilus as evaluated using 6 reference strains and 28 strains of bacteria isolated from fish sauce fermentation. The qPCR efficiencies were 101.1% for V. halodenitrificans and 90.2% for T. halophilus. The quantification limits of the assays were 10(3) CFU/mL and 10(2) CFU/mL in fish sauce samples with linear correlations over 4 Logs for V. halodenitrificans and T. halophilus, respectively. The matrix effect was not observed when evaluated using fish sauce samples fermented for 1-6 months. The developed PMA-qPCR methods were successfully applied to monitor changes of Virgibacillus sp. SK37 and T. halophilus MS33 in a mackerel fish sauce fermentation model where culture-dependent techniques failed to quantify the starter cultures. The results demonstrated the usability of the methods as practical tools for monitoring the starter cultures in fish sauce fermentation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Selection of Reliable Reference Genes for Gene Expression Studies in the Biofuel Plant Jatropha curcas Using Real-Time Quantitative PCR

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    Lu Zhang

    2013-12-01

    Full Text Available Jatropha curcas is a promising renewable feedstock for biodiesel and bio-jet fuel production. To study gene expression in Jatropha in different tissues throughout development and under stress conditions, we examined a total of 11 typical candidate reference genes using real-time quantitative polymerase chain reaction (RT-qPCR analysis, which is widely used for validating transcript levels in gene expression studies. The expression stability of these candidate reference genes was assessed across a total of 20 samples, including various tissues at vegetative and reproductive stages and under desiccation and cold stress treatments. The results obtained using software qBasePLUS showed that the top-ranked reference genes differed across the sample subsets. The combination of actin, GAPDH, and EF1α would be appropriate as a reference panel for normalizing gene expression data across samples at different developmental stages; the combination of actin, GAPDH, and TUB5 should be used as a reference panel for normalizing gene expression data across samples under various abiotic stress treatments. With regard to different developmental stages, we recommend the use of actin and TUB8 for normalization at the vegetative stage and GAPDH and EF1α for normalization at the reproductive stage. For abiotic stress treatments, we recommend the use of TUB5 and TUB8 for normalization under desiccation stress and GAPDH and actin for normalization under cold stress. These results are valuable for future research on gene expression during development or under abiotic stress in Jatropha. To our knowledge, this is the first report on the stability of reference genes in Jatropha.

  3. Identification and validation of reference genes for quantification of target gene expression with quantitative real-time PCR for tall fescue under four abiotic stresses.

    Directory of Open Access Journals (Sweden)

    Zhimin Yang

    Full Text Available Tall fescue (Festuca arundinacea Schreb. is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41 was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

  4. The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

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    Jing Cai

    Full Text Available Quantitative real-time PCR (qPCR is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD, an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA and nonparametric (Kruskal-Wallis tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

  5. Selection of Suitable Internal Control Genes for Accurate Normalization of Real-Time Quantitative PCR Data of Buffalo (Bubalus bubalis) Blastocysts Produced by SCNT and IVF.

    Science.gov (United States)

    Sood, Tanushri Jerath; Lagah, Swati Viviyan; Sharma, Ankita; Singla, Suresh Kumar; Mukesh, Manishi; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

    2017-10-01

    We evaluated the suitability of 10 candidate internal control genes (ICGs), belonging to different functional classes, namely ACTB, EEF1A1, GAPDH, HPRT1, HMBS, RPS15, RPS18, RPS23, SDHA, and UBC for normalizing the real-time quantitative polymerase chain reaction (qPCR) data of blastocyst-stage buffalo embryos produced by hand-made cloning and in vitro fertilization (IVF). Total RNA was isolated from three pools, each of cloned and IVF blastocysts (n = 50/pool) for cDNA synthesis. Two different statistical algorithms geNorm and NormFinder were used for evaluating the stability of these genes. Based on gene stability measure (M value) and pairwise variation (V value), calculated by geNorm analysis, the most stable ICGs were RPS15, HPRT1, and ACTB for cloned blastocysts, HMBS, UBC, and HPRT1 for IVF blastocysts and RPS15, GAPDH, and HPRT1 for both the embryo types analyzed together. RPS18 was the least stable gene for both cloned and IVF blastocysts. Following NormFinder analysis, the order of stability was RPS15 = HPRT1>GAPDH for cloned blastocysts, HMBS = UBC>RPS23 for IVF blastocysts, and HPRT1>GAPDH>RPS15 for cloned and IVF blastocysts together. These results suggest that despite overlapping of the three most stable ICGs between cloned and IVF blastocysts, the panel of ICGs selected for normalization of qPCR data of cloned and IVF blastocyst-stage embryos should be different.

  6. The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

    Science.gov (United States)

    Cai, Jing; Li, Tao; Huang, Bangxing; Cheng, Henghui; Ding, Hui; Dong, Weihong; Xiao, Man; Liu, Ling; Wang, Zehua

    2014-01-01

    Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

  7. Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR

    Directory of Open Access Journals (Sweden)

    Longjian Niu

    2015-06-01

    Full Text Available Real-time quantitative PCR (RT-qPCR is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis, a promising oilseed crop known for its polyunsaturated fatty acid (PUFA-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt, BestKeeper, geNorm, and NormFinder were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE, actin (ACT and phospholipase A22 (PLA were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13, cyclophilin (CYC and elongation factor-1alpha (EF1α were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.

  8. Hyperspectral Imaging Using Intracellular Spies: Quantitative Real-Time Measurement of Intracellular Parameters In Vivo during Interaction of the Pathogenic Fungus Aspergillus fumigatus with Human Monocytes.

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    Sara Mohebbi

    Full Text Available Hyperspectral imaging (HSI is a technique based on the combination of classical spectroscopy and conventional digital image processing. It is also well suited for the biological assays and quantitative real-time analysis since it provides spectral and spatial data of samples. The method grants detailed information about a sample by recording the entire spectrum in each pixel of the whole image. We applied HSI to quantify the constituent pH variation in a single infected apoptotic monocyte as a model system. Previously, we showed that the human-pathogenic fungus Aspergillus fumigatus conidia interfere with the acidification of phagolysosomes. Here, we extended this finding to monocytes and gained a more detailed analysis of this process. Our data indicate that melanised A. fumigatus conidia have the ability to interfere with apoptosis in human monocytes as they enable the apoptotic cell to recover from mitochondrial acidification and to continue with the cell cycle. We also showed that this ability of A. fumigatus is dependent on the presence of melanin, since a non-pigmented mutant did not stop the progression of apoptosis and consequently, the cell did not recover from the acidic pH. By conducting the current research based on the HSI, we could measure the intracellular pH in an apoptotic infected human monocyte and show the pattern of pH variation during 35 h of measurements. As a conclusion, we showed the importance of melanin for determining the fate of intracellular pH in a single apoptotic cell.

  9. Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients.

    Science.gov (United States)

    Ramírez, Juan Carlos; Cura, Carolina Inés; da Cruz Moreira, Otacilio; Lages-Silva, Eliane; Juiz, Natalia; Velázquez, Elsa; Ramírez, Juan David; Alberti, Anahí; Pavia, Paula; Flores-Chávez, María Delmans; Muñoz-Calderón, Arturo; Pérez-Morales, Deyanira; Santalla, José; Marcos da Matta Guedes, Paulo; Peneau, Julie; Marcet, Paula; Padilla, Carlos; Cruz-Robles, David; Valencia, Edward; Crisante, Gladys Elena; Greif, Gonzalo; Zulantay, Inés; Costales, Jaime Alfredo; Alvarez-Martínez, Miriam; Martínez, Norma Edith; Villarroel, Rodrigo; Villarroel, Sandro; Sánchez, Zunilda; Bisio, Margarita; Parrado, Rudy; Maria da Cunha Galvão, Lúcia; Jácome da Câmara, Antonia Cláudia; Espinoza, Bertha; Alarcón de Noya, Belkisyole; Puerta, Concepción; Riarte, Adelina; Diosque, Patricio; Sosa-Estani, Sergio; Guhl, Felipe; Ribeiro, Isabela; Aznar, Christine; Britto, Constança; Yadón, Zaida Estela; Schijman, Alejandro G

    2015-09-01

    An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  10. 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.

  11. Selection and validation of reference genes for gene expression analysis in switchgrass (Panicum virgatum using quantitative real-time RT-PCR.

    Directory of Open Access Journals (Sweden)

    Jacinta Gimeno

    Full Text Available Switchgrass (Panicum virgatum has received a lot of attention as a forage and bioenergy crop during the past few years. Gene expression studies are in progress to improve new traits and develop new cultivars. Quantitative real time PCR (qRT-PCR has emerged as an important technique to study gene expression analysis. For accurate and reliable results, normalization of data with reference genes is essential. In this work, we evaluate the stability of expression of genes to use as reference for qRT-PCR in the grass P. virgatum. Eleven candidate reference genes, including eEF-1α, UBQ6, ACT12, TUB6, eIF-4a, GAPDH, SAMDC, TUA6, CYP5, U2AF, and FTSH4, were validated for qRT-PCR normalization in different plant tissues and under different stress conditions. The expression stability of these genes was verified by the use of two distinct algorithms, geNorm and NormFinder. Differences were observed after comparison of the ranking of the candidate reference genes identified by both programs but eEF-1α, eIF-4a, CYP5 and U2AF are ranked as the most stable genes in the samples sets under study. Both programs discard the use of SAMDC and TUA6 for normalization. Validation of the reference genes proposed by geNorm and NormFinder were performed by normalization of transcript abundance of a group of target genes in different samples. Results show similar expression patterns when the best reference genes selected by both programs were used but differences were detected in the transcript abundance of the target genes. Based on the above research, we recommend the use of different statistical algorithms to identify the best reference genes for expression data normalization. The best genes selected in this study will help to improve the quality of gene expression data in a wide variety of samples in switchgrass.

  12. Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

    International Nuclear Information System (INIS)

    Malik, Afshan N.; Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil

    2011-01-01

    Highlights: → Mitochondrial dysfunction is central to many diseases of oxidative stress. → 95% of the mitochondrial genome is duplicated in the nuclear genome. → Dilution of untreated genomic DNA leads to dilution bias. → Unique primers and template pretreatment are needed to accurately measure mitochondrial DNA content. -- Abstract: Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as β-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a 'dilution bias' when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

  13. Selection of suitable housekeeping genes for real-time quantitative PCR in CD4(+ lymphocytes from asthmatics with or without depression.

    Directory of Open Access Journals (Sweden)

    Ting Wang

    Full Text Available OBJECTIVE: No optimal housekeeping genes (HKGs have been identified for CD4(+ T cells from non-depressive asthmatic and depressive asthmatic adults for normalizing quantitative real-time PCR (qPCR assays. The aim of present study was to select appropriate HKGs for gene expression analysis in purified CD4(+ T cells from these asthmatics. METHODS: Three groups of subjects (Non-depressive asthmatic, NDA, n = 10, Depressive asthmatic, DA, n = 11, and Healthy control, HC, n = 10 respectively were studied. qPCR for 9 potential HKGs, namely RNA, 28S ribosomal 1 (RN28S1, ribosomal protein, large, P0 (RPLP0, actin, beta (ACTB, cyclophilin A (PPIA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, phosphoglycerate kinase 1 (PGK1, beta-2-microglobulin (B2M, glucuronidase, beta (GUSB and ribosomal protein L13a (RPL13A, was performed. Then the data were analyzed with three different applications namely BestKeeper, geNorm, and NormFinder. RESULTS: The analysis of gene expression data identified B2M and RPLP0 as the most stable reference genes and showed that the level of PPIA was significantly different among subjects of three groups when the two best HKGs identified were applied. Post-hoc analysis by Student-Newman-Keuls correction shows that depressive asthmatics and non-depressive asthmatics exhibited lower expression level of PPIA than healthy controls (p<0.05. CONCLUSIONS: B2M and RPLP0 were identified as the most optimal HKGs in gene expression studies involving human blood CD4(+ T cells derived from normal, depressive asthmatics and non-depressive asthmatics. The suitability of using the PPIA gene as the HKG for such studies was questioned due to its low expression in asthmatics.

  14. Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR

    Science.gov (United States)

    Niu, Longjian; Tao, Yan-Bin; Chen, Mao-Sheng; Fu, Qiantang; Li, Chaoqiong; Dong, Yuling; Wang, Xiulan; He, Huiying; Xu, Zeng-Fu

    2015-01-01

    Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi. PMID:26047338

  15. 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.

  16. Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

    Science.gov (United States)

    Qiu, Reng; Sun, Boguang; Fang, Shasha; Sun, Li; Liu, Xiao

    2013-03-01

    Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Vibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues (digestive glands, foot muscle, gill, hemocyte, and mantle) were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-1-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

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

    Science.gov (United States)

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

    2017-11-01

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

  18. Quantitation of Marek's disease and chicken anemia viruses in organs of experimentally infected chickens and commercial chickens by multiplex real-time PCR.

    Science.gov (United States)

    Davidson, Irit; Raibshtein, I; Al-Touri, A

    2013-06-01

    The worldwide distribution of chicken anemia virus (CAV) and Marek's disease virus (MDV) is well documented. In addition to their economic significance in single- or dual-virus infections, the two viruses can often accompany various other pathogens and affect poultry health either directly, by causing tumors, anemia, and delayed growth, or indirectly, by aggravating other diseases, as a result of their immunosuppressive effects. After a decade of employing the molecular diagnosis of those viruses, which replaced conventional virus isolation, we present the development of a real-time multiplex PCR for the simultaneous detection of both viruses. The real-time PCRs for MDV and for CAV alone are more sensitive than the respective end-point PCRs. In addition, the multiplex real-time shows a similar sensitivity when compared to the single real-time PCR for each virus. The newly developed real-time multiplex PCR is of importance in terms of the diagnosis and detection of low copies of each virus, MDV and CAV in single- and in multiple-virus infections, and its applicability will be further evaluated.

  19. 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

  20. Quantitative detection of the potato cyst nematode, Globodera pallida, and the beet cyst nematode, Heterodera schachtii, using Real-Time PCR with SYBR green I dye.

    Science.gov (United States)

    Madani, Mehrdad; Subbotin, Sergei A; Moens, Maurice

    2005-04-01

    The potato cyst nematode Globodera pallida and the beet cyst nematode Heterodera schachtii are major nematode pests in world agriculture. Precise identification and knowledge about the number of nematodes in field soil are necessary to develop effective integrated pest control. Here we report the results of the Real-Time PCR assay for the rapid detection and quantification of G. pallida and H. schachtii. Using species specific primers and SYBR green I dye, we were able to detect a single second stage juvenile of cyst forming nematodes in samples. The specificity of the reaction was confirmed by the lack of amplification of DNAs from other Heterodera or Globodera species. Validation tests showed a rather high correlation between real numbers of second stage juveniles in a sample and expected numbers detected by Real-Time PCR. Reasons for observed differences in sensitivity and reliability of quantification detection for two species as well as other problems of Real-Time PCR are discussed. The Real-Time PCR assay with SYBR green I dye targeting fragments of the ITS-rDNA provided a sensitive means for the rapid and simultaneous detection and quantification of juveniles of these pests.

