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Sample records for absolute quantitative real-time

  1. A BAYESIAN METHOD FOR CALCULATING REAL-TIME QUANTITATIVE PCR CALIBRATION CURVES USING ABSOLUTE PLASMID DNA STANDARDS

    Science.gov (United States)

    In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignore...

  2. 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

  3. Quantitative Microplate Assay for Real-Time Nuclease Kinetics

    OpenAIRE

    Eriksson, Jonas; Langel, Ülo

    2016-01-01

    Utilizing the phenomenon of nucleases exposing oligonucleotide phosphate backbones to phosphatases we present a novel quantitative method for kinetics of nuclease catalysis. Inorganic phosphate released from nuclease products by phosphatases could be quantified in real-time by a fluorescent sensor of inorganic phosphate. Two different nucleases were employed, showing the versatility of this assay for multiple turnover label-free nuclease studies.

  4. A novel procedure for absolute real-time quantification of gene expression patterns

    Directory of Open Access Journals (Sweden)

    Lu Yingqing

    2012-03-01

    Full Text Available Abstract Background Temporal and tissue-specific patterns of gene expression play important roles in functionality of a biological system. Real-time quantitative polymerase chain reaction (qPCR technique has been widely applied to single gene expressions, but its potential has not been fully released as most results have been obtained as fold changes relative to control conditions. Absolute quantification of transcripts as an alternative method has yet to gain popularity because of unresolved issues. Results We propose a solution here with a novel procedure, which may accurately quantify the total cDNA conventionally prepared from a biological sample at the resolution of ~70 pg/μl, and reliably estimate the absolute numbers of transcripts in a picogram of cDNA. In comparison to the relative quantification, cDNA-based absolute (CBA qPCR method is found to be more sensitive to gene expression variations caused by factors such as developmental and environmental variations. If the number of target transcript copies is further normalized by reference transcripts, cell-level variation pattern of the target gene expression may also be detectable during a developmental process, as observed here in cases across species (Ipomoea purpurea, Nicotiana benthamiana and tissues (petals and leaves. Conclusion By allowing direct comparisons of results across experiments, the new procedure opens a window to make inferences of gene expression patterns across a broad spectrum of living systems and tissues. Such comparisons are urgently needed for biological interpretations of gene expression variations in diverse cells.

  5. Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay

    OpenAIRE

    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 qu...

  6. Real time blood testing using quantitative phase imaging.

    Science.gov (United States)

    Pham, Hoa V; Bhaduri, Basanta; Tangella, Krishnarao; Best-Popescu, Catherine; Popescu, Gabriel

    2013-01-01

    We demonstrate a real-time blood testing system that can provide remote diagnosis with minimal human intervention in economically challenged areas. Our instrument combines novel advances in label-free optical imaging with parallel computing. Specifically, we use quantitative phase imaging for extracting red blood cell morphology with nanoscale sensitivity and NVIDIA's CUDA programming language to perform real time cellular-level analysis. While the blood smear is translated through focus, our system is able to segment and analyze all the cells in the one megapixel field of view, at a rate of 40 frames/s. The variety of diagnostic parameters measured from each cell (e.g., surface area, sphericity, and minimum cylindrical diameter) are currently not available with current state of the art clinical instruments. In addition, we show that our instrument correctly recovers the red blood cell volume distribution, as evidenced by the excellent agreement with the cell counter results obtained on normal patients and those with microcytic and macrocytic anemia. The final data outputted by our instrument represent arrays of numbers associated with these morphological parameters and not images. Thus, the memory necessary to store these data is of the order of kilobytes, which allows for their remote transmission via, for example, the cellular network. We envision that such a system will dramatically increase access for blood testing and furthermore, may pave the way to digital hematology. PMID:23405194

  7. Quantitative Real-Time PCR Analysis of Gene Transcripts of Mosquito Follicles.

    Science.gov (United States)

    Telang, Aparna

    2016-01-01

    Real-time (quantitative) PCR, or QPCR, has become an indispensible tool for characterizing gene expression. Depending on the experimental design, researchers can use either the relative or absolute (standard curve) method to quantify transcript abundance. Characterizing the expression of genes in mosquito ovaries will require use of the standard curve method of quantification. Here, I describe reagents and equipment necessary to run standard curve QPCR. I also provide details on the construction of the standard linear curve and calculations required to determine transcript abundance. PMID:27557577

  8. 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

  9. Novel bioluminescent quantitative detection of nucleic acid amplification in real-time.

    Directory of Open Access Journals (Sweden)

    Olga A Gandelman

    Full Text Available BACKGROUND: The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. PRINCIPAL FINDINGS: Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. CONCLUSIONS: The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a

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

    DEFF Research Database (Denmark)

    Kumala, Lars; Hentschel, Ute; Bayer, Kristina

    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...... aerophoba. Absolute quantification of archaeal amoA-genes provided evidence of highly abundant ammonia-oxidizing archaea (AOA) in both HMA and LMA sponge species. Interestingly, detected amoA-transcripts indicated the activity of AOA only in the HMA representative. The variable abundance of AOA in chimneys......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...

  11. Quantitative Detection of Respiratory Chlamydia pneumoniae Infection by Real-Time PCR

    OpenAIRE

    Kuoppa, Yvonne; Boman, Jens; Scott, Lena; Kumlin, Urban; Eriksson, Iréne; Allard, Annika

    2002-01-01

    Real-time PCR was evaluated as a quantitative diagnostic method for Chlamydia pneumoniae infection using different respiratory samples. Real-time PCR had efficiency equal to or better than that of nested touchdown PCR. This study confirmed sputum as the best sampling material to detect an ongoing C. pneumoniae infection.

  12. Quantitative Analysis of Copy Number Variants Based on Real-Time LightCycler PCR

    OpenAIRE

    Ma, Lijiang; Chung, Wendy K.

    2014-01-01

    Quantitative real-time PCR is PCR visualized in real time by the use of fluorescent or intercalating dyes used to measure gene expression or gene quantification including including contiguous gene deletions or duplications. A simple method is described to quantify DNA copy number from human samples.

  13. Real Time Quantitative 3-D Imaging of Diffusion Flame Species

    Science.gov (United States)

    Kane, Daniel J.; Silver, Joel A.

    1997-01-01

    A low-gravity environment, in space or ground-based facilities such as drop towers, provides a unique setting for study of combustion mechanisms. Understanding the physical phenomena controlling the ignition and spread of flames in microgravity has importance for space safety as well as better characterization of dynamical and chemical combustion processes which are normally masked by buoyancy and other gravity-related effects. Even the use of so-called 'limiting cases' or the construction of 1-D or 2-D models and experiments fail to make the analysis of combustion simultaneously simple and accurate. Ideally, to bridge the gap between chemistry and fluid mechanics in microgravity combustion, species concentrations and temperature profiles are needed throughout the flame. However, restrictions associated with performing measurements in reduced gravity, especially size and weight considerations, have generally limited microgravity combustion studies to the capture of flame emissions on film or video laser Schlieren imaging and (intrusive) temperature measurements using thermocouples. Given the development of detailed theoretical models, more sophisticated studies are needed to provide the kind of quantitative data necessary to characterize the properties of microgravity combustion processes as well as provide accurate feedback to improve the predictive capabilities of the computational models. While there have been a myriad of fluid mechanical visualization studies in microgravity combustion, little experimental work has been completed to obtain reactant and product concentrations within a microgravity flame. This is largely due to the fact that traditional sampling methods (quenching microprobes using GC and/or mass spec analysis) are too heavy, slow, and cumbersome for microgravity experiments. Non-intrusive optical spectroscopic techniques have - up until now - also required excessively bulky, power hungry equipment. However, with the advent of near-IR diode

  14. Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process

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    Kohn Michael

    2007-10-01

    Full Text Available Abstract Background Since real time PCR was first developed, several approaches to estimating the initial quantity of template in an RT-PCR reaction have been tried. While initially only the early thermal cycles corresponding to exponential duplication were used, lately there has been an effort to use all of the cycles in a PCR. The efforts have included both fitting empirical sigmoid curves and more elaborate mechanistic models that explore the chemical reactions taking place during each cycle. The more elaborate mechanistic models require many more parameters than can be fit from a single amplification, while the empirical models provide little insight and are difficult to tailor to specific reactants. Results We directly estimate the initial amount of amplicon using a simplified mechanistic model based on chemical reactions in the annealing step of the PCR. The basic model includes the duplication of DNA with the digestion of Taqman probe and the re-annealing between previously synthesized DNA strands of opposite orientation. By modelling the amount of Taqman probe digested and matching that with the observed fluorescence, the conversion factor between the number of fluorescing dye molecules and observed fluorescent emission can be estimated, along with the absolute initial amount of amplicon and the rate parameter for re-annealing. The model is applied to several PCR reactions with known amounts of amplicon and is shown to work reasonably well. An expanded version of the model allows duplication of amplicon without release of fluorescent dye, by adding 1 more parameter to the model. The additional process is helpful in most cases where the initial primer concentration exceeds the initial probe concentration. Software for applying the algorithm to data may be downloaded at http://www.niehs.nih.gov/research/resources/software/pcranalyzer/ Conclusion We present proof of the principle that a mechanistically based model can be fit to observations

  15. Real-Time Bidirectional Pyrophosphorolysis-Activated Polymerization for Quantitative Detection of Somatic Mutations

    OpenAIRE

    Song, Najie; Zhong, Xueting; Li, Qingge

    2014-01-01

    Detection of somatic mutations for targeted therapy is increasingly used in clinical settings. However, due to the difficulties of detecting rare mutations in excess of wild-type DNA, current methods often lack high sensitivity, require multiple procedural steps, or fail to be quantitative. We developed real-time bidirectional pyrophosphorolysis-activated polymerization (real-time Bi-PAP) that allows quantitative detection of somatic mutations. We applied the method to quantify seven mutation...

  16. Selection and Validation of Reference Genes for Quantitative Real-time PCR in Gentiana macrophylla

    Science.gov (United States)

    He, Yihan; Yan, Hailing; Hua, Wenping; Huang, Yaya; Wang, Zhezhi

    2016-01-01

    Real time quantitative PCR (RT-qPCR or qPCR) has been extensively applied for analyzing gene expression because of its accuracy, sensitivity, and high throughput. However, the unsuitable choice of reference gene(s) can lead to a misinterpretation of results. We evaluated the stability of 10 candidates – five traditional housekeeping genes (UBC21, GAPC2, EF-1α4, UBQ10, and UBC10) and five novel genes (SAND1, FBOX, PTB1, ARP, and Expressed1) – using the transcriptome data of Gentiana macrophylla. Common statistical algorithms ΔCt, GeNorm, NormFinder, and BestKeeper were run with samples collected from plants under various experimental conditions. For normalizing expression levels from tissues at different developmental stages, GAPC2 and UBC21 had the highest rankings. Both SAND1 and GAPC2 proved to be the optimal reference genes for roots from plants exposed to abiotic stresses while EF-1α4 and SAND1 were optimal when examining expression data from the leaves of stressed plants. Based on a comprehensive ranking of stability under different experimental conditions, we recommend that SAND1 and EF-1α4 are the most suitable overall. In this study, to find a suitable reference gene and its real-time PCR assay for G. macrophylla DNA content quantification, we evaluated three target genes including WRKY30, G10H, and SLS, through qualitative and absolute quantitative PCR with leaves under elicitors stressed experimental conditions. Arbitrary use of reference genes without previous evaluation can lead to a misinterpretation of the data. Our results will benefit future research on the expression of genes related to secoiridoid biosynthesis in this species under different experimental conditions. PMID:27446172

  17. Standardization of Gene Expression Quantification by Absolute Real-Time qRT-PCR System Using a Single Standard for Marker and Reference Genes

    Directory of Open Access Journals (Sweden)

    Yi-Hong Zhou

    2010-08-01

    Full Text Available In the last decade, genome-wide gene expression data has been collected from a large number of cancer specimens. In many studies utilizing either microarray-based or knowledge-based gene expression profiling, both the validation of candidate genes and the identification and inclusion of biomarkers in prognosis-modeling has employed real-time quantitative PCR on reverse transcribed mRNA (qRT-PCR because of its inherent sensitivity and quantitative nature. In qRT-PCR data analysis, an internal reference gene is used to normalize the variation in input sample quantity. The relative quantification method used in current real-time qRT-PCR analysis fails to ensure data comparability pivotal in identification of prognostic biomarkers. By employing an absolute qRT-PCR system that uses a single standard for marker and reference genes (SSMR to achieve absolute quantification, we showed that the normalized gene expression data is comparable and independent of variations in the quantities of sample as well as the standard used for generating standard curves. We compared two sets of normalized gene expression data with same histological diagnosis of brain tumor from two labs using relative and absolute real-time qRT-PCR. Base-10 logarithms of the gene expression ratio relative to ACTB were evaluated for statistical equivalence between tumors processed by two different labs. The results showed an approximate comparability for normalized gene expression quantified using a SSMR-based qRT-PCR. Incomparable results were seen for the gene expression data using relative real-time qRT-PCR, due to inequality in molar concentration of two standards for marker and reference genes. Overall results show that SSMR-based real-time qRT-PCR ensures comparability of gene expression data much needed in establishment of prognostic/predictive models for cancer patients—a process that requires large sample sizes by combining independent sets of data.

  18. New Real-Time Quantitative PCR Procedure for Quantification of Bifidobacteria in Human Fecal Samples

    OpenAIRE

    Gueimonde, Miguel; Tölkkö, Satu; Korpimäki, Teemu; Salminen, Seppo

    2004-01-01

    The application of a real-time quantitative PCR method (5′ nuclease assay), based on the use of a probe labeled at its 5′ end with a stable, fluorescent lanthanide chelate, for the quantification of human fecal bifidobacteria was evaluated. The specificities of the primers and the primer-probe combination were evaluated by conventional PCR and real-time PCR, respectively. The results obtained by real-time PCR were compared with those obtained by fluorescent in situ hybridization, the current ...

  19. Comparison of Droplet Digital PCR to Real-Time PCR for Quantitative Detection of Cytomegalovirus

    OpenAIRE

    Hayden, R. T.; Gu, Z; Ingersoll, J; Abdul-Ali, D.; Shi, L; Pounds, S.; Caliendo, A. M.

    2013-01-01

    Quantitative real-time PCR (QRT-PCR) has been widely implemented for clinical viral load testing, but a lack of standardization and relatively poor precision have hindered its usefulness. Digital PCR offers highly precise, direct quantification without requiring a calibration curve. Performance characteristics of real-time PCR were compared to those of droplet digital PCR (ddPCR) for cytomegalovirus (CMV) load testing. Tenfold serial dilutions of the World Health Organization (WHO) and the Na...

  20. Different expression of house-keeping genes by quantitative real time RT-PCR

    Czech Academy of Sciences Publication Activity Database

    Ullmannová, V.; Truksa, Jaroslav; Haškovec, C.; Kovář, Jan

    CR: XXX , 2003, s. 1. [Functional genomics and diseases. Praha (CZ), 05.05.2003-07.05.2003] Institutional research plan: CEZ:AV0Z5052915 Keywords : quantitative real time RT-PCR * house-keeping genes expression Subject RIV: EB - Genetics ; Molecular Biology

  1. Evaluation of Quantitative Real-Time PCR Assays for Detection of Citrus Greening

    Science.gov (United States)

    Citrus huanglongbing (HLB), or citrus greening, is a serious and industry-limiting disease. Preliminary diagnoses can be made through visual symptoms, and greater certainty can be achieved through quantitative real-time PCR (qPCR). Several qPCR procedures are available including those by designed by...

  2. 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...

  3. Rapid Staphylococcus aureus agr Type Determination by a Novel Multiplex Real-Time Quantitative PCR Assay

    OpenAIRE

    Francois, Patrice; Koessler, Thibaud; Huyghe, Antoine; Harbarth, Stephan; Bento, Manuela; Lew, Daniel; Etienne, Jérôme; Pittet, Didier; Schrenzel, Jacques

    2006-01-01

    The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information.

  4. Rapid Staphylococcus aureus agr type determination by a novel multiplex real-time quantitative PCR assay.

    Science.gov (United States)

    Francois, Patrice; Koessler, Thibaud; Huyghe, Antoine; Harbarth, Stephan; Bento, Manuela; Lew, Daniel; Etienne, Jérôme; Pittet, Didier; Schrenzel, Jacques

    2006-05-01

    The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information. PMID:16672433

  5. Rapid Staphylococcus aureus agr Type Determination by a Novel Multiplex Real-Time Quantitative PCR Assay

    Science.gov (United States)

    Francois, Patrice; Koessler, Thibaud; Huyghe, Antoine; Harbarth, Stephan; Bento, Manuela; Lew, Daniel; Etienne, Jérôme; Pittet, Didier; Schrenzel, Jacques

    2006-01-01

    The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information. PMID:16672433

  6. 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...

  7. Improved quantitative real-time RT–PCR for expression profiling of individual cells

    OpenAIRE

    Liss, Birgit

    2002-01-01

    The real-time quantitative polymerase chain reaction (rtqPCR) has overcome the limitations of conventional, time-consuming quantitative PCR strategies and is maturing into a routine tool to quantify gene expression levels, following reverse transcription (RT) of mRNA into complementary DNA (cDNA). Expression profiling with single-cell resolution is highly desirable, in particular for complex tissues like the brain that contain a large variety of different cell types in close proximity. The pa...

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

    OpenAIRE

    Ana Lisa do Vale Gomes; Fábio L Melo; Roberto P Werkhauser; Frederico GC Abath

    2006-01-01

    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). Th...

  9. Quantitative assay of photoinduced DNA strand breaks by real-time PCR.

    Science.gov (United States)

    Wiczk, Justyna; Westphal, Kinga; Rak, Janusz

    2016-09-01

    Real-time PCR (qPCR) - a modern methodology primarily used for studying gene expression has been employed for the quantitative assay of an important class of DNA damage - single strand breaks. These DNA lesions which may lead to highly cytotoxic double strand breaks were quantified in a model system where double stranded DNA was sensitized to UV photons by labeling with 5-bromo-2'-deoxyuridine. The amount of breaks formed due to irradiation with several doses of 320nm photons was assayed by two independent methods: LC-MS and qPCR. A very good agreement between the relative damage measured by the two completely different analytical tools proves the applicability of qPCR for the quantitative analysis of SSBs. Our results suggest that the popularity of the hitherto underestimated though accurate and site-specific technique of real-time PCR may increase in future DNA damage studies. PMID:27371921

  10. Multiplex, Quantitative, Real-Time PCR Assay for Cytomegalovirus and Human DNA

    OpenAIRE

    Sanchez, Jason L.; Storch, Gregory A.

    2002-01-01

    We created a multiplex, quantitative, real-time PCR assay that amplifies cytomegalovirus (CMV) and human DNA in the same reaction tube, allowing for a viral load determination that is normalized to measured human DNA. The assay targets a conserved region of the CMV DNA polymerase gene that is not affected by known drug resistance mutations. All 36 strains of CMV detected by culture or qualitative PCR in a population of lung transplant recipients were detected. The assay detected 1 to 10 copie...

  11. Calibrated Real-Time PCR Assay for Quantitation of Human Herpesvirus 8 DNA in Biological Fluids

    OpenAIRE

    Broccolo, Francesco; Locatelli, Giuseppe; Sarmati, Loredana; Piergiovanni, Sara; Veglia, Fabrizio; Andreoni, Massimo; Buttò, Stefano; Ensoli, Barbara; Lusso, Paolo; Malnati, Mauro S.

    2002-01-01

    Accurate laboratory tests for the diagnosis of active human herpesvirus 8 (HHV-8) infection are becoming essential to study the pathogenesis of HHV-8-associated tumors and for the clinical management of HHV-8-infected individuals. We have developed a highly sensitive, calibrated quantitative real-time PCR assay for the measurement of cell-free HHV-8 DNA in body fluids, based on the addition of a synthetic DNA calibrator prior to DNA extraction. The calibrator controls each sample for the pres...

  12. 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.

  13. Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods.

    Science.gov (United States)

    Taverniers, Isabel; Van Bockstaele, Erik; De Loose, Marc

    2004-03-01

    Analytical real-time PCR technology is a powerful tool for implementation of the GMO labeling regulations enforced in the EU. The quality of analytical measurement data obtained by quantitative real-time PCR depends on the correct use of calibrator and reference materials (RMs). For GMO methods of analysis, the choice of appropriate RMs is currently under debate. So far, genomic DNA solutions from certified reference materials (CRMs) are most often used as calibrators for GMO quantification by means of real-time PCR. However, due to some intrinsic features of these CRMs, errors may be expected in the estimations of DNA sequence quantities. In this paper, two new real-time PCR methods are presented for Roundup Ready soybean, in which two types of plasmid DNA fragments are used as calibrators. Single-target plasmids (STPs) diluted in a background of genomic DNA were used in the first method. Multiple-target plasmids (MTPs) containing both sequences in one molecule were used as calibrators for the second method. Both methods simultaneously detect a promoter 35S sequence as GMO-specific target and a lectin gene sequence as endogenous reference target in a duplex PCR. For the estimation of relative GMO percentages both "delta C(T)" and "standard curve" approaches are tested. Delta C(T) methods are based on direct comparison of measured C(T) values of both the GMO-specific target and the endogenous target. Standard curve methods measure absolute amounts of target copies or haploid genome equivalents. A duplex delta C(T) method with STP calibrators performed at least as well as a similar method with genomic DNA calibrators from commercial CRMs. Besides this, high quality results were obtained with a standard curve method using MTP calibrators. This paper demonstrates that plasmid DNA molecules containing either one or multiple target sequences form perfect alternative calibrators for GMO quantification and are especially suitable for duplex PCR reactions. PMID:14689155

  14. 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.

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

    OpenAIRE

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

    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...

  16. Off-axis quantitative phase imaging processing using CUDA: toward real-time applications

    OpenAIRE

    Pham, Hoa; Ding, Huafeng; Sobh, Nahil; Do, Minh; Patel, Sanjay; Popescu, Gabriel

    2011-01-01

    We demonstrate real time off-axis Quantitative Phase Imaging (QPI) using a phase reconstruction algorithm based on NVIDIA’s CUDA programming model. The phase unwrapping component is based on Goldstein’s algorithm. By mapping the process of extracting phase information and unwrapping to GPU, we are able to speed up the whole procedure by more than 18.8× with respect to CPU processing and ultimately achieve video rate for mega-pixel images. Our CUDA implementation also supports processing of mu...

  17. The performance of semi-quantitative differential PCR is similar to that of real-time PCR for the detection of the MYCN gene in neuroblastomas

    OpenAIRE

    A.C.M.F. Souza; D.R.V. Souza; S.S. Sanabani; Giorgi, R.R.; Bendit, I

    2009-01-01

    Amplification of the MYCN gene in neuroblastomas is a potent biological marker of highly aggressive tumors, which are invariably fatal unless sound clinical management is applied. To determine the usefulness of semi-quantitative differential PCR (SQ-PCR) for accurate quantification of MYCN gene copy number, we evaluated the analytical performance of this method by comparing the results obtained with it for 101 tumor samples of neuroblastoma to that obtained by absolute and relative real-time ...

  18. International Ring Trial for the Validation of an Event-Specific Golden Rice 2 Quantitative Real-Time Polymerase Chain Reaction Method

    OpenAIRE

    JACCHIA SARA; NARDINI ELENA; BASSANI NICCOLO; SAVINI Cristian; SHIM Jung-Hyun; TRIJATMIKO Kurniawan; KREYSA JOACHIM; Mazzara, Marco

    2014-01-01

    This article describes the international validation of the quantitative real-time polymerase chain reaction (PCR) detection method for Golden Rice 2. The method consists of a taxon-specific assay amplifying a fragment of rice Phospholipase D α2 gene, and an event-specific assay designed on the 3′ junction between transgenic insert and plant DNA. We validated the two assays independently, with absolute quantification, and in combination, with relative quantification, on DNA samples prepared in...

  19. 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.

  20. Systematic Comparison and Validation of Quantitative Real-Time PCR Methods for the Quantitation of Adeno-Associated Viral Products

    OpenAIRE

    Werling, Natalie Jayne; Satkunanathan, Stifani; Thorpe, Robin; Zhao, Yuan

    2015-01-01

    Abstract Adeno-associated viral (AAV) vectors show great promise for gene therapy because of their excellent safety profile; however, development of robust dose-determining assays for AAV has presented a significant challenge. With the ultimate goal of future harmonization and standardization of AAV dose determination assays, we systematically analyzed the influence of key variables, including sample preparation procedure, the choice of primers, and real-time quantitative PCR (qPCR) target se...

  1. Quantitative genotyping of mouse brain-specific PEX13 gene disruption by real-time PCR.

    Science.gov (United States)

    Müller, C Catharina; Nourse, Jamie P; Nguyen, Tam H; Crane, Denis I

    2009-06-30

    The Cre/loxP-system has become an invaluable tool for the generation of tissue-specific gene disruption in mice. However, because Cre recombinase excision of individual genes can be variable, an accurate and sensitive method is necessary to determine the ultimate level of gene disruption. The analysis of gene disruption is particularly difficult for tissue that has been fixed for (immuno)histochemical analysis with paraformaldehyde. Here, we describe a simple, rapid and cost effective method for measurement of gene disruption using quantitative real-time PCR, through application to the analysis of PEX13 gene disruption in a brain-specific PEX13 mouse mutant. We show that this general protocol is suitable for both normal and paraformaldehyde-fixed tissue. PMID:19422853

  2. Evaluation of residual antibacterial potency in antibiotic production wastewater using a real-time quantitative method.

    Science.gov (United States)

    Zhang, Hong; Zhang, Yu; Yang, Min; Liu, Miaomiao

    2015-11-01

    While antibiotic pollution has attracted considerable attention due to its potential in promoting the dissemination of antibiotic resistance genes in the environment, the antibiotic activity of their related substances has been neglected, which may underestimate the environmental impacts of antibiotic wastewater discharge. In this study, a real-time quantitative approach was established to evaluate the residual antibacterial potency of antibiotics and related substances in antibiotic production wastewater (APW) by comparing the growth of a standard bacterial strain (Staphylococcus aureus) in tested water samples with a standard reference substance (e.g. oxytetracycline). Antibiotic equivalent quantity (EQ) was used to express antibacterial potency, which made it possible to assess the contribution of each compound to the antibiotic activity in APW. The real-time quantitative method showed better repeatability (Relative Standard Deviation, RSD 1.08%) compared with the conventional fixed growth time method (RSD 5.62-11.29%). And its quantification limits ranged from 0.20 to 24.00 μg L(-1), depending on the antibiotic. We applied the developed method to analyze the residual potency of water samples from four APW treatment systems, and confirmed a significant contribution from antibiotic transformation products to potent antibacterial activity. Specifically, neospiramycin, a major transformation product of spiramycin, was found to contribute 13.15-22.89% of residual potency in spiramycin production wastewater. In addition, some unknown related substances with antimicrobial activity were indicated in the effluent. This developed approach will be effective for the management of antibacterial potency discharge from antibiotic wastewater and other waste streams. PMID:26395288

  3. Gene expression analysis by quantitative real-time PCR for floral tissues.

    Science.gov (United States)

    Bustamante, Mariana; Jin, Jian; Casagran, Oriol; Nolan, Tania; Riechmann, José Luis

    2014-01-01

    Real-time, or quantitative, reverse transcription polymerase chain reaction (qRT-PCR), is a powerful method for rapid and reliable quantification of mRNA abundance. Although it has not featured prominently in flower development research in the past, the availability of novel techniques for the synchronized induction of flower development, or for the isolation of cell-specific mRNA populations, suggests that detailed quantitative analyses of gene expression over time and in specific tissues and cell types by qRT-PCR will become more widely used. In this chapter, we discuss specific considerations for studying gene expression by using qRT-PCR, such as the identification of suitable reference genes for the experimental setup used. In addition, we provide protocols for performing qRT-PCR experiments in a multiwell plate format (with the LightCycler(®) 480 system, Roche) and with nanofluidic arrays (BioMark™ system, Fluidigm), which allow the automatic combination of sets of samples with sets of assays, and significantly reduce reaction volume and the number of liquid-handling steps performed during the experiment. PMID:24395270

  4. Real-time quantitative nicking endonuclease-mediated isothermal amplification with small molecular beacons.

    Science.gov (United States)

    Xu, Wentao; Wang, Chenguang; Zhu, Pengyu; Guo, Tianxiao; Xu, Yuancong; Huang, Kunlun; Luo, Yunbo

    2016-04-21

    Techniques of isothermal amplification have recently made great strides, and have generated significant interest in the field of point-of-care detection. Nicking endonuclease-mediated isothermal amplification (NEMA) is an example of simple isothermal technology. In this paper, a real-time quantitative nicking endonuclease-mediated isothermal amplification with small molecular beacons (SMB-NEMA) of improved specificity and sensitivity is described. First, we optimized the prohibition of de novo synthesis by choosing Nt·BstNBI endonuclease. Second, the whole genome was successfully amplified with Nt·BstNBI (6 U), betaine (1 M) and trehalose (60 mM) for the first time. Third, we achieved 10 pg sensitivity for the first time after adding a small molecular beacon that spontaneously undergoes a conformational change when hybridizing to target, and the practical test validated the assay's application. The small molecular beacon has a similar melting temperature to the reaction temperature, but is approximately 10 bp shorter than the length of a traditional molecular beacon. A new threshold regulation was also established for isothermal conditions. Finally, we established a thermodynamic model for designing small molecular beacons. This multistate model is more correct than the traditional algorithm. This theoretical and practical basis will help us to monitor SMB-NEMA in a quantitative way. In summary, our SMB-NEMA method allows the simple, specific and sensitive assessment of isothermal DNA quantification. PMID:27027375

  5. The quantitative real-time PCR applications in the monitoring of marine harmful algal bloom (HAB) species

    OpenAIRE

    Antonella, Penna; Luca, Galluzzi

    2012-01-01

    In the last decade, various molecular methods (e.g., fluorescent hybridization assay, sandwich hybridization assay, automatized biosensor detection, real-time PCR assay) have been developed and implemented for accurate and specific identification and estimation of marine toxic microalgal species. This review focuses on the recent quantitative real-time PCR (qrt-PCR) technology developed for the control and monitoring of the most important taxonomic phytoplankton groups producing biotoxins wit...