  1. Quantitative Microbiological Study of Human Carious Dentine by Culture and Real-Time PCR: Association of Anaerobes with Histopathological Changes in Chronic Pulpitis

    Science.gov (United States)

    Martin, F. Elizabeth; Nadkarni, Mangala A.; Jacques, Nicholas A.; Hunter, Neil

    2002-01-01

    The bacteria found in carious dentine were correlated with the tissue response of the dental pulps of 65 teeth extracted from patients with advanced caries and pulpitis. Standardized homogenates of carious dentine were plated onto selective and nonselective media under anaerobic and microaerophilic conditions. In addition, real-time PCR was used to quantify the recovery of anaerobic bacteria. Primers and fluorogenic probes were designed to detect the total anaerobic microbial load, the genera Prevotella and Fusobacterium, and the species Prevotella melaninogenica, Porphyromonas endodontalis, Porphyromonas gingivalis, and Micromonas (formerly Peptostreptococcus) micros. The pulpal pathology was categorized according to the cellular response and degenerative changes. Analysis of cultured bacteria showed a predominance of gram-positive microorganisms, particularly lactobacilli. Gram-negative bacteria were also present in significant numbers with Prevotella spp., the most numerous anaerobic group cultured. Real-time PCR analysis indicated a greater microbial load than that determined by colony counting. The total number of anaerobes detected was 41-fold greater by real-time PCR than by colony counting, while the numbers of Prevotella and Fusobacterium spp. detected were 82- and 2.4-fold greater by real-time PCR than by colony counting, respectively. Real-time PCR also identified M. micros, P. endodontalis, and P. gingivalis in 71, 60, and 52% of carious samples, respectively. Correlation matrices of the real-time PCR data revealed significant positive associations between M. micros and P. endodontalis detection and inflammatory degeneration of pulpal tissues. These anaerobes have been strongly implicated in endodontic infections that occur as sequelae to carious pulpitis. Accordingly, the data suggest that the presence of high levels of these bacteria in carious lesions may be indicative of irreversible pulpal pathology. PMID:11980945

  2. Establishment of real time allele specific locked nucleic acid quantitative PCR for detection of HBV YIDD (ATT mutation and evaluation of its application.

    Directory of Open Access Journals (Sweden)

    Yongbin Zeng

    Full Text Available BACKGROUND: Long-term use of nucleos(tide analogues can increase risk of HBV drug-resistance mutations. The rtM204I (ATT coding for isoleucine is one of the most important resistance mutation sites. Establishing a simple, rapid, reliable and highly sensitive assay to detect the resistant mutants as early as possible is of great clinical significance. METHODS: Recombinant plasmids for HBV YMDD (tyrosine-methionine-aspartate-aspartate and YIDD (tyrosine-isoleucine-aspartate-aspartate were constructed by TA cloning. Real time allele specific locked nucleic acid quantitative PCR (RT-AS-LNA-qPCR with SYBR Green I was established by LNA-modified primers and evaluated with standard recombinant plasmids, clinical templates (the clinical wild type and mutant HBV DNA mixture and 102 serum samples from nucleos(tide analogues-experienced patients. The serum samples from a chronic hepatitis B (CHB patient firstly received LMV mono therapy and then switched to LMV + ADV combined therapy were also dynamically analyzed for 10 times. RESULTS: The linear range of the assay was between 1×10(9 copies/μl and 1 × 10(2 copies/μl. The low detection limit was 1 × 10(1 copies/μl. Sensitivity of the assay were 10(-6, 10(-4 and 10(-2 in the wild-type background of 1 × 10(9 copies/μl, 1 × 10(7 copies/μl and 1 × 10(5 copies/μl, respectively. The sensitivity of the assay in detection of clinical samples was 0.03%. The complete coincidence rate between RT-AS-LNA-qPCR and direct sequencing was 91.2% (93/102, partial coincidence rate was 8.8% (9/102, and no complete discordance was observed. The two assays showed a high concordance (Kappa = 0.676, P = 0.000. Minor variants can be detected 18 weeks earlier than the rebound of HBV DNA load and alanine aminotransferase level. CONCLUSIONS: A rapid, cost-effective, high sensitive, specific and reliable method of RT-AS-LNA-qPCR with SYBR Green I for early and absolute quantification of HBV YIDD (ATT coding for isoleucine

  3. 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.

  4. Quantitative Detection of Clostridium perfringens in Broiler Chickens by Real-Time PCR Targeting the Alpha-Toxin Gene

    DEFF Research Database (Denmark)

    Abildgaard, Lone; Engberg, Ricarda M.; Schramm, Andreas

    2006-01-01

    was developed by sequencing the α-toxin gene from ~60 strains of C. perfringens, isolated from diseased as well as healthy broilers. For its application to the chicken gastrointestinal tract (i.e., ileum), DNA extraction efficiency and potential inhibition of the real-time PCR process by ileum content...

  5. Modeling of Phenoxy Acid Herbicide Mineralization and Growth of Microbial Degraders in 15 Soils Monitored by Quantitative Real-Time PCR of the Functional tfdA Gene

    DEFF Research Database (Denmark)

    Bælum, Jacob; Prestat, Emmanuel; David, Maude M.

    2012-01-01

    continents. The mineralization patterns were fitted by zero/linear or exponential growth forms of the three-half-order models and by logarithmic (log), first-order, or zero-order kinetic models. Prior and subsequent to the mineralization event, tfdA genes were quantified using real-time PCR to estimate...

  6. Multicenter evaluation of the new Abbott RealTime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA

    NARCIS (Netherlands)

    Schutten, Martin; Peters, D; Back, N K T; Beld, M; Beuselinck, K; Foulongne, V; Geretti, A-M; Pandiani, L; Tiemann, C; Niesters, H G M

    The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS

  7. Multicenter evaluation of the new Abbott RealTime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA

    NARCIS (Netherlands)

    Schutten, M.; Peters, D.; Back, N. K. T.; Beld, M.; Beuselinck, K.; Foulongne, V.; Geretti, A.-M.; Pandiani, L.; Tiemann, C.; Niesters, H. G. M.

    2007-01-01

    The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS

  8. Quantitative real-time PCR study on persistence of pDNA vaccine pVax-Hsp60 TM814 in beef muscles

    Czech Academy of Sciences Publication Activity Database

    Orság, P.; Kvardová, V.; Raška, M.; Miller, A. D.; Ledvina, Miroslav; Turánek, J.

    2008-01-01

    Roč. 6, č. 11 (2008), s. 1-11 ISSN 1479-0556 R&D Projects: GA MZe QF3115 Institutional research plan: CEZ:AV0Z40550506 Keywords : DNA vaccine * real-time PCR * liposomes Subject RIV: CC - Organic Chemistry

  9. Evaluation of the analytical performance of the new Abbott RealTime RT-PCRs for the quantitative detection of HCV and HIV-1 RNA

    NARCIS (Netherlands)

    Schutten, Martin; Fries, E; Burghoorn-Maas, C; Niesters, H G M

    2007-01-01

    BACKGROUND: Despite FDA approval and CE marking of commercial tests, manufacturer independent testing of technical aspects is important. OBJECTIVES: To evaluate the analytical performance of the new Abbott RealTime HCV and HIV-1 viral load tests. STUDY DESIGN: Sensitivity, specificity and

  10. Development of a novel quantitative real-time RT-PCR assay for the simultaneous detection of all serotypes of Foot-and-mouth disease virus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; de Stricker, K.

    2003-01-01

    Foot-and-mouth disease virus (FMDV) spreads extremely fast and the need for rapid and robust diagnostic virus detection systems was obvious during the recent European epidemic. Using a novel real-time RT-PCR system based on primer-probe energy transfer (PriProET) we present here an assay targeting...

  11. Real-Time Enzyme Kinetics by Quantitative NMR Spectroscopy and Determination of the Michaelis-Menten Constant Using the Lambert-W Function

    Science.gov (United States)

    Her, Cheenou; Alonzo, Aaron P.; Vang, Justin Y.; Torres, Ernesto; Krishnan, V. V.

    2015-01-01

    Enzyme kinetics is an essential part of a chemistry curriculum, especially for students interested in biomedical research or in health care fields. Though the concept is routinely performed in undergraduate chemistry/biochemistry classrooms using other spectroscopic methods, we provide an optimized approach that uses a real-time monitoring of the…

  12. Quantitative analysis of aortic regurgitation: real-time 3-dimensional and 2-dimensional color Doppler echocardiographic method--a clinical and a chronic animal study

    Science.gov (United States)

    Shiota, Takahiro; Jones, Michael; Tsujino, Hiroyuki; Qin, Jian Xin; Zetts, Arthur D.; Greenberg, Neil L.; Cardon, Lisa A.; Panza, Julio A.; Thomas, James D.

    2002-01-01

    BACKGROUND: For evaluating patients with aortic regurgitation (AR), regurgitant volumes, left ventricular (LV) stroke volumes (SV), and absolute LV volumes are valuable indices. AIM: The aim of this study was to validate the combination of real-time 3-dimensional echocardiography (3DE) and semiautomated digital color Doppler cardiac flow measurement (ACM) for quantifying absolute LV volumes, LVSV, and AR volumes using an animal model of chronic AR and to investigate its clinical applicability. METHODS: In 8 sheep, a total of 26 hemodynamic states were obtained pharmacologically 20 weeks after the aortic valve noncoronary (n = 4) or right coronary (n = 4) leaflet was incised to produce AR. Reference standard LVSV and AR volume were determined using the electromagnetic flow method (EM). Simultaneous epicardial real-time 3DE studies were performed to obtain LV end-diastolic volumes (LVEDV), end-systolic volumes (LVESV), and LVSV by subtracting LVESV from LVEDV. Simultaneous ACM was performed to obtain LVSV and transmitral flows; AR volume was calculated by subtracting transmitral flow volume from LVSV. In a total of 19 patients with AR, real-time 3DE and ACM were used to obtain LVSVs and these were compared with each other. RESULTS: A strong relationship was found between LVSV derived from EM and those from the real-time 3DE (r = 0.93, P <.001, mean difference (3D - EM) = -1.0 +/- 9.8 mL). A good relationship between LVSV and AR volumes derived from EM and those by ACM was found (r = 0.88, P <.001). A good relationship between LVSV derived from real-time 3DE and that from ACM was observed (r = 0.73, P <.01, mean difference = 2.5 +/- 7.9 mL). In patients, a good relationship between LVSV obtained by real-time 3DE and ACM was found (r = 0.90, P <.001, mean difference = 0.6 +/- 9.8 mL). CONCLUSION: The combination of ACM and real-time 3DE for quantifying LV volumes, LVSV, and AR volumes was validated by the chronic animal study and was shown to be clinically applicable.

  13. Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan hulless barley.

    Directory of Open Access Journals (Sweden)

    Jing Cai

    Full Text Available Hulless barley (Hordeum vulgare L. var. nudum. hook. f. has been cultivated as a major crop in the Qinghai-Tibet plateau of China for thousands of years. Compared to other cereal crops, the Tibetan hulless barley has developed stronger endogenous resistances to survive in the severe environment of its habitat. To understand the unique resistant mechanisms of this plant, detailed genetic studies need to be performed. The quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR is the most commonly used method in detecting gene expression. However, the selection of stable reference genes under limited experimental conditions was considered to be an essential step for obtaining accurate results in qRT-PCR. In this study, 10 candidate reference genes-ACT (Actin, E2 (Ubiquitin conjugating enzyme 2, TUBα (Alpha-tubulin, TUBβ6 (Beta-tubulin 6, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase, EF-1α (Elongation factor 1-alpha, SAMDC (S-adenosylmethionine decarboxylase, PKABA1 (Gene for protein kinase HvPKABA1, PGK (Phosphoglycerate kinase, and HSP90 (Heat shock protein 90-were selected from the NCBI gene database of barley. Following qRT-PCR amplifications of all candidate reference genes in Tibetan hulless barley seedlings under various stressed conditions, the stabilities of these candidates were analyzed by three individual software packages including geNorm, NormFinder, and BestKeeper. The results demonstrated that TUBβ6, E2, TUBα, and HSP90 were generally the most suitable sets under all tested conditions; similarly, TUBα and HSP90 showed peak stability under salt stress, TUBα and EF-1α were the most suitable reference genes under cold stress, and ACT and E2 were the most stable under drought stress. Finally, a known circadian gene CCA1 was used to verify the service ability of chosen reference genes. The results confirmed that all recommended reference genes by the three software were suitable for gene expression

  14. Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate▿

    Science.gov (United States)

    Saikaly, Pascal E.; Barlaz, Morton A.; de los Reyes, Francis L.

    2007-01-01

    Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can

  15. Screening for seemingly healthy newborns with congenital cytomegalovirus infection by quantitative real-time polymerase chain reaction using newborn urine: an observational study.

    Science.gov (United States)

    Yamaguchi, Akira; Oh-Ishi, Tsutomu; Arai, Takashi; Sakata, Hideaki; Adachi, Nodoka; Asanuma, Satoshi; Oguma, Eiji; Kimoto, Hirofumi; Matsumoto, Jiro; Fujita, Hidetoshi; Uesato, Tadashi; Fujita, Jutaro; Shirato, Ken; Ohno, Hideki; Kizaki, Takako

    2017-01-20

    Approximately 8-10% of newborns with asymptomatic congenital cytomegalovirus (cCMV) infection develop sensorineural hearing loss (SNHL). However, the relationship between CMV load, SNHL and central nervous system (CNS) damage in cCMV infection remains unclear. This study aimed to examine the relationship between urinary CMV load, SNHL and CNS damage in newborns with cCMV infection. The study included 23 368 newborns from two maternity hospitals in Saitama Prefecture, Japan. Urine screening for cCMV infection (quantitative real-time PCR) and newborn hearing screening (automated auditory brainstem response (AABR) testing) were conducted within 5 days of birth to examine the incidence of cCMV infection and SNHL, respectively. CNS damage was assessed by MRI of cCMV-infected newborns. The incidence of cCMV infection was 60/23 368 (0.257%; 95% CI 0.192% to 0.322%). The geometric mean urinary CMV DNA copy number in newborns with cCMV was 1.79×10 6 copies/mL (95% CI 7.97×10 5 to 4.02×10 6 ). AABR testing revealed abnormalities in 171 of the 22 229 (0.769%) newborns whose parents approved hearing screening. Of these 171 newborns, 22 had SNHL (12.9%), and 5 of these 22 were infected with cCMV (22.7%). Newborns with both cCMV and SNHL had a higher urinary CMV DNA copy number than newborns with cCMV without SNHL (p=0.036). MRI revealed CNS damage, including white matter abnormalities, in 83.0% of newborns with cCMV. Moreover, newborns with CNS damage had a significantly greater urinary CMV load than newborns without CNS damage (p=0.013). We determined the incidence of cCMV infection and urinary CMV DNA copy number in seemingly healthy newborns from two hospitals in Saitama Prefecture. SNHL and CNS damage were associated with urinary CMV DNA copy number. Quantification of urinary CMV load may effectively predict the incidence of late-onset SNHL and neurodevelopmental disorders. Published by the BMJ Publishing Group Limited. For permission to use (where not already

  16. Quantitative real-time PCR combined with propidium monoazide for the selective quantification of viable periodontal pathogens in an in vitro subgingival biofilm model.