  6. Development of real time PCR for detection and quantitation of Dengue Viruses

    Directory of Open Access Journals (Sweden)

    Singh A

    2009-01-01

    Full Text Available Abstract Background Dengue virus (DENV, a mosquito borne flavivirus is an important pathogen causing more than 50 million infections every year around the world. Dengue diagnosis depends on serology, which is not useful in the early phase of the disease and virus isolation, which is laborious and time consuming. There is need for a rapid, sensitive and high throughput method for detection of DENV in the early stages of the disease. Several real-time PCR assays have been described for dengue viruses, but there is scope for improvement. The new generation TaqMan Minor Groove Binding (MGB probe approach was used to develop an improved real time RT-PCR (qRT-PCR for DENV in this study. Results The 3'UTR of thirteen Indian strains of DENV was sequenced and aligned with 41 representative sequences from GenBank. A region conserved in all four serotypes was used to target primers and probes for the qRT-PCR. A single MGB probe and a single primer pair for all the four serotypes of DENV were designed. The sensitivity of the two step qRT-PCR assay was10 copies of RNA molecules per reaction. The specificity and sensitivity of the assay was 100% when tested with a panel of 39 known positive and negative samples. Viral RNA could be detected and quantitated in infected mouse brain, cell cultures, mosquitoes and clinical samples. Viral RNA could be detected in patients even after seroconversion till 10 days post onset of infection. There was no signal with Japanese Encephalitis (JE, West Nile (WN, Chikungunya (CHK viruses or with Leptospira, Plasmodium vivax, Plasmodium falciparum and Rickettsia positive clinical samples. Conclusion We have developed a highly sensitive and specific qRT-PCR for detection and quantitation of dengue viruses. The assay will be a useful tool for differential diagnosis of dengue fever in a situation where a number of other clinically indistinguishable infectious diseases like malaria, Chikungunya, rickettsia and leptospira occur. The

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

    International Nuclear Information System (INIS)

    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.

  8. 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

    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 (qPCR...... 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...... normalization of the qPCR 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...

  9. Identification of five highly priced tuna species by quantitative real-time polymerase chain reaction.

    Science.gov (United States)

    Liu, Shasha; Xu, Kunhua; Wu, Zhigang; Xie, Xiao; Feng, Junli

    2016-09-01

    Tunas are economically important fishery worldwide, and are often used for commercial processed production. For effective fishery management and protection of consumers' rights, it is important to develop a molecular method to identify species in canned tuna products rapidly and reliably. Here, we have developed a duplex quantitative real-time PCR (qPCR) for identification of five highly priced tuna species (Thunnus maccoyii, Thunnus obesus, Thunnus albacares, Thunnus alalunga and Katsuwonus pelamis) from processed as well as fresh fish. After amplification and sequencing of seven genetic markers commonly used for species identification, 16S rDNA and control region (CR) of mitochondrial DNA were selected as the reference gene markers for genus Thunnus and tuna species identification, respectively. Subsequently, a 73 bp fragment of 16S rDNA and 85-99 bp fragment of CR were simultaneously amplified from each target species by qPCR. The qPCR efficiency of each reaction was calculated according to the standard curves, and the method was validated by amplification DNA extracted from single or mixed tuna specimen. The developed duplex qPCR system was applied to authenticate species of 14 commercial tuna products successfully, which demonstrated it was really a useful and academic technique to identify highly priced tuna species. PMID:25714139

  10. 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. PMID:26772407

  11. Development of Quantitative Real-time PCR Assays for Different Clades of “Candidatus Accumulibacter”

    Science.gov (United States)

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-01-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85–112% (R2 = 0.962–0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants. PMID:27142574

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

    DEFF Research Database (Denmark)

    Drag, Markus; Höglund, Johan; Nejsum, Peter;

    PCR at predetermined intervals for up to 336 h. Under aerobic conditions, L1 was observed after 24 h at 25°C, while development to L1 took 336 h at 4°C. A corresponding significant increase of the ITS2 copies was also observed (p < 0.0001). However, anaerobic conditions inhibited embryonation at both......The aims of this study were to assess how the development of Ostertagia ostertagi eggs into first-stage larva (L1) affects the copy numbers of the Internal Transcribed Spacer region 2 (ITS2) of the ribosomal DNA; and based on these results, to suggest optimal storage conditions for faecal samples...... 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...

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

    DEFF Research Database (Denmark)

    Drag, Markus; Höglund, Johan; Nejsum, Peter;

    PCR at predetermined intervals for up to 336 h. Under aerobic conditions, L1 was observed after 24 h at 25°C, while development to L1 took 336 h at 4°C. A corresponding significant increase of the ITS2 copies was also observed (p < 0.0001). However, anaerobic conditions inhibited embryonation at both......The aims of this study were to assess how the development of Ostertagia ostertagi eggs into first-stage larva (L1) affects the copy numbers of the Internal Transcribed Spacer region 2 (ITS2) of the ribosomal DNA; and based on these results, to suggest optimal storage conditions for faecal samples...... 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...

  14. Real-time polymerase chain reaction for diagnosis and quantitation of negative strand of chikungunya virus.

    Science.gov (United States)

    Chiam, Chun Wei; Chan, Yoke Fun; Loong, Shih Keng; Yong, Sara Su Jin; Hooi, Poh Sim; Sam, I-Ching

    2013-10-01

    Quantitative real-time polymerase chain reaction (qRT-PCR) is useful for diagnosis and studying virus replication. We developed positive- and negative-strand qRT-PCR assays to detect nsP3 of chikungunya virus (CHIKV), a positive-strand RNA alphavirus that causes epidemic fever, rash, and arthritis. The positive- and negative-strand qRT-PCR assays had limits of quantification of 1 and 3 log10 RNA copies/reaction, respectively. Compared to a published E1 diagnostic assay using 30 laboratory-confirmed clinical samples, the positive-strand nsP3 qRT-PCR assay had higher R(2) and efficiency and detected more positive samples. Peak viral load of 12.9 log(10) RNA copies/mL was reached on day 2 of illness, and RNA was detectable up to day 9, even in the presence of anti-CHIKV IgM. There was no correlation between viral load and persistent arthralgia. The positive-strand nsP3 assay is suitable for diagnosis, while the negative-strand nsP3 assay, which uses tagged primers to increase specificity, is useful for study of active viral replication kinetics. PMID:23886793

  15. FungiQuant: A broad-coverage fungal quantitative real-time PCR assay

    Directory of Open Access Journals (Sweden)

    Liu Cindy M

    2012-11-01

    Full Text Available Abstract Background Fungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for new broad-coverage techniques. Methods We analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid standards using a qPCR-based approach. We evaluated assay coverage against 4,968 sequences and performed assay validation following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE guidelines. Results We designed FungiQuant, a TaqMan® qPCR assay targeting a 351 bp region in the fungal 18S rRNA gene. Our in silico analysis showed that FungiQuant is a perfect sequence match to 90.0% of the 2,617 fungal species analyzed. We showed that FungiQuant’s is 100% sensitive and its amplification efficiencies ranged from 76.3% to 114.5%, with r2-values of >0.99 against the 69 fungal species tested. Additionally, FungiQuant inter- and intra-run coefficients of variance ranged from Conclusions FungiQuant has comprehensive coverage against diverse fungi and is a robust quantification and detection tool for delineating between true fungal detection and non-target human DNA.

  16. Analysis of gene expression in emerald ash borer (Agrilus planipennis) using quantitative real time-PCR.

    Science.gov (United States)

    Bhandary, Binny; Rajarapu, Swapna Priya; Rivera-Vega, Loren; Mittapalli, Omprakash

    2010-01-01

    Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1(st)-, 2(nd)-, 3(rd)-, 4(th)-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium. PMID:20445495

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

    International Nuclear Information System (INIS)

    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)

  18. Development of Quantitative Real-time PCR Assays for Different Clades of “Candidatus Accumulibacter”

    Science.gov (United States)

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-05-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85–112% (R2 = 0.962–0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants.

  19. Development of Quantitative Real-time PCR Assays for Different Clades of "Candidatus Accumulibacter".

    Science.gov (United States)

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-01-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual "Candidatus Accumulibacter" (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85-112% (R(2) = 0.962-0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants. PMID:27142574

  20. Validation of Zebrafish (Danio rerio) Reference Genes for Quantitative Real-time RT-PCR Normalization

    Institute of Scientific and Technical Information of China (English)

    Rongying TANG; Andrew DODD; Daniel LAI; Warren C.MCNABB; Donald R.LOVE

    2007-01-01

    The normalization of quantitative real time RT-PCR (qRT-PCR) is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or housekeeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions, qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The β-actin, EF1α and Rpl13α genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1α, Rpl13α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.

  1. 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.

  2. Virological Diagnosis of Herpes Simplex Virus 1 Esophagitis by Quantitative Real-Time PCR Assay

    Science.gov (United States)

    Jazeron, Jean-François; Barbe, Coralie; Frobert, Emilie; Renois, Fanny; Talmud, Déborah; Brixi-Benmansour, Hedia; Brodard, Véronique; Andréoletti, Laurent; Diebold, Marie-Danièle

    2012-01-01

    Herpes simplex virus 1 (HSV-1) esophagitis diagnosis is routinely based on the endoscopic findings confirmed by histopathological examination of the esophagitis lesions. Virological diagnosis is not systematically performed and restricted to viral culture or to qualitative PCR assay from esophagitis biopsy specimens. The aim of this study was to assess the interest of quantitative real-time PCR assay in HSV-1 esophagitis diagnosis by comparing the results obtained to those of histological examination associated with immunohistochemical staining, which is considered the “gold standard.” From 53 esophagitis biopsy specimens, the PCR assay detected HSV-1 in 18 of 19 histologically proven to have herpetic esophagitis and in 9 of 34 that had esophagitis related to other causes, demonstrating sensitivity, specificity, positive predictive value, and negative predictive value of 94.7%, 73%, 66.7%, and 96%, respectively. Interestingly, HSV-1 was not detected in 16 specimens without the histological aspect of esophagitis. The viral loads normalized per μg of total extracted DNA in each biopsy specimen detected positive by HSV PCR were then compared and appeared to be significantly higher in histopathologically positive herpetic esophagitis (median = 2.9 × 106 ± 1.1 × 108) than in histopathologically negative herpetic esophagitis (median = 3.1 × 103 ± 6.2 × 103) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 104 copies would allow an HSV-1 esophagitis diagnosis with a sensitivity and specificity of 83.3% and 100%, respectively. In conclusion, this work demonstrated that HSV quantitative PCR results for paraffin-embedded esophageal tissue was well correlated to histopathological findings for an HSV-1 esophagitis diagnosis and could be diagnostic through viral load assessment when histopathological results are missing or uncertain. PMID:22170921

  3. Virological diagnosis of herpes simplex virus 1 esophagitis by quantitative real-time PCR assay.

    Science.gov (United States)

    Jazeron, Jean-François; Barbe, Coralie; Frobert, Emilie; Renois, Fanny; Talmud, Déborah; Brixi-Benmansour, Hedia; Brodard, Véronique; Andréoletti, Laurent; Diebold, Marie-Danièle; Lévêque, Nicolas

    2012-03-01

    Herpes simplex virus 1 (HSV-1) esophagitis diagnosis is routinely based on the endoscopic findings confirmed by histopathological examination of the esophagitis lesions. Virological diagnosis is not systematically performed and restricted to viral culture or to qualitative PCR assay from esophagitis biopsy specimens. The aim of this study was to assess the interest of quantitative real-time PCR assay in HSV-1 esophagitis diagnosis by comparing the results obtained to those of histological examination associated with immunohistochemical staining, which is considered the "gold standard." From 53 esophagitis biopsy specimens, the PCR assay detected HSV-1 in 18 of 19 histologically proven to have herpetic esophagitis and in 9 of 34 that had esophagitis related to other causes, demonstrating sensitivity, specificity, positive predictive value, and negative predictive value of 94.7%, 73%, 66.7%, and 96%, respectively. Interestingly, HSV-1 was not detected in 16 specimens without the histological aspect of esophagitis. The viral loads normalized per μg of total extracted DNA in each biopsy specimen detected positive by HSV PCR were then compared and appeared to be significantly higher in histopathologically positive herpetic esophagitis (median = 2.9 × 10(6) ± 1.1 × 10(8)) than in histopathologically negative herpetic esophagitis (median = 3.1 × 10(3) ± 6.2 × 10(3)) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 10(4) copies would allow an HSV-1 esophagitis diagnosis with a sensitivity and specificity of 83.3% and 100%, respectively. In conclusion, this work demonstrated that HSV quantitative PCR results for paraffin-embedded esophageal tissue was well correlated to histopathological findings for an HSV-1 esophagitis diagnosis and could be diagnostic through viral load assessment when histopathological results are missing or uncertain. PMID:22170921

  4. 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

  5. Validation of Reference Genes for Quantitative Real-Time PCR in Laodelphax striatellus

    Institute of Scientific and Technical Information of China (English)

    HE Xiu-ting; LIU Cheng-cheng; LI Zhao-qun; ZHANG Zan; LI Guo-qing; LI Fei; DONG Shuang-lin

    2014-01-01

    The normalization of quantitative real-time PCR (qPCR) is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can be regulated under different conditions. qPCR has recently been used for gene expression study in Laodelphax striatellus, but there is no study on validation of the reference genes. In this study, ifve new housekeeping genes (LstrTUB1, LstrTUB2, LstrTUB3, LstrARF and LstrRPL9) in L. striatellus were cloned and deposited in the GenBank with accession numbers of JF728809, JF728810, JF728811, JF728807 and JF728806, respectively. Furthermore, mRNA expressions of the five genes and β-actin were measured by qPCR with insect samples of different instar at nymph stage, and the expression stabilities were determined by the software geNorm and NormFinder. As a result, ARF and RPL9 were consistently more stable thanβ-actin, while three TUB genes were less stable than β-actin. To determine the optimal number of reference genes used in qPCR, a pairwise variations analysis by geNorm indicated that two references ARF and RPL9 were required to obtain the accurate quantiifcation. These results were further conifrmed by the validation qPCR experiment with chitinase gene as the target gene, in which the standard error of the mRNA quantiifcation by using binary reference ARF-RPL9 was much lower than those by ARF, RPL9 orβ-actin alone. Taken together, our study suggested that the combination of ARF-RPL9 could replaceβ-actin as the reference genes for qPCR in L. striatellus.

  6. Estimating the number of integrations in transformed plants by quantitative real-time PCR

    Directory of Open Access Journals (Sweden)

    Vaira Anna Maria

    2002-10-01

    Full Text Available Abstract Background When generating transformed plants, a first step in their characterization is to obtain, for each new line, an estimate of how many copies of the transgene have been integrated in the plant genome because this can deeply influence the level of transgene expression and the ease of stabilizing expression in following generations. This task is normally achieved by Southern analysis, a procedure that requires relatively large amounts of plant material and is both costly and labour-intensive. Moreover, in the presence of rearranged copies the estimates are not correct. New approaches to the problem could be of great help for plant biotechnologists. Results By using a quantitative real-time PCR method that requires limited preliminary optimisation steps, we achieved statistically significant estimates of 1, 2 and 3 copies of a transgene in the primary transformants. Furthermore, by estimating the copy number of both the gene of interest and the selectable marker gene, we show that rearrangements of the T-DNA are not the exception, and probably happen more often than usually recognised. Conclusions We have developed a rapid and reliable method to estimate the number of integrated copies following genetic transformation. Unlike other similar procedures, this method is not dependent on identical amplification efficiency between the PCR systems used and does not need preliminary information on a calibrator. Its flexibility makes it appropriate in those situations where an accurate optimisation of all reaction components is impossible or impractical. Finally, the quality of the information produced is higher than what can be obtained by Southern blot analysis.

  7. Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage

    Directory of Open Access Journals (Sweden)

    McCulloch Ryan S

    2012-11-01

    Full Text Available Abstract Background Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes. Results Ten candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were: beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A (cyclophilin A, ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein—zeta polypeptide. The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were (most to least stable succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, succinate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin. Conclusions BestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species.

  8. 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.

  9. 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.

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

    International Nuclear Information System (INIS)

    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)

  11. Quantitative Detection of Perchlorate-Reducing Bacteria by Real-Time PCR Targeting the Perchlorate Reductase Gene▿

    OpenAIRE

    Nozawa-Inoue, Mamie; Jien, Mercy; Hamilton, Nicholas S.; Stewart, Valley; Scow, Kate M.; Hristova, Krassimira R.

    2008-01-01

    A quantitative real-time PCR assay targeting the pcrA gene, encoding the catalytic subunit of perchlorate reductase, detected pcrA genes from perchlorate-reducing bacteria in three different genera and from soil microbial communities. Partial pcrA sequences indicated differences in the composition of perchlorate-reducing bacterial communities following exposure to different electron donors.

  12. Duplex TaqMan real-time PCR assay for quantitative detection of Pantoea stewartii subsp. stewartii and Stenocarpella maydis

    Science.gov (United States)

    A new TaqMan real-time PCR assay was developed for the simultaneous quantitative detection of two seedborne maize pathogens in a single assay. Pantoea stewartii subsp. stewartii (Pnss) (syn. Erwinia stewartii) is the causal agent of Stewart's bacterial wilt and leaf blight of maize. Stewart's wilt i...

  13. 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...

  14. A highly sensitive quantitative real-time pcr assay for determination of mutant jak2 exon 12 allele burden

    DEFF Research Database (Denmark)

    Kjær, L.; Riley, C.H.; Westman, M.; Høgdall, E.; Bjerrum, O.W.; Hasselbalch, H.

    2012-01-01

    present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel...

  15. 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...

  16. 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...

  17. Quantitative detection of perchlorate-reducing bacteria by real-time PCR targeting the perchlorate reductase gene.

    Science.gov (United States)

    Nozawa-Inoue, Mamie; Jien, Mercy; Hamilton, Nicholas S; Stewart, Valley; Scow, Kate M; Hristova, Krassimira R

    2008-03-01

    A quantitative real-time PCR assay targeting the pcrA gene, encoding the catalytic subunit of perchlorate reductase, detected pcrA genes from perchlorate-reducing bacteria in three different genera and from soil microbial communities. Partial pcrA sequences indicated differences in the composition of perchlorate-reducing bacterial communities following exposure to different electron donors. PMID:18245250

  18. Quantitative Real-Time PCR Assay for QPX (Thraustochytriidae), a Parasite of the Hard Clam (Mercenaria mercenaria)▿

    OpenAIRE

    Liu, Qianqian; Allam, Bassem; Collier, Jackie L.

    2009-01-01

    We developed a real-time quantitative PCR (qPCR) assay targeting the rRNA internal transcribed spacer region of the hard clam pathogen QPX. The qPCR assay was more sensitive than was histology in detecting clams with light QPX infections. QPX was detected in 4 of 43 sediment samples but in none of 40 seawater samples.

  19. Testing vaccines in human experimental malaria: statistical analysis of parasitemia measured by a quantitative real-time polymerase chain reaction.

    NARCIS (Netherlands)

    Hermsen, C.C.; Vlas, S.J. de; Gemert, G.J.A. van; Telgt, D.S.C.; Verhage, D.F.; Sauerwein, R.W.

    2004-01-01

    Clinical trials are an essential step in evaluation of safety and efficacy of malaria vaccines, and human experimental malaria infections have been used for evaluation of protective immunity of Plasmodium falciparum malaria. In this study, a quantitative real-time polymerase chain reaction was used

  20. Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR

    OpenAIRE

    Hu, Meizhen; Hu, Wenbin; Xia, Zhiqiang; Zhou, Xincheng; Wang, Wenquan

    2016-01-01

    Reverse transcription quantitative real-time polymerase chain reaction (real-time PCR, also referred to as quantitative RT-PCR or RT-qPCR) is a highly sensitive and high-throughput method used to study gene expression. Despite the numerous advantages of RT-qPCR, its accuracy is strongly influenced by the stability of internal reference genes used for normalizations. To date, few studies on the identification of reference genes have been performed on cassava (Manihot esculenta Crantz). Therefo...

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

    International Nuclear Information System (INIS)

    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

  2. QUANTITATIVE ASSESSMENT OF MYOCARDIAL PERFUSION DEFECTS WITH REAL-TIME THREE-DIMENSIONAL MYOCARDIAL CONTRAST ECHOCARDIOGRAPHY

    Institute of Scientific and Technical Information of China (English)

    Lei Zhuang; Ming-xing Xie; Wei-juan Wang; Xiang-xin Yang; Tao Liu

    2006-01-01

    Objective To evaluate the feasibility and accuracy of measurement of myocardial perfusion defects with intravenous contrast-enhanced real-time three-dimensional echocardiography (CE-RT3 DE).Methods RT3DE was performed in 21 open-chest mongrel dogs undergoing acute ligation of the left anterior descending artery (LAD,n=14) or distal branch of the left circumflex artery (LCX,n=7).A perfluorocarbon microbubble contrast agent was injected intravenously to assess the resulting myocardial perfusion defects with Philips Sonos7500 ultrasound system. Evans blue dye was injected into the occluded coronary artery for subsequent anatomic identification of underperfused myocardium. In vitro anatomic measurement of myocardial mass after removal of the animal's heart was regarded as the control. Blinded off-line calculation of left ventricular mass and perfusion defect mass from RT3DE images were performed using an interactive aided-manual tracing technique.Results Total left ventricular (LV) myocardial mass ranged from 38. 9 to78.5 (mean±SD: 60.0±10.1)g. The mass of perfusion defect ranged from 0 to 21.4 (mean±SD: 12.0±5.0) g or 0 to 27% of total LV mass (mean±SD: 19%±6%). The RT3DE estimation of total LV mass (mean±SD: 59.8±9.9 g) strongly correlated with the anatomic measurement (r=0.98; y=2.01+0.96x). The CE-RT3DE calculation of the mass of underperfused myocardium (mean±SD: 12.3±5.3 g) also strongly correlated with the anatomic measurement (r=0.96; y=0.10+1.04x) and when expressed as percentage of total LV mass (r=0.95; y=-0.20+1.04x).Conclusions RT3DE with myocardial contrast opacification could accurately estimate underperfused myocardial mass in dogs of acute coronary occlusion and would play an important role in quantitative assessment of myocardial perfusion defects in patients with coronary artery disease.

  3. Quantitation of Cell-Free and Cell-Associated Kaposi's Sarcoma-Associated Herpesvirus DNA by Real-Time PCR

    OpenAIRE

    White, Irene E.; Campbell, Thomas B.

    2000-01-01

    A real-time PCR assay for quantitation of Kaposi's sarcoma-associated herpes virus (KSHV or human herpesvirus 8) DNA was evaluated. The linear dynamic range was 10 to 105 copies of KSHV DNA (r2 > 0.99). The accuracy of DNA purification and quantitation was less than ±0.4 log10 copies for samples that contained from 10 to 105 copies of KSHV DNA. Cell-associated KSHV DNA was quantitated over a range of infected cell frequencies from 0.1 to 10−5, and cell-free KSHV DNA in plasma was quantitated ...

  4. 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 model...

  5. Detection of Human Cytomegalovirus DNA by Real-Time Quantitative PCR

    OpenAIRE

    Nitsche, Andreas; Steuer, Nina; Schmidt, Christian Andreas; Landt, Olfert; Ellerbrok, Heinz; Pauli, Georg; Siegert, Wolfgang

    2000-01-01

    A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients.

  6. MOLECULAR ABO PHENOTYPING IN CYNOMOLGUS MACAQUES USING REAL TIME QUANTITATIVE PCR (QPCR)

    OpenAIRE

    Premasuthan, Amritha; Ng, Jillian; Kanthaswamy, Sreetharan; Trask, Jessica Satkoski; Houghton, Paul; Farkas, Tibor; Sestak, Karol; Smith, David Glenn

    2012-01-01

    Macaques are commonly used in biomedical research as animal models of human disease. The ABO phenotype of donors and recipients plays an important role in the success of transplantation and stem cell research of both human and macaque tissue. Traditional serological methods for ABO phenotyping can be time consuming, provide ambiguous results and/or require tissue that is unavailable or unsuitable. We developed a novel method to detect the A, B, and AB phenotypes of macaques using real-time qu...

  7. Real-time absolute frequency measurement of continuous-wave terahertz wave based on dual terahertz combs of photocarriers with different frequency spacings

    CERN Document Server

    Yasui, Takeshi; Ichikawa, Ryuji; Cahyadi, Harsono; Hsieh, Yi-Da; Mizutani, Yasuhiro; Yamamoto, Hirotsugu; Iwata, Tetsuo; Inaba, Hajime; Minoshima, Kaoru

    2015-01-01

    Real-time measurement of the absolute frequency of continuous-wave terahertz (CW-THz) waves is required for characterization and frequency calibration of practical CW-THz sources. We proposed a method for real-time monitoring of the absolute frequency of CW-THz waves involving temporally parallel, i.e., simultaneous, measurement of two pairs of beat frequencies and laser repetition frequencies based on dual THz combs of photocarriers (PC-THz combs) with different frequency spacings. To demonstrate the method, THz-comb-referenced spectrum analyzers were constructed with a dual configuration based on dual femtosecond lasers. Regardless of the presence or absence of frequency control in the PC-THz combs, a frequency precision of 10-11 was achieved at a measurement rate of 100 Hz. Furthermore, large fluctuation of the CW-THz frequencies, crossing several modes of the PC-THz combs, was correctly monitored in real time. The proposed method will be a powerful tool for the research and development of practical CW-THz...

  8. Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR

    OpenAIRE

    Ling, Hui; Wu, Qibin; Guo, Jinlong; Xu, Liping; Que, Youxiong

    2014-01-01

    The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls acro...

  9. Rapid Detection of Enteroviruses in Small Volumes of Natural Waters by Real-Time Quantitative Reverse Transcriptase PCR

    OpenAIRE

    Fuhrman, Jed A.; Liang, Xiaolin; Noble, Rachel T.

    2005-01-01

    Despite viral contamination of recreational waters, only bacterial, not viral, indicators are monitored routinely, due to a lack of rapid and cost-effective assays. We used negatively charged filters to capture enteroviruses from seawater and freshwater. Viral RNA was extracted using a commercial kit, and the viruses were quantified by real-time quantitative reverse transcriptase PCR (qRT-PCR). Poliovirus (6.6 to 330,000 virus particles/ml) was added to samples from watersheds in Los Angeles,...

  10. Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR

    OpenAIRE

    Van Bockstaele Erik; Maroufi Asad; De Loose Marc

    2010-01-01

    Abstract Background Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a sensitive technique for quantifying gene expression levels. One or more appropriate reference genes must be selected to accurately compare mRNA transcripts across different samples and tissues. Thus far, only actin-2 has been used as a reference gene for qRT-PCR in chicory, and a full comparison of several candidate reference genes in chicory has not yet been reported. Results Seven candi...

  11. Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

    OpenAIRE

    Tongeren, S. P.; Degener, J. E.; Harmsen, H. J. M.

    2011-01-01

    The development of fast and easy on-site molecular detection and quantification methods for hazardous microbes on solid surfaces is desirable for several applications where specialised laboratory facilities are absent. The quantification of bacterial contamination necessitates the assessment of the efficiency of the used methodology as a whole, including the preceding steps of sampling and sample processing. We used quantitative real-time polymerase chain reaction (qrtPCR) for Escherichia col...

  12. Validation of suitable reference genes for gene expression analysis in the halophyte Salicornia europaea by real-time quantitative PCR

    OpenAIRE

    Xiao, Xinlong; Ma, Jinbiao; Wang, Junru; Wu, Xiaomeng; Li, Pengbo; Yao, Yinan

    2015-01-01

    Real-time quantitative polymerase chain reaction (RT-qPCR), a reliable technique for quantifying gene expression, requires stable reference genes to normalize its data. Salicornia europaea, a stem succulent halophyte with remarkable salt resistance and high capacity for ion accumulation, has not been investigated with regards to the selection of appropriate reference genes for RT-qPCR. In this study, the expression of 11 candidate reference genes, GAPDH (Glyceraldehyde 3-phosphate dehydrogena...

  13. Selection and validation of reference genes for quantitative real-time PCR analysis of gene expression in Cichorium intybus

    OpenAIRE

    Delporte, Marianne; Legrand, Guillaume; Hilbert, Jean-Louis; Gagneul, David

    2015-01-01

    Plant polyphenols represent a huge reservoir of bioactive compounds. Industrial chicory, an important crop from northwestern Europe, accumulates an original combination of such compounds, i.e., chlorogenic, isochlorogenic, caftaric, and chicoric acids arising from the phenylpropanoid pathway. For a complete understanding of these biochemical pathways, analyses of gene expression using quantitative real-time PCR (qRT-PCR) should be considered. Because cell cultures are a model of choice for sp...