    Science.gov (United States)

    Sánchez, M C; Marín, M J; Figuero, E; Llama-Palacios, A; León, R; Blanc, V; Herrera, D; Sanz, M

    2014-02-01

    Differentiation of live and dead cells is an important challenge when using molecular diagnosis for microbial identification. This is particularly relevant when bacteria have been exposed to antimicrobial agents. The objective of this study was to test a method using quantitative real-time polymerase chain reaction (qPCR) combined with propidium monoazide (PMA), developed for the selective quantification of viable P. gingivalis, A. actinomycetemcomitans, F. nucleatum and total bacteria in an in vitro biofilm model after antimicrobial treatment. PMA-qPCR method was tested in an in vitro biofilm model, using isopropyl alcohol as the antimicrobial agent. Matured biofilms were exposed for 1, 5, 10 and 30 min to isopropyl alcohol by immersion. Biofilms were disrupted and PMA added (final concentration of 100 μm). After DNA isolation, qPCR was carried out using specific primers and probes for the target bacteria. The differentiation of live and dead cells was tested by analysis of variance. When PMA was used in the presence of viable target bacterial cells, no statistically significant inhibition of qPCR amplification was detected (p > 0.05 in all cases). Conversely, after immersion in isopropyl alcohol of the biofilm, PMA resulted in a significant total reduction of qPCR amplification of about 4 log10 . P. gingivalis showed a vitality reduction in the biofilm of 3 log10 , while A. actinomycetemcomitans and F. nucleatum showed a 2 log10 reduction. These results demonstrate the efficiency of PMA for differentiating viable and dead P. gingivalis, A. actinomycetemcomitans and F. nucleatum cells, as well as total bacteria, in an in vitro biofilm model, after being exposed to an antimicrobial agent. Hence, this PMA-qPCR method may be useful for studying the effect of antimicrobial agents aimed at oral biofilms. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Development and Use of a Real-Time Quantitative PCR Method for Detecting and Quantifying Equol-Producing Bacteria in Human Faecal Samples and Slurry Cultures

    Directory of Open Access Journals (Sweden)

    Lucía Vázquez

    2017-06-01

    Full Text Available This work introduces a novel real-time quantitative PCR (qPCR protocol for detecting and quantifying equol-producing bacteria. To this end, two sets of primers targeting the dihydrodaidzein reductase (ddr and tetrahydrodaidzein reductase (tdr genes, which are involved in the synthesis of equol, were designed. The primers showed high specificity and sensitivity when used to examine DNA from control bacteria, such as Slackia isoflavoniconvertens, Slackia equolifaciens, Asaccharobacter celatus, Adlercreutzia equolifaciens, and Enterorhabdus mucosicola. To demonstrate the validity and reliability of the protocol, it was used to detect and quantify equol-producing bacteria in human faecal samples and their derived slurry cultures. These samples were provided by 18 menopausal women under treatment of menopause symptoms with a soy isoflavone concentrate, among whom three were known to be equol-producers given the prior detection of the molecule in their urine. The tdr gene was detected in the faeces of all these equol-producing women at about 4–5 log10 copies per gram of faeces. In contrast, the ddr gene was only amplified in the faecal samples of two of these three women, suggesting the presence in the non-amplified sample of reductase genes unrelated to those known to be involved in equol formation and used for primer design in this study. When tdr and ddr were present in the same sample, similar copy numbers of the two genes were recorded. However, no significant increase in the copy number of equol-related genes along isoflavone treatment was observed. Surprisingly, positive amplification for both tdr and ddr genes was obtained in faecal samples and derived slurry cultures from two non-equol producing women, suggesting the genes could be non-functional or the daidzein metabolized to other compounds in samples from these two women. This novel qPCR tool provides a technique for monitoring gut microbes that produce equol in humans. Monitoring equol

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

    Science.gov (United States)

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

    2014-07-22

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

  19. Detection of infectious bronchitis virus with the use of real-time quantitative reverse transcriptase-PCR and correlation with virus detection in embryonated eggs.

    Science.gov (United States)

    Roh, Ha-Jung; Hilt, Deborah A; Jackwood, Mark W

    2014-09-01

    Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays have been used to detect the presence of challenge virus when the efficacy of infectious bronchitis virus (IBV) vaccine against field viruses is being experimentally evaluated. However, federal guidelines for licensing IBV vaccines indicate that challenge-virus detection following vaccination is to be conducted in embryonated eggs. In this study, we examined qRT-PCR data with the use of universal and type-specific primers and probe sets for IBV detection and compared those data with challenge-virus detection in embryonated eggs to determine if the two methods of evaluating vaccine efficacy are comparable. In addition, we tested the qRT-PCR assays on thermocyclers from two different manufacturers. We found the universal IBV primers and probe set to be comparable to challenge-virus detection in embryonated eggs. However, for some IBV types (Mass41 and Conn on the SmartCycler II and Ark, Mass41, Conn, and GA98 on the ABI 7500) the qRT-PCR assay was more sensitive than virus detection in embryonated eggs. This may simply be due to the universal IBV qRT-PCR assay being more sensitive than virus detection in eggs or to the assay detecting nucleic acid from nonviable virus. This finding is important and needs to be considered when evaluating challenge-virus detection for vaccination and challenge studies, because qRT-PCR could potentially identify positive birds that would otherwise be negative by virus detection in embryonated eggs; thus it could lead to a more stringent measure of vaccine efficacy. We also found that the IBV type-specific primers and probe sets designed in this study were in general less sensitive than the universal IBV primers and probe set. Only the Ark-DPI-spedcific assay on the SmartCycler II and the Ark-DPI-, Mass41-, and DE072/GA98- (for detection of GA98 virus only) specific assays on the ABI 7500 were comparable in sensitivity to virus detection in eggs. We

  20. Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan hulless barley.

    Science.gov (United States)

    Cai, Jing; Li, Pengfei; Luo, Xiao; Chang, Tianliang; Li, Jiaxing; Zhao, Yuwei; Xu, Yao

    2018-01-01

    Hulless barley (Hordeum vulgare L. var. nudum. hook. f.) has been cultivated as a major crop in the Qinghai-Tibet plateau of China for thousands of years. Compared to other cereal crops, the Tibetan hulless barley has developed stronger endogenous resistances to survive in the severe environment of its habitat. To understand the unique resistant mechanisms of this plant, detailed genetic studies need to be performed. The quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method in detecting gene expression. However, the selection of stable reference genes under limited experimental conditions was considered to be an essential step for obtaining accurate results in qRT-PCR. In this study, 10 candidate reference genes-ACT (Actin), E2 (Ubiquitin conjugating enzyme 2), TUBα (Alpha-tubulin), TUBβ6 (Beta-tubulin 6), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), EF-1α (Elongation factor 1-alpha), SAMDC (S-adenosylmethionine decarboxylase), PKABA1 (Gene for protein kinase HvPKABA1), PGK (Phosphoglycerate kinase), and HSP90 (Heat shock protein 90)-were selected from the NCBI gene database of barley. Following qRT-PCR amplifications of all candidate reference genes in Tibetan hulless barley seedlings under various stressed conditions, the stabilities of these candidates were analyzed by three individual software packages including geNorm, NormFinder, and BestKeeper. The results demonstrated that TUBβ6, E2, TUBα, and HSP90 were generally the most suitable sets under all tested conditions; similarly, TUBα and HSP90 showed peak stability under salt stress, TUBα and EF-1α were the most suitable reference genes under cold stress, and ACT and E2 were the most stable under drought stress. Finally, a known circadian gene CCA1 was used to verify the service ability of chosen reference genes. The results confirmed that all recommended reference genes by the three software were suitable for gene expression analysis

  1. Multiplex real-time quantitative PCR, microscopy and rapid diagnostic immuno-chromatographic tests for the detection of Plasmodium spp: performance, limit of detection analysis and quality assurance

    Directory of Open Access Journals (Sweden)

    Ralevski Filip

    2009-12-01

    Full Text Available Abstract Background Accurate laboratory diagnosis of malaria species in returning travelers is paramount in the treatment of this potentially fatal infectious disease. Materials and methods A total of 466 blood specimens from returning travelers to Africa, Asia, and South/Central America with suspected malaria infection were collected between 2007 and 2009 at the reference public health laboratory. These specimens were assessed by reference microscopy, multipex real-time quantitative polymerase chain reaction (QPCR, and two rapid diagnostic immuno-chromatographic tests (ICT in a blinded manner. Key clinical laboratory parameters such as limit of detection (LOD analysis on clinical specimens by parasite stage, inter-reader variability of ICTs, staffing implications, quality assurance and cost analysis were evaluated. Results QPCR is the most analytically sensitive method (sensitivity 99.41%, followed by CARESTART (sensitivity 88.24%, and BINAXNOW (sensitivity 86.47% for the diagnosis of malaria in returning travelers when compared to reference microscopy. However, microscopy was unable to specifically identify Plasmodia spp. in 18 out of 170 positive samples by QPCR. Moreover, the 17 samples that were negative by microscopy and positive by QPCR were also positive by ICTs. Quality assurance was achieved for QPCR by exchanging a blinded proficiency panel with another reference laboratory. The Kappa value of inter-reader variability among three readers for BINAXNOW and CARESTART was calculated to be 0.872 and 0.898 respectively. Serial dilution studies demonstrated that the QPCR cycle threshold correlates linearly with parasitemia (R2 = 0.9746 in a clinically relevant dynamic range and retains a LOD of 11 rDNA copies/μl for P. falciparum, which was several log lower than reference microscopy and ICTs. LOD for QPCR is affected not only by parasitemia but the parasite stage distribution of each clinical specimen. QPCR was approximately 6-fold more

  2. Real Time Baseball Database

    Science.gov (United States)

    Fukue, Yasuhiro

    The author describes the system outline, features and operations of "Nikkan Sports Realtime Basaball Database" which was developed and operated by Nikkan Sports Shimbun, K. K. The system enables to input numerical data of professional baseball games as they proceed simultaneously, and execute data updating at realtime, just-in-time. Other than serving as supporting tool for prepareing newspapers it is also available for broadcasting media, general users through NTT dial Q2 and others.

  3. 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.

  4. 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...

  5. Comparison of three-way and four-way calibration for the real-time quantitative analysis of drug hydrolysis in complex dynamic samples by excitation-emission matrix fluorescence.

    Science.gov (United States)

    Yin, Xiao-Li; Gu, Hui-Wen; Liu, Xiao-Lu; Zhang, Shan-Hui; Wu, Hai-Long

    2018-03-05

    Multiway calibration in combination with spectroscopic technique is an attractive tool for online or real-time monitoring of target analyte(s) in complex samples. However, how to choose a suitable multiway calibration method for the resolution of spectroscopic-kinetic data is a troubling problem in practical application. In this work, for the first time, three-way and four-way fluorescence-kinetic data arrays were generated during the real-time monitoring of the hydrolysis of irinotecan (CPT-11) in human plasma by excitation-emission matrix fluorescence. Alternating normalization-weighted error (ANWE) and alternating penalty trilinear decomposition (APTLD) were used as three-way calibration for the decomposition of the three-way kinetic data array, whereas alternating weighted residual constraint quadrilinear decomposition (AWRCQLD) and alternating penalty quadrilinear decomposition (APQLD) were applied as four-way calibration to the four-way kinetic data array. The quantitative results of the two kinds of calibration models were fully compared from the perspective of predicted real-time concentrations, spiked recoveries of initial concentration, and analytical figures of merit. The comparison study demonstrated that both three-way and four-way calibration models could achieve real-time quantitative analysis of the hydrolysis of CPT-11 in human plasma under certain conditions. However, it was also found that both of them possess some critical advantages and shortcomings during the process of dynamic analysis. The conclusions obtained in this paper can provide some helpful guidance for the reasonable selection of multiway calibration models to achieve the real-time quantitative analysis of target analyte(s) in complex dynamic systems. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Comparison of three-way and four-way calibration for the real-time quantitative analysis of drug hydrolysis in complex dynamic samples by excitation-emission matrix fluorescence

    Science.gov (United States)

    Yin, Xiao-Li; Gu, Hui-Wen; Liu, Xiao-Lu; Zhang, Shan-Hui; Wu, Hai-Long

    2018-03-01

    Multiway calibration in combination with spectroscopic technique is an attractive tool for online or real-time monitoring of target analyte(s) in complex samples. However, how to choose a suitable multiway calibration method for the resolution of spectroscopic-kinetic data is a troubling problem in practical application. In this work, for the first time, three-way and four-way fluorescence-kinetic data arrays were generated during the real-time monitoring of the hydrolysis of irinotecan (CPT-11) in human plasma by excitation-emission matrix fluorescence. Alternating normalization-weighted error (ANWE) and alternating penalty trilinear decomposition (APTLD) were used as three-way calibration for the decomposition of the three-way kinetic data array, whereas alternating weighted residual constraint quadrilinear decomposition (AWRCQLD) and alternating penalty quadrilinear decomposition (APQLD) were applied as four-way calibration to the four-way kinetic data array. The quantitative results of the two kinds of calibration models were fully compared from the perspective of predicted real-time concentrations, spiked recoveries of initial concentration, and analytical figures of merit. The comparison study demonstrated that both three-way and four-way calibration models could achieve real-time quantitative analysis of the hydrolysis of CPT-11 in human plasma under certain conditions. However, it was also found that both of them possess some critical advantages and shortcomings during the process of dynamic analysis. The conclusions obtained in this paper can provide some helpful guidance for the reasonable selection of multiway calibration models to achieve the real-time quantitative analysis of target analyte(s) in complex dynamic systems.

  7. 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 ...

  8. Time-resolved quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Verano-Braga, Thiago; Schwämmle, Veit; Sylvester, Marc

    2012-01-01

    proteins involved in the Ang-(1-7) signaling, we performed a mass spectrometry-based time-resolved quantitative phosphoproteome study of human aortic endothelial cells (HAEC) treated with Ang-(1-7). We identified 1288 unique phosphosites on 699 different proteins with 99% certainty of correct peptide...

  9. 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.

  10. Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data

    Directory of Open Access Journals (Sweden)

    Alves-Ferreira Marcio

    2010-03-01

    Full Text Available Abstract Background Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR. Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes. Results By the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1α5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhβTUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development. Conclusion We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references

  11. Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data.

    Science.gov (United States)

    Artico, Sinara; Nardeli, Sarah M; Brilhante, Osmundo; Grossi-de-Sa, Maria Fátima; Alves-Ferreira, Marcio

    2010-03-21

    Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes. By the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1alpha5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhbetaTUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development. We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references for normalization of gene expression measures in

  12. Evaluation and validation of a real-time PCR assay for detection and quantitation of human adenovirus 14 from clinical samples.

    Directory of Open Access Journals (Sweden)

    David Metzgar

    Full Text Available In 2007, the Centers for Disease Control and Prevention (CDC reported that Human adenovirus type 14 (HAdV-14 infected 106 military personnel and was responsible for the death of one U.S. soldier at Lackland Air Force Base in Texas. Identification of the responsible adenovirus, which had not previously been seen in North America and for which rapid diagnostic tools were unavailable, required retrospective analysis at reference laboratories. Initial quarantine measures were also reliant on relatively slow traditional PCR analysis at other locations. To address this problem, we developed a real-time PCR assay that detects a 225 base pair sequence in the HAdV-14a hexon gene. Fifty-one oropharyngeal swab specimens from the Naval Health Research Center, San Diego, CA and Advanced Diagnostic Laboratory, Lackland AFB, TX were used to validate the new assay. The described assay detected eight of eight and 19 of 19 confirmed HAdV-14a clinical isolates in two separate cohorts from respiratory disease outbreaks. The real-time PCR assay had a wide dynamic range, detecting from 10(2 to 10(7 copies of genomic DNA per reaction. The assay did not cross-react with other adenoviruses, influenza, respiratory syncytial virus, or common respiratory tract bacteria. The described assay is easy to use, sensitive and specific for HAdV-14a in clinical throat swab specimens, and very rapid since turnaround time is less than four hours to obtain an answer.

  13. 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

  14. 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

  15. 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

  16. 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

  17. A simple method for normalization of DNA extraction to improve the quantitative detection of soil-borne plant pathogenic oomycetes by real-time PCR.

    Science.gov (United States)

    Li, M; Ishiguro, Y; Kageyama, K; Zhu, Z

    2015-08-01

    Most of the current research into the quantification of soil-borne pathogenic oomycetes lacks determination of DNA extraction efficiency, probably leading to an incorrect estimation of DNA quantity. In this study, we developed a convenient method by using a 100 bp artificially synthesized DNA sequence derived from the mitochondrion NADH dehydrogenase subunit 2 gene of Thunnus thynnus as a control to determine the DNA extraction efficiency. The control DNA was added to soils and then co-extracted along with soil genomic DNA. DNA extraction efficiency was determined by the control DNA. Two different DNA extraction methods were compared and evaluated using different types of soils, and the commercial kit was proved to give more consistent results. We used the control DNA combined with real-time PCR to quantify the oomycete DNAs from 12 naturally infested soils. Detectable target DNA concentrations were three to five times higher after normalization. Our tests also showed that the extraction efficiencies varied on a sample-to-sample basis and were simple and useful for the accurate quantification of soil-borne pathogenic oomycetes. Oomycetes include many important plant pathogens. Accurate quantification of these pathogens is essential in the management of diseases. This study reports an easy method utilizing an external DNA control for the normalization of DNA extraction by real-time PCR. By combining two different efficient soil DNA extraction methods, the developed quantification method dramatically improved the results. This study also proves that the developed normalization method is necessary and useful for the accurate quantification of soil-borne plant pathogenic oomycetes. © 2015 The Society for Applied Microbiology.

  18. Primary quantitative analysis of the mtDNA4977bp deletion induced by lonizing radiation in human peripheral blood u-sing real-time PCR

    International Nuclear Information System (INIS)

    Duan Zhikai; Liu Jiangong; Guo Wanlong; Zhang Shuxian

    2011-01-01

    Objective: To observe the influence of mtDNA4977bp deletion induced by different dose of γ ray in human peripheral blood in order to explore the feasibility of mtDNA4977bp deletion as biodosimeter. Methods: Human peripheral blood samples were collected from three healthy donors and irradiated by γ ray, MtDNA4977bp deletion was detected by real-time PCR. Results: It indicated that that from the range of 0 ∼ 8 Gy, the relationship between mtDNA4977bp deletion and irradiation dose represents certain curvilinear correlation (Y=1.2693+1.0660X+0.0198X 2 ). Conclusion: We find that γ ray has influence on the mtDNA4977bp deletion, so it may be an important biodosmeter in future. (authors)

  19. Reference gene selection for quantitative real-time PCR analysis in virus infected cells: SARS corona virus, Yellow fever virus, Human Herpesvirus-6, Camelpox virus and Cytomegalovirus infections

    Directory of Open Access Journals (Sweden)

    Müller Marcel A

    2005-02-01

    Full Text Available Abstract Ten potential reference genes were compared for their use in experiments investigating cellular mRNA expression of virus infected cells. Human cell lines were infected with Cytomegalovirus, Human Herpesvirus-6, Camelpox virus, SARS coronavirus or Yellow fever virus. The expression levels of these genes and the viral replication were determined by real-time PCR. Genes were ranked by the BestKeeper tool, the GeNorm tool and by criteria we reported previously. Ranking lists of the genes tested were tool dependent. However, over all, β-actin is an unsuitable as reference gene, whereas TATA-Box binding protein and peptidyl-prolyl-isomerase A are stable reference genes for expression studies in virus infected cells.

  20. Real time automatic discriminating of ultrasonic flaws

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  1. Real time automatic scene classification

    NARCIS (Netherlands)

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

    2004-01-01

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

  2. Real time freeway incident detection.

    Science.gov (United States)

    2014-04-01

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

  3. 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.

  4. Designing Real Time Assistive Technologies

    DEFF Research Database (Denmark)

    Sonne, Tobias; Obel, Carsten; Grønbæk, Kaj

    2015-01-01

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

  5. 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.

  6. Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India.

    Science.gov (United States)

    Yadav, Reena; Paria, Anutosh; Mankame, Smruti; Makesh, M; Chaudhari, Aparna; Rajendran, K V

    2015-12-01

    Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10(9) copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10(9) to 10 copies) with coefficient of regression (R(2)) > 0.99, calculated average slope -3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and -3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Screening and Validation of Housekeeping Genes of the Root and Cotyledon of Cunninghamia lanceolata under Abiotic Stresses by Using Quantitative Real-Time PCR

    Directory of Open Access Journals (Sweden)

    Wenlong Bao

    2016-07-01

    Full Text Available Cunninghamia lanceolata (Chinese fir is a fast-growing and commercially important conifer of the Cupressaceae family. Due to the unavailability of complete genome sequences and relatively poor genetic background information of the Chinese fir, it is necessary to identify and analyze the expression levels of suitable housekeeping genes (HKGs as internal reference for precise analysis. Based on the results of database analysis and transcriptome sequencing, we have chosen five candidate HKGs (Actin, GAPDH, EF1a, 18S rRNA, and UBQ with conservative sequences in the Chinese fir and related species for quantitative analysis. The expression levels of these HKGs in roots and cotyledons under five different abiotic stresses in different time intervals were measured by qRT-PCR. The data were statistically analyzed using the following algorithms: NormFinder, BestKeeper, and geNorm. Finally, RankAggreg was applied to merge the sequences generated from three programs and rank these according to consensus sequences. The expression levels of these HKGs showed variable stabilities under different abiotic stresses. Among these, Actin was the most stable internal control in root, and GAPDH was the most stable housekeeping gene in cotyledon. We have also described an experimental procedure for selecting HKGs based on the de novo sequencing database of other non-model plants.

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

    Directory of Open Access Journals (Sweden)

    Andreas Garcia-Bardon

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

  9. Real time psychrometric data collection

    International Nuclear Information System (INIS)

    McDaniel, K.H.

    1996-01-01

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

  10. 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.

  11. Species level identification of coagulase negative Staphylococcus spp. from buffalo using matrix-assisted laser desorption ionization-time of flight mass spectrometry and cydB real-time quantitative PCR.

    Science.gov (United States)

    Pizauro, Lucas J L; de Almeida, Camila C; Soltes, Glenn A; Slavic, Durda; Rossi-Junior, Oswaldo D; de Ávila, Fernando A; Zafalon, Luiz F; MacInnes, Janet I

    2017-05-01

    Incorrect identification of Staphylococcus spp. can have serious clinical and zoonotic repercussions. Accordingly, the aim of this study was to determine if matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or cydB real- time quantitative PCR (qPCR) could be used to accurately identify coagulase negative Staphylococcus spp. (CoNS) obtained from buffalo milk and milking environment samples. Seventy-five of 84 CoNS isolates could be identified to the species level (score value >1.99) using MALDI-TOF MS. However, as determined by cytochrome d ubiquinol oxidase subunit II (cydB) qPCR and by 16S RNA and cydB gene sequencing, 10S. agnetis strains were wrongly identified as S. hyicus by MALDI-TOF MS. In addition, 9 isolates identified by MALDI-TOF only to the genus level (score values between 1.70 and 1.99) could be identified to species by cydB qPCR. Our findings suggest that MALDI-TOF MS is a reliable method for rapid identification of S. chromogenes and S. epidermidis (species of interest both in human and veterinary medicine) and may be able to correctly identify other Staphylococcus spp. However, at present not all Staphylococcus spp. found in buffalo milk can be accurately identified by MALDI-TOF MS and for these organisms, the cydB qPCR developed in the current study may provide a reliable alternative method for rapid identification of CoNS species. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Rapid Quantification of Viable Campylobacter Bacteria on Chicken Carcasses, Using Real-Time PCR and Propidium Monoazide Treatment, as a Tool for Quantitative Risk Assessment

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Löfström, Charlotta; Hansen, Tina Beck

    2010-01-01

    A number of intervention strategies against Campylobacter contaminated poultry focus on post-slaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter. We present a new and rapid quantitative approach for enumeration...... method does not detect DNA from dead Campylobacter, but recognises the infectious potential of the VBNC state, and is thereby able to assess the effect of control strategies, and provide trustworthy data for risk assessment....

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Xin Zhang

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

  15. Real-time imaging of quantum entanglement.

    Science.gov (United States)

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

    2013-01-01

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

  16. 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...

  17. A novel strategy to obtain quantitative data for modelling: Combined enrichment and real-time PCR for enumeration of salmonellae from pig carcasses

    DEFF Research Database (Denmark)

    Malorny, Burkhard; Krämer, Nadine; Vigre, Håkan

    2011-01-01

    The primary sources for the major zoonotic pathogen Salmonella are food-producing animals such as pigs and poultry. For risk assessment and hazard analysis and critical control point (HACCP) concepts, it is essential to produce large amounts of quantitative data, which is currently not achievable...

  18. The real time rolling shutter

    OpenAIRE

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

    2015-01-01

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

  19. Development of an event-specific hydrolysis probe quantitative real-time polymerase chain reaction assay for Embrapa 5.1 genetically modified common bean (Phaseolus vulgaris).

    Science.gov (United States)

    Treml, Diana; Venturelli, Gustavo L; Brod, Fábio C A; Faria, Josias C; Arisi, Ana C M

    2014-12-10

    A genetically modified (GM) common bean event, namely Embrapa 5.1, resistant to the bean golden mosaic virus (BGMV), was approved for commercialization in Brazil. Brazilian regulation for genetically modified organism (GMO) labeling requires that any food containing more than 1% GMO be labeled. The event-specific polymerase chain reaction (PCR) method has been the primary trend for GMO identification and quantitation because of its high specificity based on the flanking sequence. This work reports the development of an event-specific assay, named FGM, for Embrapa 5.1 detection and quantitation by use of SYBR Green or hydrolysis probe. The FGM assay specificity was tested for Embrapa 2.3 event (a noncommercial GM common bean also resistant to BGMV), 46 non-GM common bean varieties, and other crop species including maize, GM maize, soybean, and GM soybean. The FGM assay showed high specificity to detect the Embrapa 5.1 event. Standard curves for the FGM assay presented a mean efficiency of 95% and a limit of detection (LOD) of 100 genome copies in the presence of background DNA. The primers and probe developed are suitable for the detection and quantitation of Embrapa 5.1.

  20. Real time analysis under EDS

    International Nuclear Information System (INIS)

    Schneberk, D.

    1985-07-01

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

  1. Molecular sensitivity threshold of wet mount and an immunochromatographic assay evaluated by quantitative real-time PCR for diagnosis of Trichomonas vaginalis infection in a low-risk population of childbearing women.

    Science.gov (United States)

    Leli, Christian; Castronari, Roberto; Levorato, Lucia; Luciano, Eugenio; Pistoni, Eleonora; Perito, Stefano; Bozza, Silvia; Mencacci, Antonella

    2016-06-01

    Vaginal trichomoniasis is a sexually transmitted infection caused by Trichomonas vaginalis, a flagellated protozoan. Diagnosis of T. vaginalis infection is mainly performed by wet mount microscopy, with a sensitivity ranging from 38% to 82%, compared to culture, still considered the gold standard. Commercial immunochromatographic tests for monoclonal-antibody-based detection have been introduced as alternative methods for diagnosis of T. vaginalis infection and have been reported in some studies to be more sensitive than wet mount. Real-time PCR methods have been recently developed, with optimal sensitivity and specificity. The aim of this study was to evaluate whether there is a molecular sensitivity threshold for both wet mount and imunochromatographic assays. To this aim, a total of 1487 low-risk childbearing women (median age 32 years, interquartile range 27-37) were included in the study, and underwent vaginal swab for T. vaginalis detection by means of a quantitative real-time PCR assay, wet mount and an immunochromatographic test. Upon comparing the results, prevalence values observed were 1.3% for real-time PCR, 0.5% for microscopic examination, and 0.8% for the immunochromatographic test. Compared to real-time PCR, wet mount sensitivity was 40% (95% confidence interval 19.1% to 63.9%) and specificity was 100% (95% CI 99.7% to 100%). The sensitivity and specificity of the immunochromatographic assay were 57.9% (95% CI 33.5% to 79.8%) and 99.9% (95% CI 99.6% to 100%), respectively. Evaluation of the wet mount results and those of immunochromatographic assay detection in relation to the number of T. vaginalis DNA copies detected in vaginal samples showed that the lower identification threshold for both wet mount (chi-square 6.1; P = 0.016) and the immunochromatographic assay (chi-square 10.7; P = 0.002) was ≥100 copies of T. vaginalis DNA/5 mcl of eluted DNA.

  2. A novel strategy to obtain quantitative data for modelling: Combined enrichment and real-time PCR for enumeration of salmonellae from pig carcasses

    DEFF Research Database (Denmark)

    Burkhard, Malorny; Krämer, Nadine; Vigre, Håkan

    2011-01-01

    The primary sources for the major zoonotic pathogen Salmonella are food-producing animals such as pigs and poultry. For risk assessment and hazard analysis and critical control point (HACCP) concepts, it is essential to produce large amounts of quantitative data, which is currently not achievable....../sample. The precision was similar to the standard reference most probable number (MPN) method. A screening of 200 potentially naturally contaminated cork borer samples obtained over seven weeks in a slaughterhouse resulted in 25 Salmonella-positive samples. The analysis of salmonellae within these samples showed...