  14. Use of Quantitative Real-Time PCR for Direct Detection of Serratia marcescens in Marine and Other Aquatic Environments

    OpenAIRE

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

    2014-01-01

    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 ...

  15. Molecular detection of Mycoplasma pneumoniae by quantitative real-time PCR in patients with community acquired pneumonia

    OpenAIRE

    Rama Chaudhry; Sutikshan Sharma; Sabah Javed; Kapil Passi; Dey, A. B.; Pawan Malhotra

    2013-01-01

    Background & objectives: Mycoplasma pneumoniae is the most important and common cause of community-acquired pneumonia (CAP). The conventional detection methods (culture and serology) lack sensitivity. PCR offers a better approach for rapid detection but is prone to carry over contamination during manipulation of amplification products. Quantitative real-time PCR (qRT-PCR) method offers an attractive alternative detection method. In the present study, qRT-PCR, PCR and serology methods were use...

  16. Automated and Manual Methods of DNA Extraction for Aspergillus fumigatus and Rhizopus oryzae Analyzed by Quantitative Real-Time PCR▿

    OpenAIRE

    Francesconi, Andrea; Kasai, Miki; Harrington, Susan M.; Beveridge, Mara G; Petraitiene, Ruta; Petraitis, Vidmantas; Schaufele, Robert L.; Walsh, Thomas J.

    2008-01-01

    Quantitative real-time PCR (qPCR) may improve the detection of fungal pathogens. Extraction of DNA from fungal pathogens is fundamental to optimization of qPCR; however, the loss of fungal DNA during the extraction process is a major limitation to molecular diagnostic tools for pathogenic fungi. We therefore studied representative automated and manual extraction methods for Aspergillus fumigatus and Rhizopus oryzae. Both were analyzed by qPCR for their ability to extract DNA from propagules a...

  17. Identification and validation of suitable housekeeping genes for normalizing quantitative real-time PCR assays in injured peripheral nerves

    OpenAIRE

    Gambarotta, Giovanna; Ronchi, Giulia; Friard, Olivier; Galletta, Pantaleo; Perroteau, Isabelle; GEUNA, Stefano

    2014-01-01

    Injury to the peripheral nerve induces dramatic changes in terms of cellular composition that are reflected on RNA quality and quantity, making messenger RNA expression analysis very complex. Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not suitable for quantitative real-time PCR normalization; moreover, the presence of pseudogenes in some of them impairs their use. To deal with this problem, we have developed a new method to identify n...

  18. Quantitative real-time PCR detection of Zika virus and evaluation with field-caught Mosquitoes

    OpenAIRE

    Faye, Oumar; Faye, Ousmane; Diallo, Diawo; Diallo, Mawlouth; Weidmann, Manfred; Sall, Amadou A.

    2013-01-01

    Background Zika virus (ZIKV), a mosquito borne flavivirus is a pathogen affecting humans in Asia and Africa. ZIKV infection diagnosis relies on serology–which is challenging due to cross-reactions with other flaviviruses and/or absence or low titer of IgM and IgG antibodies at early phase of infection- virus isolation, which is labor intensive, time consuming and requires appropriate containment. Therefore, real-time RT-PCR (rRT-PCR) is an appealing option as a rapid, sensitive and spec...

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

    International Nuclear Information System (INIS)

    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 (CT) 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 CT 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

  20. Real-time probe based quantitative determination of material properties at the nanoscale

    International Nuclear Information System (INIS)

    Tailoring the properties of a material at the nanoscale holds the promise of achieving hitherto unparalleled specificity of the desired behavior of the material. Key to realizing this potential of tailoring materials at the nanoscale are methods for rapidly estimating physical properties of the material at the nanoscale. In this paper, we report a method for simultaneously determining the topography, stiffness and dissipative properties of materials at the nanoscale in a probe based dynamic mode operation. The method is particularly suited for investigating soft-matter such as polymers and bio-matter. We use perturbation analysis tools for mapping dissipative and stiffness properties of material into parameters of an equivalent linear time-invariant model. Parameters of the equivalent model are adaptively estimated, where, for robust estimation, a multi-frequency excitation of the probe is introduced. We demonstrate that the reported method of simultaneously determining multiple material properties can be implemented in real-time on existing probe based instruments. We further demonstrate the effectiveness of the method by investigating properties of a polymer blend in real-time. (paper)

  1. Optimization of a duplex real-time PCR method for relative quantitation of infectious laryngotracheitis virus.

    Science.gov (United States)

    Vagnozzi, Ariel; Riblet, Sylva M; Zavala, Guillermo; García, Maricarmen

    2012-06-01

    Infectious laryngotracheitis is a highly contagious respiratory disease of chickens controlled by biosecurity and vaccination with live attenuated or recombinant vaccines. Infectious laryngotracheitis virus (ILTV) infections are characterized by a peak of viral replication in the trachea followed by a steady decrease in replication that results in the establishment of latency. Estimation of viral load is an important tool to determine the stage of ILTV infection. Here, a multiplex real-time PCR was optimized for the quantification of ILTV genomes. Quantification of viral genomes was based on the amplification of the ILTV UL44 gene, and sample variability was normalized using the chicken (Gallusgallus domesticus) alpha2-collagen gene as an endogenous control in a duplex reaction. PMID:22856202

  2. Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR

    Science.gov (United States)

    Bushon, R.N.; Kephart, C.M.; Koltun, G.F.; Francy, D.S.; Schaefer, F. W., III; Lindquist, H.D. Alan

    2010-01-01

    Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms. ?? 2010 The Society for Applied Microbiology.

  3. International ring trial for the validation of an event-specific Golden Rice 2 quantitative real-time polymerase chain reaction method.

    Science.gov (United States)

    Jacchia, Sara; Nardini, Elena; Bassani, Niccolò; Savini, Christian; Shim, Jung-Hyun; Trijatmiko, Kurniawan; Kreysa, Joachim; Mazzara, Marco

    2015-05-27

    This article describes the international validation of the quantitative real-time polymerase chain reaction (PCR) detection method for Golden Rice 2. The method consists of a taxon-specific assay amplifying a fragment of rice Phospholipase D α2 gene, and an event-specific assay designed on the 3' junction between transgenic insert and plant DNA. We validated the two assays independently, with absolute quantification, and in combination, with relative quantification, on DNA samples prepared in haploid genome equivalents. We assessed trueness, precision, efficiency, and linearity of the two assays, and the results demonstrate that both the assays independently assessed and the entire method fulfill European and international requirements for methods for genetically modified organism (GMO) testing, within the dynamic range tested. The homogeneity of the results of the collaborative trial between Europe and Asia is a good indicator of the robustness of the method. PMID:25946377

  4. Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection.

    Science.gov (United States)

    Buh Gasparic, Meti; Tengs, Torstein; La Paz, Jose Luis; Holst-Jensen, Arne; Pla, Maria; Esteve, Teresa; Zel, Jana; Gruden, Kristina

    2010-03-01

    Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use. PMID:20087729

  5. Real-time quantitative polymerase chain reaction methods for four genetically modified maize varieties and maize DNA content in food.

    Science.gov (United States)

    Brodmann, Peter D; Ilg, Evelyn C; Berthoud, Hélène; Herrmann, Andre

    2002-01-01

    Quantitative detection methods are needed for enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients. This labeling threshold, which is set to 1% in the European Union and Switzerland, must be applied to all approved GMOs. Four different varieties of maize are approved in the European Union: the insect-resistant Bt176 maize (Maximizer), Btl 1 maize, Mon810 (YieldGard) maize, and the herbicide-tolerant T25 (Liberty Link) maize. Because the labeling must be considered individually for each ingredient, a quantitation system for the endogenous maize content is needed in addition to the GMO-specific detection systems. Quantitative real-time polymerase chain reaction detection methods were developed for the 4 approved genetically modified maize varieties and for an endogenous maize (invertase) gene system. PMID:12083257

  6. Real-time fluorescent quantitative PCR assay for measuring cytomegalovirus DNA load in patients after haematopoietic stem cell transplantation

    Institute of Scientific and Technical Information of China (English)

    FAN Jun; MA Wei-hang; YANG Mei-fang; XUE Han; GAO Hai-nü; LI Lan-juan

    2006-01-01

    @@ Cytomegalovirus (CMV) infection is a major and often deadly complication of haematopoietic stem cell (HSC) transplantation.1 Successful preemptive CMV therapy in transplant patients depends on the availability of sensitive, specific, and timely diagnostic tests for CMV infections.2 The pp65antigenemia assay has been used for this purposewith considerable success but has disadvantages of being time-consuming and labor-intensive.3 Recently,commercial quantitative polymerase chain reaction (PCR) methods based on TaqMan technique have become available and proven to be useful in the diagnosis of microbial infection as well as the determination of viral load.4 In this study, we developed a fluorescent-based quantitative real-time PCR (RT-FQ PCR) assay using TaqMan chemistry for rapid and quantitative detection of CMV DNA and assessed its clinic value for monitoring the reinfection of CMV in patients after HSC transplantion.

  7. Real-time Quantitative RT-PCR for CT9 Level in Human Cancer

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    CT9 is a recently cloned cancer-testis antigen, which is a member of the bromodomain and extraterminal family.Each member of this protein family contains two N-terminal bromodomain motifs. We investigated the distribution of CT9 in different tissues and the possibility for it to be used as a potential therapeutic target in cancer treament. By using the real-time RT-PCR method and 18SrRNA as an internal standard, we analyzed the CT9 expression in several normal human tissues and in the tissues of patients suffering from cancer. The result of this study shows that the highest level of mRNA is only present in testis tissue because the CT9 expression has not been detected in other normal tissues. In 6 of 10 cases of gastric adenocarcinoma, in 3 of 10 cases of esophageal squamous cell carcinoma, in 2 of 9 cases of endometrial carcinoma and only in 1 of 12 cases of brain cancer, the low level expression of CT9 was detected. In none of the 12 cases of cervical squamous cell carcinoma, the expression of CT9 was detected. Since the high level expression of CT9 is only found in the normal testis tissue, but the low expression in cancer tissues, for example tissues of cervical squamous cell carcinoma, brain cancer, endometrial adenocarcinoma, esophageal squamous cell carcinoma, we conclude that CT9 cannot be used as a cancer therapeutic target molecule for cervical squamous cell carcinoma, brain cancer, endometrial adenocarcinoma, esophageal squamous cell carcinoma.

  8. Application of real time PCR for the quantitative detection of radiation-induced genomic DNA strand breaks

    International Nuclear Information System (INIS)

    The frequency of single strand breaks (SSBs) occurring on both strands of the pBR322 plasmid DNA region flanked by a pair of primers used for polymerase chain reaction (PCR) amplifications was determined after irradiation with sup(137)Cs gamma rays. We verified that real time PCR is suitable for the detection and quantitative analysis of SSBs caused by gamma ray irradiation. The utility of this approach was also supported by the comparison of the practical experimental data with the Monte Carlo simulation. The potential application of this PCR method for the detection of genomic DNA damage was also confirmed

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

    OpenAIRE

    Costa Elena; Donnangelo Anita; Carminati Mario; Valaperta Rea; Rusconi Daniela; de Filippis Tiziana; Passeri Elena; Frigerio Marcello; Persani Luca; Finelli Palma; Corbetta Sabrina

    2011-01-01

    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...

  10. Identification and Validation of Reference Genes for Quantitative Real-Time PCR in Drosophila suzukii (Diptera: Drosophilidae)

    OpenAIRE

    Zhai, Yifan; Lin, Qingcai; Zhou, Xianhong; Zhang, Xiaoyan; Liu, Tingli; Yu, Yi

    2014-01-01

    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 evalu...

  11. Rapid and Sensitive Detection of Physical Status of Human Papillomavirus Type 16 DNA by Quantitative Real-Time PCR

    OpenAIRE

    Nagao, Shoji; Yoshinouchi, Mitsuo; Miyagi, Yasunari; Hongo, Atsushi; Kodama, Junichi; Itoh, Sachio; Kudo,Takafumi

    2002-01-01

    A rapid quantitative real-time PCR method was employed to quantify the copy number of E2 and E6 genes for analysis of the physical status of human papillomavirus type 16 (HPV-16) DNA. Significant differences with respect to both copy numbers were found when more than 40% of HPV-16 DNA was integrated with disruption of the E2 gene in an experimental model. The physical status of HPV-16 DNA in 50 clinical samples was exclusively episomal in 21 cases (42%), concomitant in 14 cases (28%), and int...

  12. Quantitation of Viral DNA by Real-Time PCR Applying Duplex Amplification, Internal Standardization, and Two-Color Fluorescence Detection

    OpenAIRE

    Gruber, Franz; Falkner, Falko G.; Dorner, Friedrich; Hämmerle, Thomas

    2001-01-01

    A real-time PCR method was developed to quantitate viral DNA that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve. Applied to human parvovirus B19 DNA, the linear range was from 102 to at least 5 × 106 copies per ml of sample. The coefficient of variation was 0.29 using a run control of 2,876 copies per ml. The method reduces the risk of false-negative results, yields high precision, a...

  13. 实时定量PCR技术及其应用%Real-time Quantitative PCR and Its Applications

    Institute of Scientific and Technical Information of China (English)

    王梁燕; 洪奇华; 张耀洲

    2004-01-01

    实时定量PCR(Real-time Quantitative Polymerase Chain Reaction,RQ-PCR)技术是20世纪90年代中期发展起来的一种新型核酸定量技术.该技术具有实时监测、快速、灵敏、精确等特点,是对原有PCR技术的革新,扩大了PCR的应用范围.本文综述了RQ-PCR技术的原理、RQ-PCR仪、RQ-PCR实时定量检测系统及其应用.

  14. Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of Serotypes 40 and 41

    OpenAIRE

    Jothikumar, Narayanan; Cromeans, Theresa L.; Hill, Vincent R.; Lu, Xiaoyan; Sobsey, Mark D.; Erdman, Dean D.

    2005-01-01

    A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV4...

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

    OpenAIRE

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

    2006-01-01

    Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and specie...

  16. Real-time Quantitative Analysis of Mixed Lanthanides for an Assessment of Material Stream in Pyroprocessing

    Energy Technology Data Exchange (ETDEWEB)

    Jee, Young Taek; Yun, Jong Il [KAIST (Korea, Republic of); Kim, Bong Young [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2015-05-15

    In this work, a computational tool for quantitative analysis of absorption and fluorescence spectra of lanthanides in high temperature LiCl-KCl molten eutectic has been successfully developed. The results produced from the code showed precise and reliable deconvolution of both absorption and fluorescence spectra for the Nd-Pr-Sm-Dy mixed lanthanide sample. The relative concentration errors obtained from the quantitative analysis of absorption spectrum were less than 10 %, and the qualitative analysis achieved with fluorescence spectrum revealed the fluorescence resonant energy transfer behavior in the mixed lanthanide samples. In this research, four representative lanthanides, which are neutron-poisoning due to their extraordinarily high neutron capture cross sections, were quantitatively analyzed by using UV/Vis absorption and fluorescence spectra. To achieve a condition of continuous monitoring, spectroscopic techniques were employed for the in-situ analysis. The measured spectra were then immediately deconvoluted into reference spectra of each lanthanide, and by investigating deconvoluted spectra, quantitative analysis could be completed.