  3. A real time monitoring system

    International Nuclear Information System (INIS)

    Fontanini, Horacio; Galdoz, Erwin

    1989-01-01

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

  4. Autonomous Real Time Requirements Tracing

    Science.gov (United States)

    Plattsmier, George; Stetson, Howard

    2014-01-01

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

  5. Real time 3D photometry

    Science.gov (United States)

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

    2017-09-01

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

  6. Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

    International Nuclear Information System (INIS)

    Cicinnati, Vito R; Shen, Qingli; Sotiropoulos, Georgios C; Radtke, Arnold; Gerken, Guido; Beckebaum, Susanne

    2008-01-01

    Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented. Six genes, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues or cells under investigation

  7. Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

    Directory of Open Access Journals (Sweden)

    Radtke Arnold

    2008-11-01

    Full Text Available Abstract Background Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR. The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC is presented. Methods Six genes, beta-2-microglobulin (B2M, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, hydroxymethyl-bilane synthase (HMBS, hypoxanthine phosphoribosyl-transferase 1 (HPRT1, succinate dehydrogenase complex, subunit A (SDHA and ubiquitin C (UBC, with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. Results HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Conclusion Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the

  8. Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

    Science.gov (United States)

    Cicinnati, Vito R; Shen, Qingli; Sotiropoulos, Georgios C; Radtke, Arnold; Gerken, Guido; Beckebaum, Susanne

    2008-01-01

    Background Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented. Methods Six genes, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. Results HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Conclusion Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues

  9. Creation of a bovine herpes virus 1 (BoHV-1) quantitative particle standard by transmission electron microscopy and comparison with established standards for use in real-time PCR.

    Science.gov (United States)

    Hoferer, Marc; Braun, Anne; Sting, Reinhard

    2017-07-01

    Standards are pivotal for pathogen quantification by real-time PCR (qPCR); however, the creation of a complete and universally applicable virus particle standard is challenging. In the present study a procedure based on purification of bovine herpes virus type 1 (BoHV-1) and subsequent quantification by transmission electron microscopy (TEM) is described. Accompanying quantitative quality controls of the TEM preparation procedure using qPCR yielded recovery rates of more than 95% of the BoHV-1 virus particles on the grid used for virus counting, which was attributed to pre-treatment of the grid with 5% bovine albumin. To compare the value of the new virus particle standard for use in qPCR, virus counter based quantification and established pure DNA standards represented by a plasmid and an oligonucleotide were included. It could be shown that the numbers of virus particles, plasmid and oligonucleotide equivalents were within one log10 range determined on the basis of standard curves indicating that different approaches provide comparable quantitative values. However, only virus particles represent a complete, universally applicable quantitative virus standard that meets the high requirements of an RNA and DNA virus gold standard. In contrast, standards based on pure DNA have to be considered as sub-standard due to limited applications. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  10. Chromogenic in situ Hybridization Compared with Real time Quantitative Polymerase Chain Reaction to Evaluate HER2/neu Status in Breast Cancer.

    Science.gov (United States)

    Ayatollahi, Hossein; Fani, Azar; Ghayoor Karimiani, Ehsan; Homaee, Fateme; Shajiei, Arezoo; Sheikh, Maryam; Shakeri, Sepideh; Shams, Seyyede Fatemeh

    2017-01-01

    The assessment of human epidermal growth factor receptor 2 (HER2) status has become of great importance in the diagnosis of breast cancer. The aim of this study was to investigate the diagnostic value of quantitative Polymerase Chain Reaction (qPCR) and Chromogenic In Situ Hybridization (CISH) to assess HER2 status of biopsy specimens. To elucidate the status of HER2 gene amplification, biopsies of breast carcinoma from 120 patients with 2+ IHC status were analyzed by qPCR and CISH. The results of the two experiments were compared, and it was depicted that the concordance rate between CISH and qPCR assays was 88.1%.The quantification of HER2 gene with CISH and qPCR showed that there was a significant correlation (p value= 0.0001 and r= 0.808). The results of this research support the idea that qPCR is a precise and reproducible technique, which can be employed as a supplementary method to evaluate HER2 status.

  11. Quantitative real-time PCR analysis of Anopheles dirus TEP1 and NOS during Plasmodium berghei infection, using three reference genes

    Directory of Open Access Journals (Sweden)

    Jonathan W.K. Liew

    2017-07-01

    Full Text Available Quantitative reverse transcription PCR (qRT-PCR has been an integral part of characterizing the immunity of Anopheles mosquitoes towards Plasmodium invasion. Two anti-Plasmodium factors of Anopheles, thioester-containing protein 1 (TEP1 and nitric oxide synthase (NOS, play a role in the refractoriness of Anopheles towards Plasmodium infection and are generally expressed during infection. However, these are less studied in Anopheles dirus, a dominant malaria vector in Southeast Asia. Furthermore, most studies used a single reference gene for normalization during gene expression analysis without proper validation. This may lead to erroneous quantification of expression levels. Therefore, the present study characterized and investigated the expression profiles of TEP1 and NOS of Anopheles dirus during P. berghei infection. Prior to that, the elongation factor 1-alpha (EF1, actin 1 (Act and ribosomal protein S7 (S7 genes were validated for their suitability as a set of reference genes. TEP1 and NOS expressions in An. dirus were found to be significantly induced after P. berghei infection.

  12. Evaluation of RNA extraction methods and identification of putative reference genes for real-time quantitative polymerase chain reaction expression studies on olive (Olea europaea L.) fruits.

    Science.gov (United States)

    Nonis, Alberto; Vezzaro, Alice; Ruperti, Benedetto

    2012-07-11

    Genome wide transcriptomic surveys together with targeted molecular studies are uncovering an ever increasing number of differentially expressed genes in relation to agriculturally relevant processes in olive (Olea europaea L). These data need to be supported by quantitative approaches enabling the precise estimation of transcript abundance. qPCR being the most widely adopted technique for mRNA quantification, preliminary work needs to be done to set up robust methods for extraction of fully functional RNA and for the identification of the best reference genes to obtain reliable quantification of transcripts. In this work, we have assessed different methods for their suitability for RNA extraction from olive fruits and leaves and we have evaluated thirteen potential candidate reference genes on 21 RNA samples belonging to fruit developmental/ripening series and to leaves subjected to wounding. By using two different algorithms, GAPDH2 and PP2A1 were identified as the best reference genes for olive fruit development and ripening, and their effectiveness for normalization of expression of two ripening marker genes was demonstrated.

  13. Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples

    Directory of Open Access Journals (Sweden)

    Reis Patricia P

    2010-06-01

    Full Text Available Abstract Background MicroRNAs (miRs are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE tissue. Results Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p 35, we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p Conclusion Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.

  14. Quantitative Real-Time Polymerase Chain Reaction for the Diagnosis of Mycoplasma genitalium Infection in South African Men With and Without Symptoms of Urethritis.

    Science.gov (United States)

    le Roux, Marie Cecilia; Hoosen, Anwar Ahmed

    2017-01-01

    This study was done to diagnose Mycoplasma genitalium infection based on bacterial load in urine specimens from symptomatic and asymptomatic men. Urine specimens from 94 men with visible urethral discharge, 206 with burning on micturition and 75 without symptoms presenting to a family practitioner were tested for M. genitalium as well as Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis by transcription-mediated amplification assays. A quantitative polymerase chain reaction assay was used to determine the bacterial load for all specimens in which M. genitalium was the only organism detected. Among the 375 specimens collected, M. genitalium was detected in 59 (15.7%) men (both symptomatic and asymptomatic) using the transcription-mediated amplification assay, and in 45 (12.0%) of the total population, it was the only pathogen detected. One or more pathogens were detected in 129 (43%) of the symptomatic men, with N. gonorrhoeae in 50 (16.7%); C. trachomatis in 37 (12.3%) and T. vaginalis present in 24 (8.0%) patients. Among the 17 patients where mixed infections were detected, M. genitalium with N. gonorrhoeae was the most common (11/17; 64.7%). Patients with visible urethral discharge had significantly higher M. genitalium concentrations than those with burning on micturition. The median M. genitalium load in symptomatic men was significantly higher than that in asymptomatic men. This study confirms the high prevalence of M. genitalium among men with urethritis in South Africa and demonstrates that there is a strong association with M. genitalium bacterial load and clinical urethritis. As the number of organisms increased, the severity of the symptoms increased, an indication of the role that the organism plays in disease progression.

  15. Development, validation and field evaluation of a quantitative real-time PCR able to differentiate between field Mycoplasma synoviae and the MS-H-live vaccine strain.

    Science.gov (United States)

    Dijkman, R; Feberwee, A; Landman, W J M

    2017-08-01

    A quantitative PCR (qPCR) able to differentiate between field Mycoplasma synoviae and MS-H vaccine strain was developed, validated and evaluated. It was developed using nucleotide differences in the obg gene. Analytical specificity and sensitivity assessed using DNA from 194 M. synoviae field samples, three different batches of MS-H vaccine and from 43 samples representing four other avian Mycoplasma species proved to be 100%. The detection limit for field M. synoviae and MS-H vaccine strain was 10 2-3 and 10 2 colony-forming units PCR equivalents/g trachea mucus, respectively. The qPCR was able to detect both, field M. synoviae and MS-H vaccine strain in ratios of 1:100 determined both using spiked and field samples. One hundred and twenty samples from M. synoviae-infected non-vaccinated birds, 110 samples from M. synoviae-vaccinated birds from a bird experiment and 224 samples from M. synoviae negative (serology and PCR) birds were used to determine the relative sensitivity and specificity using a previously described M. synoviae PCR as reference. The relative sensitivity and specificity for field M. synoviae were 95.0% and 99.6%, respectively, and 94.6% and 100% for the MS-H-live vaccine, respectively. Field validation and confirmation by multi locus sequence typing revealed that the qPCR correctly distinguished between MS-H and field M. synoviae. Evaluation of the differentiating M. synoviae qPCR in three commercial flocks suggested transmission of MS-H-live vaccine from vaccinated to non-vaccinated flocks at the same farm. Furthermore, it showed evidence for the colonization with field M. synoviae in MS-H-vaccinated flocks.

  16. 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...

  17. Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia

    DEFF Research Database (Denmark)

    Jamal, Syed M.; Belsham, Graham

    2015-01-01

    Asia,A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 subline-agewere also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV....... Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysableprobe-based real time reverse transcription quantitative polymerase chain reaction (RTqPCR) assays were developed for specific detection...... and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnosticassays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of OPan...

  18. Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR

    Science.gov (United States)

    2014-01-01

    Background Gene expression analysis using quantitative reverse transcription PCR (qRT-PCR) is a robust method wherein the expression levels of target genes are normalised using internal control genes, known as reference genes, to derive changes in gene expression levels. Although reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. Therefore, an assessment of reference genes was required to validate the recent findings and select stably expressed genes across different olive cultivars. Results A total of eight candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine/threonine-protein phosphatase catalytic subunit (PP2A), elongation factor 1 alpha (EF1-alpha), polyubiquitin (OUB2), aquaporin tonoplast intrinsic protein (TIP2), tubulin alpha (TUBA), 60S ribosomal protein L18-3 (60S RBP L18-3) and polypyrimidine tract-binding protein homolog 3 (PTB)] were chosen based on their stability in olive tissues as well as in other plants. Expression stability was examined by qRT-PCR across 12 biological samples, representing mesocarp tissues at various developmental stages in three different olive cultivars, Barnea, Frantoio and Picual, independently and together during the 2009 season with two software programs, GeNorm and BestKeeper. Both software packages identified GAPDH, EF1-alpha and PP2A as the three most stable reference genes across the three cultivars and in the cultivar, Barnea. GAPDH, EF1-alpha and 60S RBP L18-3 were found to be most stable reference genes in the cultivar Frantoio while 60S RBP L18-3, OUB2 and PP2A were found to be most stable reference genes in the cultivar Picual. Conclusions The analyses of expression stability of reference genes using qRT-PCR revealed that GAPDH, EF1-alpha, PP2A, 60S RBP L18-3 and OUB2 are suitable reference genes for expression analysis in developing Olea europaea mesocarp tissues, displaying the highest level

  19. Ultrafast Screening and Quantitation of Pesticides in Food and Environmental Matrices by Solid-Phase Microextraction-Transmission Mode (SPME-TM) and Direct Analysis in Real Time (DART).

    Science.gov (United States)

    Gómez-Ríos, Germán Augusto; Gionfriddo, Emanuela; Poole, Justen; Pawliszyn, Janusz

    2017-07-05

    The direct interface of microextraction technologies to mass spectrometry (MS) has unquestionably revolutionized the speed and efficacy at which complex matrices are analyzed. Solid Phase Micro Extraction-Transmission Mode (SPME-TM) is a technology conceived as an effective synergy between sample preparation and ambient ionization. Succinctly, the device consists of a mesh coated with polymeric particles that extracts analytes of interest present in a given sample matrix. This coated mesh acts as a transmission-mode substrate for Direct Analysis in Real Time (DART), allowing for rapid and efficient thermal desorption/ionization of analytes previously concentrated on the coating, and dramatically lowering the limits of detection attained by sole DART analysis. In this study, we present SPME-TM as a novel tool for the ultrafast enrichment of pesticides present in food and environmental matrices and their quantitative determination by MS via DART ionization. Limits of quantitation in the subnanogram per milliliter range can be attained, while total analysis time does not exceed 2 min per sample. In addition to target information obtained via tandem MS, retrospective studies of the same sample via high-resolution mass spectrometry (HRMS) were accomplished by thermally desorbing a different segment of the microextraction device.

  20. Effect of food matrix and thermal processing on the performance of a normalised quantitative real-time PCR approach for lupine (Lupinus albus) detection as a potential allergenic food.

    Science.gov (United States)

    Villa, Caterina; Costa, Joana; Gondar, Cristina; Oliveira, M Beatriz P P; Mafra, Isabel

    2018-10-01

    Lupine is widely used as an ingredient in diverse food products, but it is also a source of allergens. This work aimed at proposing a method to detect/quantify lupine as an allergen in processed foods based on a normalised real-time PCR assay targeting the Lup a 4 allergen-encoding gene of Lupinus albus. Sensitivities down to 0.0005%, 0.01% and 0.05% (w/w) of lupine in rice flour, wheat flour and bread, respectively, and 1 pg of L. albus DNA were obtained, with adequate real-time PCR performance parameters using the ΔCt method. Both food matrix and processing affected negatively the quantitative performance of the assay. The method was successfully validated with blind samples and applied to processed foods. Lupine was estimated between 4.12 and 22.9% in foods, with some results suggesting the common practice of precautionary labelling. In this work, useful and effective tools were proposed for the detection/quantification of lupine in food products. Copyright © 2018 Elsevier Ltd. All rights reserved.

  1. Hard Real-Time Networking on Firewire

    NARCIS (Netherlands)

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

    2005-01-01

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

  2. Modular specification of real-time systems

    DEFF Research Database (Denmark)

    Inal, Recep

    1994-01-01

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

  3. Selection of reference genes for quantitative real-time PCR expression studies of microdissected reproductive tissues in apomictic and sexual Boechera

    Directory of Open Access Journals (Sweden)

    Amiteye Samuel

    2011-08-01

    Full Text Available Abstract Background Apomixis, a natural form of asexual seed production in plants, is considered to have great biotechnological potential for agriculture. It has been hypothesised that de-regulation of the sexual developmental pathway could trigger apomictic reproduction. The genus Boechera represents an interesting model system for understanding apomixis, having both sexual and apomictic genotypes at the diploid level. Quantitative qRT-PCR is the most extensively used method for validating genome-wide gene expression analyses, but in order to obtain reliable results, suitable reference genes are necessary. In this work we have evaluated six potential reference genes isolated from a 454 (FLX derived cDNA library of Boechera. RNA from live microdissected ovules and anthers at different developmental stages, as well as vegetative tissues of apomictic and sexual Boechera, were used to validate the candidates. Results Based on homologies with Arabidopsis, six genes were selected from a 454 cDNA library of Boechera: RPS18 (Ribosomal sub protein 18, Efalpha1 (Elongation factor 1 alpha, ACT 2 (Actin2, UBQ (polyubiquitin, PEX4 (Peroxisomal ubiquitin conjugating enzyme and At1g09770.1 (Arabidopsis thaliana cell division cycle 5. Total RNA was extracted from 17 different tissues, qRT-PCRs were performed, and raw Ct values were analyzed for primer efficiencies and gene ratios. The geNorm and normFinder applications were used for selecting the most stable genes among all tissues and specific tissue groups (ovule, anthers and vegetative tissues in both apomictic and sexual plants separately. Our results show that BoechRPS18, BoechEfα1, BoechACT2 and BoechUBQ were the most stable genes. Based on geNorm, the combinations of BoechRPS18 and BoechEfα1 or BoechUBQ and BoechEfα1 were the most stable in the apomictic plant, while BoechRPS18 and BoechACT2 or BoechUBQ and BoechACT2 performed best in the sexual plant. When subgroups of tissue samples were analyzed

  4. Mobile real time radiography system

    Energy Technology Data Exchange (ETDEWEB)

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

    1997-11-01

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

  5. Mobile real time radiography system

    International Nuclear Information System (INIS)

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

    1997-01-01

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

  6. 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

  7. Students Collecting Real time Data

    Science.gov (United States)

    Miller, P.

    2006-05-01

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

  8. Evaluation of Rice Resistance to Southern Rice Black-Streaked Dwarf Virus and Rice Ragged Stunt Virus through Combined Field Tests, Quantitative Real-Time PCR, and Proteome Analysis.

    Science.gov (United States)

    Wang, Zhenchao; Yu, Lu; Jin, Linhong; Wang, Wenli; Zhao, Qi; Ran, Longlu; Li, Xiangyang; Chen, Zhuo; Guo, Rong; Wei, Yongtian; Yang, Zhongcheng; Liu, Enlong; Hu, Deyu; Song, Baoan

    2017-02-22

    Diseases caused by southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) considerably decrease grain yield. Therefore, determining rice cultivars with high resistance to SRBSDV and RRSV is necessary. In this study, rice cultivars with high resistance to SRBSDV and RRSV were evaluated through field trials in Shidian and Mangshi county, Yunnan province, China. SYBR Green I-based quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to quantitatively detect virus gene expression levels in different rice varieties. The following parameters were applied to evaluate rice resistance: acre yield (A.Y.), incidence of infected plants (I.I.P.), virus load (V.L.), disease index (D.I.), and insect quantity (I.Q.) per 100 clusters. Zhongzheyou1 (Z1) and Liangyou2186 (L2186) were considered the most suitable varieties with integrated higher A.Y., lower I.I.P., V.L., D.I. and I.Q. In order to investigate the mechanism of rice resistance, comparative label-free shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) proteomic approaches were applied to comprehensively describe the proteomics of rice varieties' SRBSDV tolerance. Systemic acquired resistance (SAR)-related proteins in Z1 and L2186 may result in the superior resistance of these varieties compared with Fengyouxiangzhan (FYXZ).

  9. LTR real-time PCR for HIV-1 DNA quantitation in blood cells for early diagnosis in infants born to seropositive mothers treated in HAART area (ANRS CO 01).

    Science.gov (United States)

    Avettand-Fènoël, Véronique; Chaix, Marie-Laure; Blanche, Stéphane; Burgard, Marianne; Floch, Corinne; Toure, Kadidia; Allemon, Marie-Christine; Warszawski, Josiane; Rouzioux, Christine

    2009-02-01

    HIV-1 diagnosis in babies born to seropositive mothers is one of the challenges of HIV epidemics in children. A simple, rapid protocol was developed for quantifying HIV-1 DNA in whole blood samples and was used in the ANRS French pediatric cohort in conditions of prevention of mother-to-child transmission. A quantitative HIV-1 DNA protocol (LTR real-time PCR) requiring small blood volumes was developed. First, analytical reproducibility was evaluated on 172 samples. Results obtained on blood cell pellets and Ficoll-Hypaque separated mononuclear cells were compared in 48 adult HIV-1 samples. Second, the protocol was applied to HIV-1 diagnosis in infants in parallel with plasma HIV-RNA quantitation. This prospective study was performed in children born between May 2005 and April 2007 included in the ANRS cohort. The assay showed good reproducibility. The 95% detection cut-off value was 6 copies/PCR, that is, 40 copies/10(6) leukocytes. HIV-DNA levels in whole blood were highly correlated with those obtained after Ficoll-Hypaque separation (r = 0.900, P mothers have received HAART. (c) 2008 Wiley-Liss, Inc.

  10. 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.

  11. 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.

  12. Quantitative Evaluation of Fire and EMS Mobilization Times

    CERN Document Server

    Upson, Robert

    2010-01-01

    Quantitative Evaluation of Fire and EMS Mobilization Times presents comprehensive empirical data on fire emergency and EMS call processing and turnout times, and aims to improve the operational benchmarks of NFPA peer consensus standards through a close examination of real-world data. The book also identifies and analyzes the elements that can influence EMS mobilization response times. Quantitative Evaluation of Fire and EMS Mobilization Times is intended for practitioners as a tool for analyzing fire emergency response times and developing methods for improving them. Researchers working in a

  13. Coordinating Transit Transfers in Real Time

    Science.gov (United States)

    2016-05-06

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

  14. Touch-down reverse transcriptase-PCR detection of IgV(H) rearrangement and Sybr-Green-based real-time RT-PCR quantitation of minimal residual disease in patients with chronic lymphocytic leukemia.

    Science.gov (United States)

    Peková, Sona; Marková, Jana; Pajer, Petr; Dvorák, Michal; Cetkovský, Petr; Schwarz, Jirí

    2005-01-01

    Patients with chronic lymphocytic leukemia (CLL) can relapse even after aggressive therapy and autografts. It is commonly assumed that to prevent relapse the level of minimal residual disease (MRD) should be as low as possible. To evaluate MRD, highly sensitive quantitative assays are needed. The aim of the study was to develop a robust and sensitive method for detection of the clonal immunoglobulin heavy-chain variable (IgV(H)) rearrangement in CLL and to introduce a highly sensitive and specific methodology for MRD monitoring in patients with CLL who undergo intensive treatment. As a prerequisite for MRD detection, touch-down reverse transcriptase (RT)-PCR using degenerate primers were used for the diagnostic identification of (H) gene rearrangement(s). For quantitative MRD detection in 18 patients, we employed a real-time RT-PCR assay (RQ-PCR) making use of patient-specific primers and the cost-saving Sybr-Green reporter dye (SG). For precise calibration of RQ-PCR, patient-specific IgV(H) sequences were cloned. Touch-down RT-PCR with degenerate primers allowed the successful detection of IgV(H) clonal rearrangement(s) in 252 of 257 (98.1%) diagnostic samples. Biallelic rearrangements were found in 27 of 252 (10.7%) cases. Degenerate primers used for the identification of clonal expansion at diagnosis were not sensitive enough for MRD detection. In contrast, our RQ-PCR assay using patient-specific primers and SG reached the sensitivity of 10(-)(6). We demonstrated MRD in each patient tested, including four of four patients in complete remission following autologous hematopoietic stem cell transplantation (HSCT) and three of three following allogeneic 'mini'-HSCT. Increments in MRD might herald relapse; aggressive chemotherapy could induce molecular remission. Our touch-down RT-PCR has higher efficiency to detect clonal IgV(H) rearrangements including the biallelic ones. MRD quantitation of IgV(H) expression using SG-based RQ-PCR represents a highly specific

  15. 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.

  16. 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...

  17. 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

  18. 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,

  19. Establishment of a minor groove binder-probe based quantitative real time PCR to detect Borrelia burgdorferi sensu lato and differentiation of Borrelia spielmanii by ospA-specific conventional PCR

    Directory of Open Access Journals (Sweden)

    Strube Christina

    2010-08-01

    Full Text Available Abstract Background Borrelia burgdorferi sensu lato (sl, the causative agent of Lyme borreliosis, is transmitted by ticks of the genus Ixodes as vector. For identification of Borrelia infections in ticks a TaqMan™ minor groove binder (MGB probe-based quantitative real time PCR (qPCR was established targeting the 5S-23S intergenic spacer. Extension to a duplex qPCR included an Ixodes spp. positive control to verify successful DNA isolation. Besides qPCR, an ospA-specific conventional PCR for species-specific identification of B. spielmanii was established. Afterwards 1000 I. ricinus flagged in the city of Hanover, Germany, were investigated for B. burgdorferi sl infections followed by species identification. Furthermore, I. hexagonus ticks were investigated to proof applicability of the PCRs. Results Quantitative real time PCR (qPCR identifying B. burgdorferi sl in ticks was able to detect 1-10 copies per reaction. B. spielmanii ospA-specific conventional PCR was also highly specific and showed no cross reactions with the other tested Borrelia species. From 1000 hanoveranian ticks 24.3% were positive compared to only 7.4% positives by dark-field microscopy. Related to tick stage 1.7% larvae, 18.1% nymphs, and 34.6% adults were positive. The most frequent species was B. garinii, followed by B. afzelii, B. spielmanii, B. valaisiana and B. burgdorferi sensu stricto (ss. 70.6% of I. ricinus were mono-infected, whereas 28.0% and 1.4% were infected with two and three Borrelia species, respectively. From 232 I. hexagonus collected from hedgehogs in different sites of Germany, qPCR detected 5.7% to be infected with B. burgdorferi sl, which were identified as B. afzelii, B. garinii and B. spielmanii. Conclusions The evaluated qPCR to detect B. burgdorferi sl in Ixodes spp. is highly specific and sensitive. As a duplex qPCR including detection of Ixodes spp. DNA it is the first DNA based technique incorporating a control for successful DNA isolation from

  20. Model Checking Real-Time Systems

    DEFF Research Database (Denmark)

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

    2018-01-01

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

  1. 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...

  2. 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.

  3. 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...

  4. 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...

  5. Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients.

    Science.gov (United States)

    Bravo, Dayana; Clari, María Ángeles; Costa, Elisa; Muñoz-Cobo, Beatriz; Solano, Carlos; José Remigia, María; Navarro, David

    2011-08-01

    Limited data are available on the performance of different automated extraction platforms and commercially available quantitative real-time PCR (QRT-PCR) methods for the quantitation of cytomegalovirus (CMV) DNA in plasma. We compared the performance characteristics of the Abbott mSample preparation system DNA kit on the m24 SP instrument (Abbott), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (Roche), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (Qiagen) coupled with the Abbott CMV PCR kit, the LightCycler CMV Quant kit (Roche), and the Q-CMV complete kit (Nanogen), for both plasma specimens from allogeneic stem cell transplant (Allo-SCT) recipients (n = 42) and the OptiQuant CMV DNA panel (AcroMetrix). The EZ1 system displayed the highest extraction efficiency over a wide range of CMV plasma DNA loads, followed by the m24 and the AmpliPrep methods. The Nanogen PCR assay yielded higher mean CMV plasma DNA values than the Abbott and the Roche PCR assays, regardless of the platform used for DNA extraction. Overall, the effects of the extraction method and the QRT-PCR used on CMV plasma DNA load measurements were less pronounced for specimens with high CMV DNA content (>10,000 copies/ml). The performance characteristics of the extraction methods and QRT-PCR assays evaluated herein for clinical samples were extensible at cell-based standards from AcroMetrix. In conclusion, different automated systems are not equally efficient for CMV DNA extraction from plasma specimens, and the plasma CMV DNA loads measured by commercially available QRT-PCRs can differ significantly. The above findings should be taken into consideration for the establishment of cutoff values for the initiation or cessation of preemptive antiviral therapies and for the interpretation of data from clinical studies in the Allo-SCT setting.

  6. Multiplexing real-time timed events

    NARCIS (Netherlands)

    Holenderski, M.J.; Cools, W.A.; Bril, R.J.; Lukkien, J.J.

    2009-01-01

    This paper presents the design and implementation of RELTEQ, a timed event management algorithm based on relative event times, supporting long event interarrival time, long lifetime of the event queue, no drift and low overhead. It is targeted at embedded operating systems. RELTEQ has been conceived

  7. Expression patterns of WRKY genes in di-haploid Populus simonii × P. nigra in response to salinity stress revealed by quantitative real-time PCR and RNA sequencing.

    Science.gov (United States)

    Wang, Shengji; Wang, Jiying; Yao, Wenjing; Zhou, Boru; Li, Renhua; Jiang, Tingbo

    2014-10-01

    Spatio-temporal expression patterns of 13 out of 119 poplar WRKY genes indicated dynamic and tissue-specific roles of WRKY family proteins in salinity stress tolerance. To understand the expression patterns of poplar WRKY genes under salinity stress, 51 of the 119 WRKY genes were selected from di-haploid Populus simonii × P. nigra by quantitative real-time PCR (qRT-PCR). We used qRT-PCR to profile the expression of the top 13 genes under salinity stress across seven time points, and employed RNA-Seq platforms to cross-validate it. Results demonstrated that all the 13 WRKY genes were expressed in root, stem, and leaf tissues, but their expression levels and overall patterns varied notably in these tissues. Regarding overall gene expression in roots, the 13 genes were significantly highly expressed at all six time points after the treatment, reaching the plateau of expression at hour 9. In leaves, the 13 genes were similarly up-regulated from 3 to 12 h in response to NaCl treatment. In stems, however, expression levels of the 13 genes did not show significant changes after the NaCl treatment. Regarding individual gene expression across the time points and the three tissues, the 13 genes can be classified into three clusters: the lowly expressed Cluster 1 containing PthWRKY28, 45 and 105; intermediately expressed Clusters 2 including PthWRKY56, 88 and 116; and highly expressed Cluster 3 consisting of PthWRKY41, 44, 51, 61, 62, 75 and 106. In general, genes in Cluster 2 and 3 displayed a dynamic pattern of "induced amplification-recovering", suggesting that these WRKY genes and corresponding pathways may play a critical role in mediating salt response and tolerance in a dynamic and tissue-specific manner.