  17. Real-time Quantitative Analysis of Mixed Lanthanides for an Assessment of Material Stream in Pyroprocessing

    International Nuclear Information System (INIS)

    In this work, a computational tool for quantitative analysis of absorption and fluorescence spectra of lanthanides in high temperature LiCl-KCl molten eutectic has been successfully developed. The results produced from the code showed precise and reliable deconvolution of both absorption and fluorescence spectra for the Nd-Pr-Sm-Dy mixed lanthanide sample. The relative concentration errors obtained from the quantitative analysis of absorption spectrum were less than 10 %, and the qualitative analysis achieved with fluorescence spectrum revealed the fluorescence resonant energy transfer behavior in the mixed lanthanide samples. In this research, four representative lanthanides, which are neutron-poisoning due to their extraordinarily high neutron capture cross sections, were quantitatively analyzed by using UV/Vis absorption and fluorescence spectra. To achieve a condition of continuous monitoring, spectroscopic techniques were employed for the in-situ analysis. The measured spectra were then immediately deconvoluted into reference spectra of each lanthanide, and by investigating deconvoluted spectra, quantitative analysis could be completed

  18. Real-Time Determination of Absolute Frequency in Continuous-Wave Terahertz Radiation with a Photocarrier Terahertz Frequency Comb Induced by an Unstabilized Femtosecond Laser

    Science.gov (United States)

    Minamikawa, Takeo; Hayashi, Kenta; Mizuguchi, Tatsuya; Hsieh, Yi-Da; Abdelsalam, Dahi Ghareab; Mizutani, Yasuhiro; Yamamoto, Hirotsugu; Iwata, Tetsuo; Yasui, Takeshi

    2016-05-01

    A practical method for the absolute frequency measurement of continuous-wave terahertz (CW-THz) radiation uses a photocarrier terahertz frequency comb (PC-THz comb) because of its ability to realize real-time, precise measurement without the need for cryogenic cooling. However, the requirement for precise stabilization of the repetition frequency ( f rep) and/or use of dual femtosecond lasers hinders its practical use. In this article, based on the fact that an equal interval between PC-THz comb modes is always maintained regardless of the fluctuation in f rep, the PC-THz comb induced by an unstabilized laser was used to determine the absolute frequency f THz of CW-THz radiation. Using an f rep-free-running PC-THz comb, the f THz of the frequency-fixed or frequency-fluctuated active frequency multiplier chain CW-THz source was determined at a measurement rate of 10 Hz with a relative accuracy of 8.2 × 10-13 and a relative precision of 8.8 × 10-12 to a rubidium frequency standard. Furthermore, f THz was correctly determined even when fluctuating over a range of 20 GHz. The proposed method enables the use of any commercial femtosecond laser for the absolute frequency measurement of CW-THz radiation.

  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. 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

  1. Validation of reference genes for real-time quantitative RT-PCR studies in Talaromyces marneffei.

    Science.gov (United States)

    Dankai, Wiyada; Pongpom, Monsicha; Vanittanakom, Nongnuch

    2015-11-01

    Talaromyces marneffei (or Penicillium marneffei) is an opportunistic pathogen that can cause disseminated disease in human immunodeficiency virus (HIV)-infected patients, especially in Southeast Asia. T. marneffei is a thermally dimorphic fungus. Typically, T. marneffei has an adaptive morphology. It grows in a filamentous form (mould) at 25°C and can differentiate to produce asexual spores (conidia). In contrast, at 37°C, it grows as yeast cells that divide by fission. This study aimed to validate a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for gene expression analysis in T. marneffei. Analysis of relative gene expression by qRT-PCR requires normalization of data using a proper reference gene. However, suitable reference genes have not been identified in gene expression studies across different cell types or under different experimental conditions in T. marneffei. In this study, four housekeeping genes were selected for analysis: β-actin (act); glyceraldehyde-3-phosphate dehydrogenase (gapdh); β-tubulin (benA) and 18S rRNA. Two analysis programs; BestKeeper and geNorm software tools were used to validate the expression of the candidate normalized genes. The results indicated that the actin gene was the one which was most stably expressed and was recommended for use as the endogenous control for gene expression analysis of all growth forms in T. marneffei by qRT-PCR under normal and stress conditions. PMID:26327538

  2. Development of a quantitative real-time PCR for the detection of Tenacibaculum maritimum and its application to field samples.

    Science.gov (United States)

    Fringuelli, E; Savage, P D; Gordon, A; Baxter, E J; Rodger, H D; Graham, D A

    2012-08-01

    The development and the application of a quantitative real-time PCR for the detection of Tenacibaculum maritimum are described. A set of primers and probe was designed to amplify a 155-bp fragment specific to the T. maritimum 16S rRNA gene. The test was shown to be very sensitive, able to detect as little as 4.8 DNA copies number μL(-1) . In addition, the assay was found to have a high degree of repeatability and reproducibility, with a linear dynamic range (R(2)  = 0.999) extending over 6 log(10) dilutions and a high efficiency (100%). The assay was applied to DNA samples extracted from 48 formalin-fixed paraffin-embedded (FFPE) Atlantic salmon, Salmo salar, gill tissues showing varying degrees of gill pathology (scored 0-3) and from 26 jellyfish samples belonging to the species Phialella quadrata and Muggiaea atlantica. For each sample, the bacterial load was normalised against the level of the salmonid elongation factor alpha 1 (ELF) detected by a second real-time PCR using previously published primers and probe. Tenacibaculum maritimum DNA was detected in 89% of the blocks with no signs of gill disease as well as in 95% of the blocks with mild-to-severe gill pathology. Association between bacterial load and gill pathology severity was investigated. T. maritimum DNA was detected at low level in four of the 26 jellyfish tested. PMID:22724390

  3. Quantitative real-time PCR of enteric viruses in influent and effluent samples from wastewater treatment plants in Italy

    Directory of Open Access Journals (Sweden)

    Giuseppina La Rosa

    2010-01-01

    Full Text Available The prevalence of enteric viruses in wastewater, the efficacy of wastewater treatments in eliminating such viruses, and potential health risks from their release into the environment or by recycling of treated wastewaters, are very important issues in environmental microbiology. In this study we performed a quantitative TaqMan real-time PCR (polymerase chain reaction analysis of enteric viruses on samples of influents and effluents from 5 wastewater treatment plants in and around Rome. Three epidemiologically important, waterborne enteric viruses were analyzed: adenoviruses, enteroviruses and noroviruses (GI and GII and compared to classical bacterial indicators of fecal contamination. The concentration of adenoviruses was the highest, in both raw and treated waters. Mean values in influents were ranked as follows: adenovirus > norovirus GI > norovirus GII > enterovirus. In effluents, the ranking was: adenovirus > norovirus GI > enterovirus > norovirus GII. Removal efficiencies ranged from 35% (enterovirus to 78% (norovirus GI, while removal efficiency for bacterial indicators was up to 99%. Since molecular quantification does not necessarily indicate an actual threat to human health, we proceeded to evaluate the infectivity of enterovirus particles in treated effluents through integrated cell culture and real-time PCR. Infectivity assays detected live virions in treated water, pointing to potential public health risks through the release of these viruses into the environment. A better understanding of viral presence and resistance to sewage purification processes have the potential of contributing to the effective management of risks linked to the recycling of treated wastewater, and its discharge into the environment.

  4. 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. PMID:19446978

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

    Science.gov (United States)

    Chase, Dorothy M; Elliott, Diane G; Pascho, Ronald J

    2006-07-01

    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. PMID:16921877

  6. Real-time molecular profiling of photochemically induced rat thrombosis in vivo through quantitative Raman analysis of blood

    Science.gov (United States)

    Lin, M. M.; Shen, A. G.; Yao, H. L.; Zhang, Z. Z.; Hu, J. M.

    2014-11-01

    A device of an animal thrombosis model in vivo coupled with a Raman system for near-surface blood vessels is proposed in this letter. The dual-function set up is capable of simultaneously establishing a photochemically induced artificial thrombus model and collecting in vivo Raman data of both arterial and venous blood, and it provides the first observation of rat thrombosis under the physiological conditions from the beginning to the final form. The real-time and quantitative molecular profiling of flowing blood and the spectra of blood cells in the process of thrombosis provides an insight into the occurring mechanism of thrombosis and a promising method for the in vivo screening of new antithrombotic and thrombolytic drugs.

  7. Real-time molecular profiling of photochemically induced rat thrombosis in vivo through quantitative Raman analysis of blood

    International Nuclear Information System (INIS)

    A device of an animal thrombosis model in vivo coupled with a Raman system for near-surface blood vessels is proposed in this letter. The dual-function set up is capable of simultaneously establishing a photochemically induced artificial thrombus model and collecting in vivo Raman data of both arterial and venous blood, and it provides the first observation of rat thrombosis under the physiological conditions from the beginning to the final form. The real-time and quantitative molecular profiling of flowing blood and the spectra of blood cells in the process of thrombosis provides an insight into the occurring mechanism of thrombosis and a promising method for the in vivo screening of new antithrombotic and thrombolytic drugs. (letter)

  8. Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of Serotypes 40 and 41

    Science.gov (United States)

    Jothikumar, Narayanan; Cromeans, Theresa L.; Hill, Vincent R.; Lu, Xiaoyan; Sobsey, Mark D.; Erdman, Dean D.

    2005-01-01

    A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples. PMID:15933012

  9. 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. PMID:24375136

  10. Molecular detection of Mycoplasma pneumoniae by quantitative real-time PCR in patients with community acquired pneumonia

    Directory of Open Access Journals (Sweden)

    Rama Chaudhry

    2013-01-01

    Full Text Available Background & objectives: Mycoplasma pneumoniae is the most important and common cause of community-acquired pneumonia (CAP. The conventional detection methods (culture and serology lack sensitivity. PCR offers a better approach for rapid detection but is prone to carry over contamination during manipulation of amplification products. Quantitative real-time PCR (qRT-PCR method offers an attractive alternative detection method. In the present study, qRT-PCR, PCR and serology methods were used to detect M. pneumoniae infection in cases of pneumonias and findings compared. Methods: A total of 134 samples consisting of blood (for serology and respiratory secretions (for PCR and qRT-PCR from 134 patients were collected. The blood samples were tested for IgG, IgM and IgA using commercially available kits. For standardization of PCR of M. pneumoniae P1 gene was cloned in pGEMTEasy vector. Specific primers and reporter sequence were designed and procured for this fragment. The qRT-PCR assay was performed to prepare the standard curve for M. pneumoniae positive control DNA template and detection in patient samples. Results: Of the 134 patients, 26 (19% were positive for antibodies against M. pneumoniae. IgG was positive in 14.92 per cent (20 cases, IgM in 4.47 per cent (6 and IgA was positive in 5.22 per cent (7 cases. In the qRT-PCR assay 19 per cent (26 samples were positive. Of the 26 qRT-PCR positive samples, nine could be detected by serology. PCR was positive for 25 samples. An extra sample negative by PCR was detected by qRT-PCR. Thus, real-time PCR assay, PCR and serology in combination could detect M. pneumoniae infection in 43 patients. Interpretation & conclusions: The study shows that 17 patients were detected by serology alone, 17 were detected by qRT-PCR only and nine patients were positive by both serology and real-time PCR. Of the 134 samples tested, 25 were positive by conventional PCR, but qRT-PCR could detect one more sample that was

  11. Comparison of quantitative HCMV DNAemia in whole blood and leukocytes, and Real-Time PCR in a BMT patient

    Directory of Open Access Journals (Sweden)

    Elio De Nisco

    2012-03-01

    Full Text Available Background. HCMV is the most opportunistic viral agent in the transplantation of bone marrow and solid organ. Early therapy based on the detection of HCMV pp65 antigen in peripheral blood leukocytes, has led to a significant reduction in the incidence of related HCMV diseases.The pp65 antigenemia is difficult to standardize, while the quantification of HCMV DNA by Real-Time PCR is an alternative diagnostic approach with greater sensitivity and reproducibility, providing important information in the management of infected patients. Objectives. The aim of this study was the comparative analysis of quantitative HCMV DNAemia in leukocytes and in whole blood, with Real-time PCR, in a BMT patient with HCMV post-transplant reactivation, in order to analyze the relationship between levels of viral DNA at different time-points obtained in the two biological matrices. Study Design. The presence of HCMV DNA was detected in whole blood and peripheral blood leukocytes in a patient who underwent allogeneic marrow transplant for Ph1 + acute lymphoblastic leukemia in molecular remission. After 5-6 months, the patient has increased molecular Bcr-Abl (Philadelphia chromosome. It was activated immune reaction by means of tapering (lowering the dose of cyclosporine that uses the GvL effect to turn negative the Philadelphia chromosome positivity (Bcr/Abl negativity. Later, the patient develops GvHD and cortisone is administered. The persistence of grafts treated with immunosuppressants periodically reactivates HCMV infection. The DNA extraction from whole-blood was performed by automatic extractor QIACUBE (Qiagen, while extraction from leucocytes was performed on a standard number of leukocytes (EXTRAcell- Nanogen.The extracted DNA was amplified by Real-Time Alert Q-PCR (Nanogen.The samples were analyzed weekly for about 5 months from 1 year after transplantation. Results. The patient at 1 year after transplantation, has three HCMV reactivation at 56th, 62th and 67

  12. 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.

  13. 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.

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

    International Nuclear Information System (INIS)

    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 103A. 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

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

    Science.gov (United States)

    Lee, Dae-Young; Lauder, Heather; Cruwys, Heather; Falletta, Patricia; Beaudette, Lee A

    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(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. PMID:18423816

  16. Isotope dilution strategies for absolute quantitative proteomics

    International Nuclear Information System (INIS)

    The development of mass spectrometry (MS)-based methodologies for high-throughput protein identification has generated a concomitant need for protein quantification. Numerous MS-based relative quantification methodologies have been dedicated to the extensive comparison of multiple proteomes. On the other hand, absolute quantification methodologies, which allow the determination of protein concentrations in biological samples, are generally restricted to defined sets of proteins. Depending on the selected analytical procedure, absolute quantification approaches can provide accurate and precise estimations. These analytical performances are crucial for specific applications such as the evaluation of clinical bio-marker candidates. According to bioanalytical guidelines, accurate analytical processes require internal standards and quality controls. Regarding MS-based analysis of small molecules, isotope dilution has been recognized as the reference method for internal standardization. However, protein quantification methodologies which rely on the isotope dilution principle have been implemented in the proteomic field only recently. In these approaches, the sample is spiked with defined amounts of isotope-labeled analogue(s) of specific proteolytic peptide(s) (AQUA and QconCAT strategies) or protein(s) (PSAQ strategy). In this review, we present a critical overview of these isotope dilution methodologies. (authors)

  17. 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.