  8. 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.

  9. 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

  10. 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

  11. 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,

  12. Real time refractive index measurement by ESPI

    International Nuclear Information System (INIS)

    Torroba, R.; Joenathan, C.

    1991-01-01

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

  13. 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

  14. 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

  15. Validation of RNAi by real time PCR

    DEFF Research Database (Denmark)

    Josefsen, Knud; Lee, Ying Chiu

    2011-01-01

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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Real Time Earthquake Information System in Japan

    Science.gov (United States)

    Doi, K.; Kato, T.

    2003-12-01

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

  1. Optofluidic time-stretch quantitative phase microscopy.

    Science.gov (United States)

    Guo, Baoshan; Lei, Cheng; Wu, Yi; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Lee, Sangwook; Isozaki, Akihiro; Li, Ming; Jiang, Yiyue; Yasumoto, Atsushi; Di Carlo, Dino; Tanaka, Yo; Yatomi, Yutaka; Ozeki, Yasuyuki; Goda, Keisuke

    2018-03-01

    Innovations in optical microscopy have opened new windows onto scientific research, industrial quality control, and medical practice over the last few decades. One of such innovations is optofluidic time-stretch quantitative phase microscopy - an emerging method for high-throughput quantitative phase imaging that builds on the interference between temporally stretched signal and reference pulses by using dispersive properties of light in both spatial and temporal domains in an interferometric configuration on a microfluidic platform. It achieves the continuous acquisition of both intensity and phase images with a high throughput of more than 10,000 particles or cells per second by overcoming speed limitations that exist in conventional quantitative phase imaging methods. Applications enabled by such capabilities are versatile and include characterization of cancer cells and microalgal cultures. In this paper, we review the principles and applications of optofluidic time-stretch quantitative phase microscopy and discuss its future perspective. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Oxidative stress provokes distinct transcriptional responses in the stress-tolerant atr7 and stress-sensitive loh2 Arabidopsis thaliana mutants as revealed by multi-parallel quantitative real-time PCR analysis of ROS marker and antioxidant genes.

    Science.gov (United States)

    Mehterov, Nikolay; Balazadeh, Salma; Hille, Jacques; Toneva, Valentina; Mueller-Roeber, Bernd; Gechev, Tsanko

    2012-10-01

    The Arabidopsis thaliana atr7 mutant is tolerant to oxidative stress induced by paraquat (PQ) or the catalase inhibitor aminotriazole (AT), while its original background loh2 and wild-type plants are sensitive. Both, AT and PQ, which stimulate the intracellular formation of H₂O₂ or superoxide anions, respectively, trigger cell death in loh2 but do not lead to visible damage in atr7. To study gene expression during oxidative stress and ROS-induced programmed cell death, two platforms for multi-parallel quantitative real-time PCR (qRT-PCR) analysis of 217 antioxidant and 180 ROS marker genes were employed. The qRT-PCR analyses revealed AT- and PQ-induced expression of many ROS-responsive genes mainly in loh2, confirming that an oxidative burst plays a role in the activation of the cell death in this mutant. Some of the genes were specifically regulated by either AT or PQ, serving as markers for particular types of ROS. Genes significantly induced by both AT and PQ in loh2 included transcription factors (ANAC042/JUB1, ANAC102, DREB19, HSFA2, RRTF1, ZAT10, ZAT12, ethylene-responsive factors), signaling compounds, ferritins, alternative oxidases, and antioxidant enzymes. Many of these genes were upregulated in atr7 compared to loh2 under non-stress conditions at the first time point, indicating that higher basal levels of ROS and higher antioxidant capacity in atr7 are responsible for the enhanced tolerance to oxidative stress and suggesting a possible tolerance against multiple stresses of this mutant. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  3. 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.

  4. 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.

  5. 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

  6. Real time sensor for therapeutic radiation delivery

    International Nuclear Information System (INIS)

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

    1998-01-01

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

  7. 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.

  8. Expression of the Long Intergenic Non-Protein Coding RNA 665 (LINC00665) Gene and the Cell Cycle in Hepatocellular Carcinoma Using The Cancer Genome Atlas, the Gene Expression Omnibus, and Quantitative Real-Time Polymerase Chain Reaction.

    Science.gov (United States)

    Wen, Dong-Yue; Lin, Peng; Pang, Yu-Yan; Chen, Gang; He, Yun; Dang, Yi-Wu; Yang, Hong

    2018-05-05

    BACKGROUND Long non-coding RNAs (lncRNAs) have a role in physiological and pathological processes, including cancer. The aim of this study was to investigate the expression of the long intergenic non-protein coding RNA 665 (LINC00665) gene and the cell cycle in hepatocellular carcinoma (HCC) using database analysis including The Cancer Genome Atlas (TCGA), the Gene Expression Omnibus (GEO), and quantitative real-time polymerase chain reaction (qPCR). MATERIAL AND METHODS Expression levels of LINC00665 were compared between human tissue samples of HCC and adjacent normal liver, clinicopathological correlations were made using TCGA and the GEO, and qPCR was performed to validate the findings. Other public databases were searched for other genes associated with LINC00665 expression, including The Atlas of Noncoding RNAs in Cancer (TANRIC), the Multi Experiment Matrix (MEM), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) networks. RESULTS Overexpression of LINC00665 in patients with HCC was significantly associated with gender, tumor grade, stage, and tumor cell type. Overexpression of LINC00665 in patients with HCC was significantly associated with overall survival (OS) (HR=1.47795%; CI: 1.046-2.086). Bioinformatics analysis identified 469 related genes and further analysis supported a hypothesis that LINC00665 regulates pathways in the cell cycle to facilitate the development and progression of HCC through ten identified core genes: CDK1, BUB1B, BUB1, PLK1, CCNB2, CCNB1, CDC20, ESPL1, MAD2L1, and CCNA2. CONCLUSIONS Overexpression of the lncRNA, LINC00665 may be involved in the regulation of cell cycle pathways in HCC through ten identified hub genes.

  9. Overestimation of the Legionella spp. load in environmental samples by quantitative real-time PCR: pretreatment with propidium monoazide as a tool for the assessment of an association between Legionella concentration and sanitary risk.

    Science.gov (United States)

    Ditommaso, Savina; Ricciardi, Elisa; Giacomuzzi, Monica; Arauco Rivera, Susan R; Ceccarelli, Adriano; Zotti, Carla M

    2014-12-01

    Quantitative polymerase chain reaction (qPCR) offers rapid, sensitive, and specific detection of Legionella in environmental water samples. In this study, qPCR and qPCR combined with propidium monoazide (PMA-qPCR) were both applied to hot-water system samples and compared to traditional culture techniques. In addition, we evaluated the ability of PMA-qPCR to monitor the efficacy of different disinfection strategies. Comparison between the quantification obtained by culture and by qPCR or PMA-qPCR on environmental water samples confirms that the concentration of Legionella estimated by GU/L is generally higher than that estimated in CFU/L. Our results on 57 hot-water-system samples collected from 3 different sites show that: i) qPCR results were on average 178-fold higher than the culture results (Δ log10=2.25), ii) PMA-qPCR results were on average 27-fold higher than the culture results (Δ log10=1.43), iii) propidium monoazide-induced signal reduction in qPCR were nearly 10-fold (Δ log10=0.95), and that iv) different degrees of correlations between the 3 methods might be explained by different matrix properties, but also by different disinfection methods affecting cultivability of Legionella. In our study, we calculated the logarithmic differences between the results obtained by PMA-qPCR and those obtained by culture, and we suggested an algorithm for the interpretation of PMA-qPCR results for the routine monitoring of healthcare water systems using a commercial qPCR system (iQ-check real-time PCR kit; Bio-Rad, Marnes-la-Coquette, France). Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Single-Use Poly(etheretherketone) Solid-Phase Microextraction-Transmission Mode Devices for Rapid Screening and Quantitation of Drugs of Abuse in Oral Fluid and Urine via Direct Analysis in Real-Time Tandem Mass Spectrometry.

    Science.gov (United States)

    Vasiljevic, Tijana; Gómez-Ríos, Germán Augusto; Pawliszyn, Janusz

    2018-01-02

    The analysis of oral fluid (OF) and urine samples to detect drug consumption has garnered considerable attention as alternative biomatrices. Efficient implementation of microextraction and ambient ionization technologies for rapid detection of target compounds in such biomatrices creates a need for biocompatible devices which can be implemented for in vivo sampling and easily interfaced with mass spectrometry (MS) analyzers. This study introduces a novel solid-phase microextraction-transmission mode (SPME-TM) device made of poly(etheretherketone) (PEEK) mesh that can rapidly detect prohibited substances in biofluids via direct analysis in real-time tandem MS (DART-MS/MS). PEEK mesh was selected due to its biocompatibility, excellent resistance to various organic solvents, and its ability to withstand relatively high temperatures (≤350 °C). The meshes were coated with hydrophilic-lipophilic-balance particle-poly(acrylonitrile) (HLB-PAN) slurry. The robustness of the coated meshes was tested by performing rapid vortex agitation (≥3200 rpm) in LC/MS-grade solvents and by exposing them to the DART source jet stream at typical operational temperatures (∼250-350 °C). PEEK SPME-TM devices proved to be robust and were therefore used to perform ex vivo analysis of drugs of abuse spiked in urine and OF samples. Excellent results were obtained for all analytes under study; furthermore, the tests yielded satisfactory limits of quantitation (median, ∼0.5 ng mL -1 ), linearity (≥0.99), and accuracy (80-120%) over the evaluated range (0.5-200 ng mL -1 ). This research highlights plastic SPME-TM's potential usefulness as a method for rapidly screening for prohibited substances in on-site/in vivo scenarios, such as roadside or workplace drug testing, antidoping controls, and pain management programs.

  11. Development of a Rapid and Sensitive Method Combining a Cellulose Ester Microfilter and a Real-Time Quantitative PCR Assay To Detect Campylobacter jejuni and Campylobacter coli in 20 Liters of Drinking Water or Low-Turbidity Waters

    Science.gov (United States)

    Tissier, Adeline; Denis, Martine; Hartemann, Philippe

    2012-01-01

    Investigations of Campylobacter jejuni and Campylobacter coli in samples of drinking water suspected of being at the origin of an outbreak very often lead to negative results. One of the reasons for this failure is the small volume of water typically used for detecting these pathogens (10 to 1,000 ml). The efficiencies of three microfilters and different elution procedures were determined using real-time quantitative PCR to propose a procedure allowing detection of Campylobacter in 20 liters of drinking water or low-turbidity water samples. The results showed that more than 80% of the bacteria inoculated in 1 liter of drinking water were retained on each microfilter. An elution with a solution containing 3% beef extract, 0.05 M glycine at pH 9, combined with direct extraction of the bacterial genomes retained on the cellulose ester microfilter, allowed recovery of 87.3% (±22% [standard deviation]) of Campylobacter per 1 liter of tap water. Recoveries obtained from 20-liter volumes of tap water spiked with a C. coli strain were 69.5% (±10.3%) and 78.5% (±15.1%) for 91 CFU and 36 CFU, respectively. Finally, tests performed on eight samples of 20 liters of groundwater collected from an alluvial well used for the production of drinking water revealed the presence of C. jejuni and C. coli genomes, whereas no bacteria were detected with the normative culture method in volumes ranging from 10 to 1,000 ml. In the absence of available epidemiological data and information on bacterial viability, these last results indicate only that the water resource is not protected from contamination by Campylobacter. PMID:22138985

  12. 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

  13. Real-Time Penetrating Particle Analyzer (PAN)

    Science.gov (United States)

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

    2018-02-01

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

  14. 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....

  15. Real time monitoring of electron processors

    International Nuclear Information System (INIS)

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

    1995-01-01

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

  16. Real-time Social Internet Data to Guide Forecasting Models

    Energy Technology Data Exchange (ETDEWEB)

    Del Valle, Sara Y. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2016-09-20

    Our goal is to improve decision support by monitoring and forecasting events using social media, mathematical models, and quantifying model uncertainty. Our approach is real-time, data-driven forecasts with quantified uncertainty: Not just for weather anymore. Information flow from human observations of events through an Internet system and classification algorithms is used to produce quantitatively uncertain forecast. In summary, we want to develop new tools to extract useful information from Internet data streams, develop new approaches to assimilate real-time information into predictive models, validate approaches by forecasting events, and our ultimate goal is to develop an event forecasting system using mathematical approaches and heterogeneous data streams.

  17. Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs.

    Science.gov (United States)

    Sehata, Go; Sato, Hiroaki; Ito, Toshihiro; Imaizumi, Yoshitaka; Noro, Taichi; Oishi, Eiji

    2015-07-01

    We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication.

  18. Towards the production of reliable quantitative microbiological data for risk assessment: Direct quantification of Campylobacter in naturally infected chicken fecal samples using selective culture and real-time PCR

    DEFF Research Database (Denmark)

    Garcia Clavero, Ana Belén; Vigre, Håkan; Josefsen, Mathilde Hasseldam

    2015-01-01

    of Campylobacter by real-time PCR was performed using standard curves designed for two different DNA extraction methods: Easy-DNA™ Kit from Invitrogen (Easy-DNA) and NucliSENS® MiniMAG® from bioMérieux (MiniMAG). Results indicated that the estimation of the numbers of Campylobacter present in chicken fecal samples...... and for the evaluation of control strategies implemented in poultry production. The aim of this study was to compare estimates of the numbers of Campylobacter spp. in naturally infected chicken fecal samples obtained using direct quantification by selective culture and by real-time PCR. Absolute quantification....... Although there were differences in terms of estimates of Campylobacter numbers between the methods and samples, the differences between culture and real-time PCR were not statistically significant for most of the samples used in this study....

  19. Analytical characteristics and comparative evaluation of Aptima HCV quant Dx assay with the Abbott RealTime HCV assay and Roche COBAS AmpliPrep/COBAS TaqMan HCV quantitative test v2.0.