  18. Quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real-time PCR.

    Science.gov (United States)

    Collantes-Fernández, Esther; Zaballos, Angel; Alvarez-García, Gema; Ortega-Mora, Luis M

    2002-04-01

    We report the development of a real-time PCR assay for the quantitative detection of Neospora caninum in infected host tissues. The assay uses the double-stranded DNA-binding dye SYBR Green I to continuously monitor product formation. Oligonucleotide primers were designed to amplify a 76-bp DNA fragment corresponding to the Nc5 sequence of N. caninum. A similar method was developed to quantify the 28S rRNA host gene in order to compare the parasite load of different samples and to correct for the presence of potential PCR-inhibiting compounds in the DNA samples. A linear quantitative detection range of 6 logs with a calculated detection limit of 10(-1) tachyzoite per assay was observed with excellent linearity (R(2) = 0.998). Assay specificity was confirmed by using DNA from the closely related parasite Toxoplasma gondii. The applicability of the technique was successfully tested in a variety of host brain tissues: (i) aborted bovine fetuses classified into negative or positive Neospora-infected animals according to the observation of compatible lesions by histopathological study and (ii) experimentally infected BALB/c mice, divided into three groups, inoculated animals with or without compatible lesions and negative controls. All samples were also tested by ITS1 Neospora nested PCR and a high degree of agreement was shown between both PCR techniques (kappa = 0.86). This technique represents a useful quantitative diagnostic tool to be used in the study of the pathogenicity, immunoprophylaxis, and treatment of Neospora infection. PMID:11923330

  19. Establishment of Real-Time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of Porcine Lipoprotein Lipase mRNA

    Institute of Scientific and Technical Information of China (English)

    LIAN Hong-xia; LU De-xun; GAO Min

    2009-01-01

    Porcine lipoprotein lipase (LPL) cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Longissimus dorsi of porcine was reverse-transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed into Escherichia coli TOP 10. Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced. LPL was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The concentration of DNA template purified was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for fluorescence quantitative PCR (FQ-PCR). The method of LPL mRNA real-time PCR was well established, which detected as low as 103 with the linear range 103 to 1010 copies. The standard curves showed high correlations (R2=0.9871). A series of standards for real-time PCR analysis have been constructed successfully, and real-time TaqMan-fluorescence quantitative RT-PCR is reliable to quantitatively evaluate FQ-PCR mRNA in L. dorsi of porcine.

  20. Rapid detection of enteroviruses in small volumes of natural waters by real-time quantitative reverse transcriptase PCR.

    Science.gov (United States)

    Fuhrman, Jed A; Liang, Xiaolin; Noble, Rachel T

    2005-08-01

    Despite viral contamination of recreational waters, only bacterial, not viral, indicators are monitored routinely, due to a lack of rapid and cost-effective assays. We used negatively charged filters to capture enteroviruses from seawater and freshwater. Viral RNA was extracted using a commercial kit, and the viruses were quantified by real-time quantitative reverse transcriptase PCR (qRT-PCR). Poliovirus (6.6 to 330,000 virus particles/ml) was added to samples from watersheds in Los Angeles, California, and analysis showed that with 50-ml samples, a cellulose acetate/nitrate (HA) filter yielded final recovery of 51% (r2= 0.99) in fresh water and 23% (r2= 0.90) in seawater. However, for additions of low levels of virus (more likely to represent field samples; California: Topanga Creek (TC), a relatively pristine mountain creek, and Ballona Creek (BC), a concrete-lined urban storm drain. One TC site out of 10 and 2 BC sites out of 7 tested significantly positive for enteroviruses, with higher enterovirus concentrations in BC than in TC (ca. 10 to 25 versus 1 equivalent enterovirus particle/ml). The presented filtration-qRT-PCR approach is fast (contamination in environmental water samples. PMID:16085845

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. Validation of a primer optimisation matrix to improve the performance of reverse transcription – quantitative real-time PCR assays

    Directory of Open Access Journals (Sweden)

    Dobrovic Alexander

    2009-06-01

    Full Text Available Abstract Background The development of reverse transcription – quantitative real-time PCR (RT-qPCR platforms that can simultaneously measure the expression of multiple genes is dependent on robust assays that function under identical thermal cycling conditions. The use of a primer optimisation matrix to improve the performance of RT-qPCR assays is often recommended in technical bulletins and manuals. Despite this recommendation, a comprehensive introduction to and evaluation of this approach has been absent from the literature. Therefore, we investigated the impact of varying the primer concentration, leaving all the other reaction conditions unchanged, on a large number of RT-qPCR assays which in this case were designed to be monitored using hydrolysis probes from the Universal Probe Library (UPL library. Findings Optimal RT-qPCR conditions were determined for 60 newly designed assays. The calculated Cq (Quantification Cycle difference, non-specific amplification, and primer dimer formation for a given assay was often dependent on primer concentration. The chosen conditions were further optimised by testing two different probe concentrations. Varying the primer concentrations had a greater effect on the performance of a RT-qPCR assay than varying the probe concentrations. Conclusion Primer optimisation is important for improving the performance of RT-qPCR assays monitored by UPL probes. This approach would also be beneficial to the performance of other RT-qPCR assays such as those using other types of probes or fluorescent intercalating dyes.

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

    International Nuclear Information System (INIS)

    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)

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

    Science.gov (United States)

    Zhai, Yifan; Lin, Qingcai; Zhou, Xianhong; Zhang, Xiaoyan; Liu, Tingli; Yu, Yi

    2014-01-01

    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. PMID:25198611

  6. 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.

  7. Universal real-time PCR assay for quantitation and size evaluation of residual cell DNA in human viral vaccines.

    Science.gov (United States)

    André, Murielle; Reghin, Sylviane; Boussard, Estelle; Lempereur, Laurent; Maisonneuve, Stéphane

    2016-05-01

    Residual host cellular DNA (rcDNA) is one of the principal risk associated with continuous cell lines derived medicines such as viral vaccines. To assess rcDNA degradation, we suggest two quantitative real-time PCR assays designed to separately quantify target sequences shorter and longer than the 200 bp risk limit, the relative abundance of both targets reflecting the extent of rcDNA fragmentation. The conserved multicopy ribosomal 18S RNA gene was targeted to detect host cell templates from most mammalian cell substrates commonly used in the manufacture of human viral vaccines. The detection range of the method was assessed on purified DNA templates from different animal origins. The standard calibrator origin and structural conformation were shown crucial to achieve accurate quantification. Artificial mixtures of PCR products shorter and longer than 200 bp were used as a model to check the ability of the assay to estimate the fragment size distribution. The method was successfully applied to a panel of Vero cell derived vaccines and could be used as a universal method for determination of both content and size distribution of rcDNA in vaccines. PMID:27033773

  8. Influences of bracket bonding on mutans streptococcus in plaque detected by real time fluorescence-quantitative polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    AI Hong; LU Hong-fei; LIANG Huan-you; WU Jian; LI Ruo-lan; LIU Guo-ping; XI Yun

    2005-01-01

    Background Enamel demineralization occurs frequently during orthodontic treatment. In this study, we evaluated the changes of the density of mutans streptococcus (MS) in plaque after bracket bonding and using fluoride adhesive on maxillary incisors by real time fluorescence-quantitative polymerase chain reaction (RT-FQ PCR).Methods The study was designed as a self-paired test. Brackets were bonded with fluoride adhesive on the left side, while non-fluoride adhesive on the right side for each patient. Plaque samples were taken from the surfaces around the brackets of four maxillary incisors before brackets bonding and after the bonding 4 weeks later. The amount of MS was measured by RT-FQ PCR. The data obtained were analyzed statistically using the SPSS 11.5 version and the alpha level was set at 0.05 (2-tailed).Results The amount of MS in plaque increased significantly after bracket bonding (P0.05), and among the incisors using and not using fluoride adhesive (P>0.05).Conclusions The increase of the density of MS in plaque after bracket bonding is one of the etiological factors for enamel demineralization in orthodontic patients. The result of this study did not support what we observed clinically that the incidence of enamel demineralization for lateral incisors was higher than that for central incisors. Using fluoride adhesive for bonding did not affect the amount of MS in plaque in our study. Further study is needed.

  9. Quantitative analysis of herpes virus sequences from normal tissue and fibropapillomas of marine turtles with real-time PCR

    Science.gov (United States)

    Quackenbush, S.L.; Casey, R.N.; Murcek, R.J.; Paul, T.A.; Work, T.M.; Limpus, C.J.; Chaves, A.; duToit, L.; Perez, J.V.; Aguirre, A.A.; Spraker, T.R.; Horrocks, J.A.; Vermeer, L.A.; Balazs, G.S.; Casey, J.W.

    2001-01-01

    Quantitative real-time PCR has been used to measure fibropapilloma-associated turtle herpesvirus (FPTHV) pol DNA loads in fibropapillomas, fibromas, and uninvolved tissues of green, loggerhead, and olive ridley turtles from Hawaii, Florida, Costa Rica, Australia, Mexico, and the West Indies. The viral DNA loads from tumors obtained from terminal animals were relatively homogenous (range 2a??20 copies/cell), whereas DNA copy numbers from biopsied tumors and skin of otherwise healthy turtles displayed a wide variation (range 0.001a??170 copies/cell) and may reflect the stage of tumor development. FPTHV DNA loads in tumors were 2.5a??4.5 logs higher than in uninvolved skin from the same animal regardless of geographic location, further implying a role for FPTHV in the etiology of fibropapillomatosis. Although FPTHV pol sequences amplified from tumors are highly related to each other, single signature amino acid substitutions distinguish the Australia/Hawaii, Mexico/Costa Rica, and Florida/Caribbean groups.

  10. Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment.

    Science.gov (United States)

    Shannon, K E; Lee, D-Y; Trevors, J T; Beaudette, L A

    2007-08-15

    Bacteria were detected at five stages of municipal wastewater treatment using TaqMan(R) real-time quantitative PCR (qPCR). Thirteen probe and primer sets were tested for diverse pathogens that may be present in wastewater, including Aeromonas hydrophila, Bacillus cereus, Clostridium perfringens, Enterococcus faecalis, Escherichia coli, E. coli O157:H7, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella sp., and Staphylococcus aureus. The sensitivity of the assay was 100 fg of genomic DNA (=22 gene copies), based on a standard curve generated using A. hydrophila purified DNA. Samples from five stages of wastewater treatment were collected, including raw wastewater, primary effluents, mixed liquor, waste activated sludge and final effluents. In duplicate samples, E. coli, K. pneumoniae, C. perfringens and E. faecalis were detected throughout the wastewater process, and their numbers decreased by 3.52-3.98, 4.23-4.33, 3.15-3.39, and 3.24 orders of magnitude respectively, between the raw wastewater and final effluent stage. This qPCR method was effective for the detection of pathogens in wastewater and confirmed that the risk of exposure to pathogens in the wastewater discharge was well within the Environment Canada guidelines. PMID:17462712

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  12. Real-time PCR快速检测钩端螺旋体方法的建立%Rapid detection of pathogenic leptospira spp by quantitative real-time PCR

    Institute of Scientific and Technical Information of China (English)

    谈忠鸣; 汤奋扬; 周璐

    2011-01-01

    目的:采用新型TaqMan探针建立检测钩端螺旋体的实时荧光定量PCR.方法:根据钩端螺旋体16s rDNA设计的一对引物及探针,以钩体标准菌株的核酸为模板,在荧光定量PCR检测仪(ABI 7500)建立实时荧光定量PCR.结果:建立的定量标准曲线的循环CT值与模板拷贝数呈现良好的线性关系(r=0.998),与普通PCR相比,荧光PCR的灵敏度是其1000倍,检测其他细菌DNA,未检测到信号.结论:本研究中检测钩端螺旋体的实时荧光定量PCR具有高度的特异性和敏感性,特别适合检测样本中微量的钩端螺旋体.%Objective: To establish quantitative real - time PCR method for the detection of leptospira spp by using new Taq-Man probe. Methods: According to the 16s rDNA to design a pair of primers and probes, and then take the nuclear acid of standard strain of leptospira spp as template to set up quantitative real - time PCR on detector( ABI 7500). Results: The relationship between the values of threshold cycle( CT) and the DNA copy number was found to be linear( r = 0.998 ). The sensitivity of real - time PCR was about 1000 times of the convention PCR when used to detect Leptospira DNA, accompanied by high species - specificity. Conclusion: This results suggest that the quantitative real - time PCR is hightly specific and sensitive for detection of Leptospira, especially useful for detection of tiny leptospira spp of samples.

  13. Development and Application of Real-Time Quantitative Polymerase Chain Reaction Technique Using SYBR Green I in the Diagnosis of Down Syndrome

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    Shirin Shahbazi

    2010-01-01

    Full Text Available Objective: Rapid diagnosis of Trisomy 21 Syndrome (Down Syndrome patients usingReal-Time quantitative Polymerase Chain Reaction (Real-Time qPCR in order to establisha novel method for prenatal diagnosis in the future.Materials and Methods: A total of 5 ml of peripheral blood was obtained from eachpatient and normal controls (NR. Then, genomic DNA from lymphocytes was extractedusing the salting out procedure. Gene dosage levels of DSCAM and (PMP22, DSCAM inDown Syndrome and NR were analyzed using real-time quantitative PCR. The DSCAM/PMP22 ratio was calculated according to the 2-ΔΔCt formula for all samples.Results: Real-time PCR showed a DSCAM/PMP22 ratio of 1.48 ± 0.18 and 1.01 ± 0.10(p<0.001 in Down Syndrome and normal samples, respectively, demonstrating threecopies of the target (DSCAM gene in Trisomy 21 Syndrome.Conclusion: DSCAM/PMP22 ratio is increased significantly in Down Syndrome patientsthan NR (1.5 times. Therefore, the real-time quantitative PCR technique can be used asa sensitive, accurate and reliable technique for rapid and prenatal diagnosis of Trisomy21 Syndrome.

  14. 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 clinically applicable.

  15. Comprehensive selection of reference genes for gene expression normalization in sugarcane by real time quantitative rt-PCR.

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    Hui Ling

    Full Text Available The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR method for gene expression analysis requires one or several reference gene(s acting as normalization factor(s. In order to facilitate gene expression studies in sugarcane (Saccharum officinarum, a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls across different tissues and under various experimental treatments. These results revealed that, among these 13 candidate reference genes, GAPDH, eEF-1a and eIF-4α were the most stable and suitable for use as normalization factors across all various experimental samples. In addition, APRT could be a candidate for examining the relationship between gene copy number and transcript levels in sugarcane tissue samples. According to the results evaluated by geNorm, combining CUL and eEF-1α in hormone treatment experiments; CAC and CUL in abiotic stress tests; GAPDH, eEF-1a and CUL in all treatment samples plus CAC, CUL, APRT and TIPS-41 in cultivar tissues as groups for normalization would lead to more accurate and reliable expression quantification in sugarcane. This is the first systematic validation of reference genes for quantification of transcript expression profiles in sugarcane. This study should provide useful information for selecting reference genes for more accurate quantification of gene expression in sugarcane and other plant species.