    Science.gov (United States)

    Worlock, A; Blair, D; Hunsicker, M; Le-Nguyen, T; Motta, C; Nguyen, C; Papachristou, E; Pham, J; Williams, A; Vi, M; Vinluan, B; Hatzakis, A

    2017-04-04

    The Aptima HCV Quant Dx assay (Aptima assay) is a fully automated quantitative assay on the Panther® system. This assay is intended for confirmation of diagnosis and monitoring of HCV RNA in plasma and serum specimens. The purpose of the testing described in this paper was to evaluate the performance of the Aptima assay. The analytical sensitivity, analytical specificity, precision, and linearity of the Aptima assay were assessed. The performance of the Aptima assay was compared to two commercially available HCV assays; the Abbott RealTime HCV assay (Abbott assay, Abbott Labs Illinois, USA) and the Roche COBAS Ampliprep/COBAS Taqman HCV Quantitative Test v2.0 (Roche Assay, Roche Molecular Systems, Pleasanton CA, USA). The 95% Lower Limit of Detection (LoD) of the assay was determined from dilutions of the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) and HCV positive clinical specimens in HCV negative human plasma and serum. Probit analysis was performed to generate the 95% predicted detection limits. The Lower Limit of Quantitation (LLoQ) was established for each genotype by diluting clinical specimens and the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) in HCV negative human plasma and serum. Specificity was determined using 200 fresh and 536 frozen HCV RNA negative clinical specimens including 370 plasma specimens and 366 serum specimens. Linearity for genotypes 1 to 6 was established by diluting armored RNA or HCV positive clinical specimens in HCV negative serum or plasma from 8.08 log IU/mL to below 1 log IU/mL. Precision was tested using a 10 member panel made by diluting HCV positive clinical specimens or spiking armored RNA into HCV negative plasma and serum. A method comparison was conducted against the Abbott assay using 1058 clinical specimens and against the Roche assay using 608 clinical specimens from HCV infected patients. In addition, agreement between the Roche assay and the Aptima assay using specimens with low

  20. 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.

  1. 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

  2. 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.

  3. Refactoring Real-Time Java Profiles

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  4. Feedback as Real-Time Constructions

    Science.gov (United States)

    Keiding, Tina Bering; Qvortrup, Ane

    2014-01-01

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

  5. Testing Real-Time Systems Using UPPAAL

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  6. Long-Term Prognostic Effects of Plasma Epstein-Barr Virus DNA by Minor Groove Binder-Probe Real-Time Quantitative PCR on Nasopharyngeal Carcinoma Patients Receiving Concurrent Chemoradiotherapy

    International Nuclear Information System (INIS)

    Lin, J.-C.; Wang, W.-Y.; Liang, W.-M.; Chou, H.-Y.; Jan, J.-S.; Jiang, R.-S.; Wang, J.-Y.; Twu, C.-W.; Liang, K.-L.; Chao, Jeffrey; Shen, W.-C.

    2007-01-01

    Purpose: To evaluate the long-term prognostic impact of plasma Epstein-Barr virus (EBV) DNA concentration measured by real-time quantitative polymerase chain reaction (RTQ-PCR) in nasopharyngeal carcinoma (NPC) patients receiving concurrent chemoradiotherapy (CCRT). Methods and Materials: Epstein-Barr virus DNA was retrospectively measured from stock plasma of 152 biopsy-proven NPC patients with Stage II-IV (M0) disease with a RTQ-PCR using the minor groove binder-probe. All patients received CCRT with a median follow-up of 78 months. We divided patients into three subgroups: (1) low pretreatment EBV DNA (<1,500 copies/mL) and undetectable posttreatment EBV DNA (pre-L/post-U) (2) high pretreatment EBV DNA (≥1,500 copies/mL) and undetectable posttreatment EBV DNA (pre-H/post-U), and (3) low or high pretreatment EBV DNA and detectable posttreatment EBV DNA (pre-L or H/post-D) for prognostic analyses. Results: Epstein-Barr virus DNA (median concentration, 573 copies/mL; interquartile range, 197-3,074) was detected in the pretreatment plasma of 94.1% (143/152) of patients. After treatment, plasma EBV DNA decreased or remained 0 for all patients and was detectable in 31 patients (20.4%) with a median concentration 0 copy/mL (interquartile range, 0-0). The 5-year overall survival rates of the pre-L/post-U, pre-H/post-U, and pre-L or H/post-D subgroups were 87.2%, 71.0%, and 38.7%, respectively (p < 0.0001). The relapse-free survival showed similar results with corresponding rates of 85.6%, 75.9%, and 26.9%, respectively (p < 0.0001). Multivariate Cox analysis confirmed the superior effects of plasma EBV DNA compared to other clinical parameters in prognosis prediction. Conclusion: Plasma EBV DNA is the most valuable prognostic factor for NPC. More chemotherapy should be considered for patients with persistently detectable EBV DNA after CCRT

  7. Clinical value of miR-452-5p expression in lung adenocarcinoma: A retrospective quantitative real-time polymerase chain reaction study and verification based on The Cancer Genome Atlas and Gene Expression Omnibus databases.

    Science.gov (United States)

    Gan, Xiao-Ning; Luo, Jie; Tang, Rui-Xue; Wang, Han-Lin; Zhou, Hong; Qin, Hui; Gan, Ting-Qing; Chen, Gang

    2017-05-01

    The role and mechanism of miR-452-5p in lung adenocarcinoma remain unclear. In this study, we performed a systematic study to investigate the clinical value of miR-452-5p expression in lung adenocarcinoma. The expression of miR-452-5p in 101 lung adenocarcinoma patients was detected by quantitative real-time polymerase chain reaction. The Cancer Genome Atlas and Gene Expression Omnibus databases were joined to verify the expression level of miR-452-5p in lung adenocarcinoma. Via several online prediction databases and bioinformatics software, pathway and network analyses of miR-452-5p target genes were performed to explore its prospective molecular mechanism. The expression of miR-452-5p in lung adenocarcinoma in house was significantly lower than that in adjacent tissues (p < 0.001). Additionally, the expression level of miR-452-5p was negatively correlated with several clinicopathological parameters including the tumor size (p = 0.014), lymph node metastasis (p = 0.032), and tumor-node-metastasis stage (p = 0.036). Data from The Cancer Genome Atlas also confirmed the low expression of miR-452 in lung adenocarcinoma (p < 0.001). Furthermore, reduced expression of miR-452-5p in lung adenocarcinoma (standard mean deviations = -0.393, 95% confidence interval: -0.774 to -0.011, p = 0.044) was validated by a meta-analysis. Five hub genes targeted by miR-452-5p, including SMAD family member 4, SMAD family member 2, cyclin-dependent kinase inhibitor 1B, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta, were significantly enriched in the cell-cycle pathway. In conclusion, low expression of miR-452-5p tends to play an essential role in lung adenocarcinoma. Bioinformatics analysis might be beneficial to reveal the potential mechanism of miR-452-5p in lung adenocarcinoma.

  8. Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia.

    Science.gov (United States)

    Jamal, Syed M; Belsham, Graham J

    2015-01-01

    Rapid and accurate diagnosis of foot-and-mouth disease (FMD) and virus serotyping are of paramount importance for control of this disease in endemic areas where vaccination is practiced. Ideally this virus characterization should be achieved without the need for virus amplification in cell culture. Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysable probe-based real time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were developed for specific detection and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnostic assays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of O-PanAsia, A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 sublineage were also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV and detected the RNA from the target viruses with cycle threshold (CT) values comparable with those obtained with the serotype-independent pan-FMDV diagnostic assays. No cross-reactivity was observed in these assays between the heterotypic viruses circulating in the region. The assays reported here have higher diagnostic sensitivity (100% each for serotypes O and Asia-1, and 92% [95% CI = 81.4-100%] for serotype A positive samples) and specificity (100% each for serotypes O, A and Asia-1 positive samples) for the viruses currently circulating in West Eurasia compared to the serotyping assays reported earlier. Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for the poor

  9. Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia.

    Directory of Open Access Journals (Sweden)

    Syed M Jamal

    Full Text Available Rapid and accurate diagnosis of foot-and-mouth disease (FMD and virus serotyping are of paramount importance for control of this disease in endemic areas where vaccination is practiced. Ideally this virus characterization should be achieved without the need for virus amplification in cell culture. Due to the heterogeneity of FMD viruses (FMDVs in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysable probe-based real time reverse transcription quantitative polymerase chain reaction (RT-qPCR assays were developed for specific detection and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnostic assays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of O-PanAsia, A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08. In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 sublineage were also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV and detected the RNA from the target viruses with cycle threshold (CT values comparable with those obtained with the serotype-independent pan-FMDV diagnostic assays. No cross-reactivity was observed in these assays between the heterotypic viruses circulating in the region. The assays reported here have higher diagnostic sensitivity (100% each for serotypes O and Asia-1, and 92% [95% CI = 81.4-100%] for serotype A positive samples and specificity (100% each for serotypes O, A and Asia-1 positive samples for the viruses currently circulating in West Eurasia compared to the serotyping assays reported earlier. Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for

  10. 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.

  11. 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

  12. 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)

  13. 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

  14. Axial Tomography from Digitized Real Time Radiography

    Science.gov (United States)

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

    1985-01-18

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

  15. Replacing OSE with Real Time capable Linux

    OpenAIRE

    Boman, Simon; Rutgersson, Olof

    2009-01-01

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

  16. SignalR real time application development

    CERN Document Server

    Ingebrigtsen, Einar

    2013-01-01

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

  17. 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.

  18. Mobile waste inspection real time radiography system

    International Nuclear Information System (INIS)

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

    1995-01-01

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

  19. 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.

  20. Real-Time Imaging System for the OpenPET

    Science.gov (United States)

    Tashima, Hideaki; Yoshida, Eiji; Kinouchi, Shoko; Nishikido, Fumihiko; Inadama, Naoko; Murayama, Hideo; Suga, Mikio; Haneishi, Hideaki; Yamaya, Taiga

    2012-02-01

    The OpenPET and its real-time imaging capability have great potential for real-time tumor tracking in medical procedures such as biopsy and radiation therapy. For the real-time imaging system, we intend to use the one-pass list-mode dynamic row-action maximum likelihood algorithm (DRAMA) and implement it using general-purpose computing on graphics processing units (GPGPU) techniques. However, it is difficult to make consistent reconstructions in real-time because the amount of list-mode data acquired in PET scans may be large depending on the level of radioactivity, and the reconstruction speed depends on the amount of the list-mode data. In this study, we developed a system to control the data used in the reconstruction step while retaining quantitative performance. In the proposed system, the data transfer control system limits the event counts to be used in the reconstruction step according to the reconstruction speed, and the reconstructed images are properly intensified by using the ratio of the used counts to the total counts. We implemented the system on a small OpenPET prototype system and evaluated the performance in terms of the real-time tracking ability by displaying reconstructed images in which the intensity was compensated. The intensity of the displayed images correlated properly with the original count rate and a frame rate of 2 frames per second was achieved with average delay time of 2.1 s.

  1. True Time API Link (real time arrival info)

    Data.gov (United States)

    Allegheny County / City of Pittsburgh / Western PA Regional Data Center — This link will take you to the site where you can create an account to access Port Authority's real time arrival information. To request access to Port Authority's...

  2. Real Estate Financing and Interest Rate Hedging : A quantitative real estate investment case study

    OpenAIRE

    van de Wiel, Wimjan; Kristopher Bock, Felix

    2017-01-01

    Background: The expansive monetary policy of the European Central Bank has been leading to all-time-low interest rates and to a strong move into real estate investment. Low interest rates can work in favor of the investor (due to low interest rate expenditures), but increasing interest rates can jeopardize real estate investments. Since changes in interest rates are unpredictable, an investor needs to deal with this volatility. The capital market offers several financial instruments (so-calle...

  3. Improving clinical laboratory efficiency: a time-motion evaluation of the Abbott m2000 RealTime and Roche COBAS AmpliPrep/COBAS TaqMan PCR systems for the simultaneous quantitation of HIV-1 RNA and HCV RNA.

    Science.gov (United States)

    Amendola, Alessandra; Coen, Sabrina; Belladonna, Stefano; Pulvirenti, F Renato; Clemens, John M; Capobianchi, M Rosaria

    2011-08-01

    Diagnostic laboratories need automation that facilitates efficient processing and workflow management to meet today's challenges for expanding services and reducing cost, yet maintaining the highest levels of quality. Processing efficiency of two commercially available automated systems for quantifying HIV-1 and HCV RNA, Abbott m2000 system and Roche COBAS Ampliprep/COBAS TaqMan 96 (docked) systems (CAP/CTM), was evaluated in a mid/high throughput workflow laboratory using a representative daily workload of 24 HCV and 72 HIV samples. Three test scenarios were evaluated: A) one run with four batches on the CAP/CTM system, B) two runs on the Abbott m2000 and C) one run using the Abbott m2000 maxCycle feature (maxCycle) for co-processing these assays. Cycle times for processing, throughput and hands-on time were evaluated. Overall processing cycle time was 10.3, 9.1 and 7.6 h for Scenarios A), B) and C), respectively. Total hands-on time for each scenario was, in order, 100.0 (A), 90.3 (B) and 61.4 min (C). The interface of an automated analyzer to the laboratory workflow, notably system set up for samples and reagents and clean up functions, are as important as the automation capability of the analyzer for the overall impact to processing efficiency and operator hands-on time.

  4. Real-Time Video Stylization Using Object Flows.

    Science.gov (United States)

    Lu, Cewu; Xiao, Yao; Tang, Chi-Keung

    2017-05-05

    We present a real-time video stylization system and demonstrate a variety of painterly styles rendered on real video inputs. The key technical contribution lies on the object flow, which is robust to inaccurate optical flow, unknown object transformation and partial occlusion as well. Since object flows relate regions of the same object across frames, shower-door effect can be effectively reduced where painterly strokes and textures are rendered on video objects. The construction of object flows is performed in real time and automatically after applying metric learning. To reduce temporal flickering, we extend the bilateral filtering into motion bilateral filtering. We propose quantitative metrics to measure the temporal coherence on structures and textures of our stylized videos, and perform extensive experiments to compare our stylized results with baseline systems and prior works specializing in watercolor and abstraction.

  5. Compiling models into real-time systems

    International Nuclear Information System (INIS)

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

    1992-08-01

    This paper presents an architecture for building real-time systems from models, and model-compiling techniques. This has been applied for building a real-time model-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

  6. Real-Time Operating System/360

    Science.gov (United States)

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

    1969-01-01

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

  7. Advances in Real-Time Systems

    CERN Document Server

    Chakraborty, Samarjit

    2012-01-01

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

  8. Collecting data in real time with postcards

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  9. Studying Complex Interactions in Real Time

    DEFF Research Database (Denmark)

    Mønster, Dan

    2017-01-01

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

  10. Interactive Real-time Magnetic Resonance Imaging

    DEFF Research Database (Denmark)

    Brix, Lau

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

  11. Real-time PCR gene expression profiling

    Czech Academy of Sciences Publication Activity Database

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

    2007-01-01

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

  12. 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.

  13. Real-time systems design and analysis

    CERN Document Server

    Laplante, Phillip A

    2004-01-01

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

  14. Temporal logics and real time expert systems

    NARCIS (Netherlands)

    Blom, J.A.

    1996-01-01

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