  16. Very High Resolution Ultrasound Imaging for Real-Time Quantitative Visualization of Vascular Disruption after Spinal Cord Injury

    Science.gov (United States)

    Soubeyrand, Marc; Badner, Anna; Vawda, Reaz; Chung, Young Sun

    2014-01-01

    Abstract Spinal cord injury (SCI) is characterized by vascular disruption with intramedullary hemorrhage, alterations in blood-spinal cord barrier integrity, and perilesional ischemia. A safe and easily applied imaging technique to quantify evolving intraspinal vascular changes after SCI is lacking. We evaluated the utility of very high resolution ultrasound (VHRUS) imaging to assess SCI-induced vascular disruption in a clinically relevant rodent model. The spinal cords of Wistar rats were lesioned at the 11th thoracic vertebra (Th11) by a 35 g 1-minute clip compression. Three-dimensional quantification of intraspinal hemorrhage using VHRUS (at an acute 90-min and subacute 24-h time point post-SCI) was compared with lesional hemoglobin and extravasated Evans blue dye measured spectrophotometrically. The anatomy of hemorrhage was comparatively assessed using VHRUS and histology. Time-lapse videos demonstrated the evolution of parenchymal hemorrhage. VHRUS accurately depicted the structural (gray and white matter) and vascular anatomy of the spinal cord (after laminectomy) and was safely repeated in the same animal. After SCI, a hyperechoic signal extended from the lesion epicenter. Significant correlations were found between VHRUS signal and hemorrhage in the acute (r=0.88, p<0.0001) and subacute (r=0.85, p<0.0001) phases and extravasated Evans blue (a measure of vascular disruption) in the subacute phase (r=0.94, p<0.0001). Time-lapse videos demonstrated that the expanding parenchymal hemorrhage is preceded by new perilesional hemorrhagic foci. VHRUS enables real-time quantitative live anatomical imaging of acute and subacute vascular disruption after SCI in rats. This technique has important scientific and clinical translational applications. PMID:24831774

  17. Selection and validation of reference genes for quantitative real-time PCR analysis of gene expression in Cichorium intybus.

    Science.gov (United States)

    Delporte, Marianne; Legrand, Guillaume; Hilbert, Jean-Louis; Gagneul, David

    2015-01-01

    Plant polyphenols represent a huge reservoir of bioactive compounds. Industrial chicory, an important crop from northwestern Europe, accumulates an original combination of such compounds, i.e., chlorogenic, isochlorogenic, caftaric, and chicoric acids arising from the phenylpropanoid pathway. For a complete understanding of these biochemical pathways, analyses of gene expression using quantitative real-time PCR (qRT-PCR) should be considered. Because cell cultures are a model of choice for specialized metabolism investigations, this study described for the first time the validation of reference genes for this system in chicory. Eighteen potential reference genes were obtained by mining expressed sequence tag databases of chicory for orthologs of Arabidopsis thaliana genes currently used as reference genes. Twelve genes passed the qRT-PCR standard requirements and their expression stability across different samples was tested using three distinct softwares: geNorm, NormFinder, and BestKeeper. In cell cultures grown under various conditions, TIP41 (TIP41 like protein) was shown to be the most stable gene. Further validation of the proposed reference genes was done by normalization of expression levels of a group of genes of interest. In order to assess the potentiality of the proposed list of candidate reference genes, theses genes were in parallel tested on another experimental design, i.e., chicory seedlings. In this case, the best reference gene identified was Clath (Clathrin adaptator complex subunit). The results highlight the importance of the use of properly validated reference genes to achieve relevant interpretation of qRT-PCR analyses. Here, we provide a list of reference genes suitable for future gene expression studies in chicory. PMID:26347767

  18. Selection and validation of reference genes for quantitative real-time PCR analysis of gene expression in Cichorium intybus

    Directory of Open Access Journals (Sweden)

    Marianne eDelporte

    2015-08-01

    Full Text Available Plant polyphenols represent a huge reservoir of bioactive compounds. Industrial chicory, an important crop from northwestern Europe, accumulates an original combination of such compounds i.e. chlorogenic, isochlorogenic, caftaric and chicoric acids arising from the phenylpropanoid pathway. For a complete understanding of these biochemical pathways, analyses of gene expression using quantitative real-time PCR (qRT-PCR should be considered. Because cell cultures are a model of choice for secondary metabolism investigations, this study described for the first time the validation of reference genes for this system in chicory. Eighteen potential reference genes were obtained by mining expressed sequence tag databases of chicory for orthologs of Arabidopsis thaliana genes currently used as reference genes. Twelve genes passed the qRT-PCR standard requirements and their expression stability across different samples was tested using three distinct softwares: geNorm, NormFinder and BestKeeper. In cell cultures grown under various conditions, TIP41 (TIP41 like protein was shown to be the most stable gene. Further validation of the proposed reference genes was done by normalization of expression levels of a group of genes of interest. In order to assess the potentiality of the proposed list of candidate reference genes, theses genes were in parallel tested on another experimental design i.e. chicory seedlings. In this case, the best reference gene identified was Clath (Clathrin adaptator complex subunit. The results highlight the importance of the use of properly validated reference genes to achieve relevant interpretation of qRT-PCR analyses. Here, we provide a list of reference genes suitable for future gene expression studies in chicory.

  19. Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae Using real-time PCR.

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    Ermei Chang

    Full Text Available Platycladus orientalis is a tree species that is highly resistant, widely adaptable, and long-lived, with lifespans of even thousands of years. To explore the mechanisms underlying these characteristics, gene expressions have been investigated at the transcriptome level by RNA-seq combined with a digital gene expression (DGE technique. So, it is crucial to have a reliable set of reference genes to normalize the expressions of genes in P. orientalis under various conditions using the most accurate and sensitive method of quantitative real-time PCR (qRT-PCR. In this study, we selected 10 reference gene candidates from transcriptome data of P. orientalis, and examined their expression profiles by qRT-PCR using 29 different samples of P. orientalis, which were collected from plants of different ages, different tissues, and plants subjected to different treatments including cold, heat, salinity, polyethylene glycol (PEG, and abscisic acid (ABA. Three analytical software packages (geNorm, Bestkeeper, and NormFinder were used to assess the stability of gene expression. The results showed that ubiquitin-conjugating enzyme E2 (UBC and alpha-tubulin (aTUB were the optimum pair of reference genes at all developmental stages and under all stress conditions. ACT7 was the most stable gene across different tissues and cold-treated samples, while UBQ was the most stably expressed reference gene for NaCl- and ABA-treated samples. In parallel, aTUB and UBC were used singly or in combination as reference genes to examine the expression levels of NAC (a homolog of AtNAC2 in plants subjected to various treatments with qRT-PCR. The results further proved the reliability of the two selected reference genes. Our study will benefit future research on the expression of genes in response to stress/senescence in P. orientalis and other members of the Cupressaceae.

  20. Reference gene selection for quantitative real-time reverse-transcriptase PCR in orchardgrass subjected to various abiotic stresses.

    Science.gov (United States)

    Huang, Linkai; Yan, Haidong; Jiang, Xiaomei; Zhang, Yu; Zhang, Xinquan; Ji, Yang; Zeng, Bing; Xu, Bin; Yin, Guohua; Lee, Samantha; Yan, Yanhong; Ma, Xiao; Peng, Yan

    2014-12-15

    Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is a powerful tool for the measurement of gene expression; however, the accuracy of this approach depends on the stability of reference genes. The objective of the present study was to identify the stable reference genes in orchardgrass (Dactylis glomerata L.), a principal cool-season forage grass in the world. Ten candidate reference genes were selected in this study including ATP-binding [ABC], actin [ACTIN], cyclophilin [CYP2], glyceraldehyde 3-phosphate dehydrogenase [GAPDH], beta-amylase 4 [BAM4], zeitlupe [ZTL], MAP Kinase 4 [MPK4], ubiquitin-conjugating enzyme [UBC], S-adenosylmethionine decarboxylase [SAMDC], and translationally controlled tumor protein [TCTP]. The candidate genes were assessed in orchardgrass leaves and roots under conditions of drought, high salinity, heat, waterlogging, and abscisic acid (ABA) treatments. We used GeNorm, BestKeeper, NormFinder, and RefFinder for qRT-PCR normalization and validation to determine that the expression of these reference genes was stress-dependent. ACTIN, CYP2, and ABC were found to be the most stably expressed genes for drought stress while ACTIN, TCTP, and ABC were the most stable under salt stress. ACTIN, CYP2, and ABC were all found to be good reference genes for studying heat stress. Likewise, CYP2, MPK4, and ABC were most suitable to study waterlogging, and ACTIN, CYP2, and MPK4 were determined as the three best reference genes for ABA studies. Our study identified and validated the possible reference genes in orchardgrass that may be used for quantification of target gene expression under various abiotic stresses. PMID:25307767

  1. Validation of reference genes for gene expression analysis in chicory (Cichorium intybus using quantitative real-time PCR

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    Van Bockstaele Erik

    2010-02-01

    Full Text Available Abstract Background Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR is a sensitive technique for quantifying gene expression levels. One or more appropriate reference genes must be selected to accurately compare mRNA transcripts across different samples and tissues. Thus far, only actin-2 has been used as a reference gene for qRT-PCR in chicory, and a full comparison of several candidate reference genes in chicory has not yet been reported. Results Seven candidate reference genes, including nicotinamide adenine dinucleotide dehydrogenase (NADHD, actin (ACT, β-tubulin (TUB, glyceraldehyde-3-phosphate-dehydrogenase (GADPH, histone H3 (H3, elongation factor 1-alpha (EF and 18S rRNA (rRNA were selected to study the expression stability for normalisation of gene expression in chicory. Primer specificity and amplification efficiency were verified for each gene. The expression stability of these genes was analysed across chicory root and leaf tissues using geNorm, NormFinder and BestKeeper software. ACT, EF, and rRNA were the most stable genes as identified by the three different analysis methods. In addition, the use of ACT, EF and GAPDH as reference genes was illustrated by analysing 1-FEHII (FEHII expression in chicory root and leaf tissues. These analyses revealed the biological variation in FEHII transcript expression among the tissues studied, and between individual plants. Conclusions geNorm, NormFinder, and BestKeeper analyses indicated that ACT, EF and rRNA had the highest expression stability across leaf and root tissues, while GAPDH and NADHD showed relatively low expression stability. The results of this study emphasise the importance of validating reference genes for qRT-PCR analysis in chicory. The use of the most stable reference genes such as ACT and EF allows accurate normalisation of gene expression in chicory leaf and root tissues.

  2. In vitro evaluation of five different herbal extracts as an antimicrobial endodontic irrigant using real time quantitative polymerase chain reaction

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    Thilla S Vinothkumar

    2013-01-01

    Full Text Available Context: Sodium hypochlorite is the most commonly used irrigant but it has disadvantage like high cytotoxicity. So there is a need to find an alternative to 5.25% Sodium hypochlorite against microorganism Enterococcus faecalis and Candida albicans. Literature has shown that these 5 extracts namely Terminalia chebula, Myristica frangrans, Aloe barbadensis, Curcuma longa and Azadaricta indica has good properties which can be used as a potential endodontic irrigant. Aims: To evaluate the antimicrobial efficacy of various herbal extracts namely Curcuma longa (CL, Azadiracta indica (AI, Aloe barbadensis (AV, Myristica fragrans (MF and Terminalia chebula (TC as endodontic irrigant against Enterococcus faecalis and Candida albicans using real-time quantitative polymerase chain reaction (qPCR. Materials and Methods: Eighty-four teeth were extracted and suspended with Enterococcus faecalis and Candida albicans. A preliminary study was first performed to determine the minimum inhibitory concentration of extracts. The irrigating groups were divided into five herbal groups and 2 control groups. After irrigating the teeth the remaining microbial load was determined using qPCR. Statistical Analysis Used: Statistical analysis was performed using Oneway Anova/Kruskal-Wallis test with post-hoc Tukey′s HSD and was statistically significant ( P < 0.05. Results: It was shown that Neem was highly efficient to 5.25% NaOCl in reducing Enterococcus faecalis and Candida albicans within the root canals when compared with other extracts. Conclusions: Neem leaf extract has a significant antimicrobial efficacy against Enterococcus faecalis and Candida albicans compared to 5.25% sodium hypochlorite.

  3. Validation of suitable reference genes for gene expression analysis in the halophyte Salicornia europaea by real-time quantitative PCR

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    Xinlong eXiao

    2015-01-01

    Full Text Available Real-time quantitative polymerase chain reaction (RT-qPCR, a reliable technique for quantifying gene expression, requires stable reference genes to normalize its data. Salicornia europaea, a stem succulent halophyte with remarkable salt resistance and high capacity for ion accumulation, has not been investigated with regards to the selection of appropriate reference genes for RT-qPCR. In this study, the expression of 11 candidate reference genes, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase, Actin, α-Tub (α-tubulin, β-Tub (β-tubulin, EF1-α (Elongation factor 1-α, UBC (Ubiquitin-conjugating enzyme, UBQ (Polyubiquitin, CYP (Cyclophilin, TIP41 (TIP41-like protein, CAC (Clathrin adaptor complexes, and DNAJ (DnaJ-like protein, was analyzed in S. europaea samples, which were classified into groups according to various abiotic stresses (NaCl, nitrogen, drought, cold and heat, tissues and ages. Three commonly used software programs (geNorm, NormFinder and BestKeeper were applied to evaluate the stability of gene expression, and comprehensive ranks of stability were generated by aggregate analysis. The results show that the relatively stable genes for each group are the following: (1 CAC and UBC for whole samples; (2 CAC and UBC for NaCl stress; (3 Actin and α-Tub for for nitrogen treatment; (4 Actin and GAPDH for drought stress; (5 α-Tub and UBC for cold stress; (6 TIP41 and DNAJ for heat stress; (7 UBC and UBQ for different tissues; and (8 UBC and Actin for various developmental stages. These genes were validated by comparing transcriptome profiles. Using two stable reference genes was recommended in the normalization of RT-qPCR data. This study identifies optimal reference genes for RT-qPCR in S. europaea, which will benefit gene expression analysis under these conditions.

  4. Exploring the Bacterial Diversity of Belgian Steak Tartare Using Metagenetics and Quantitative Real-Time PCR Analysis.

    Science.gov (United States)

    Delhalle, L; Korsak, N; Taminiau, B; Nezer, C; Burteau, S; Delcenserie, V; Poullet, J B; Daube, G

    2016-02-01

    Steak tartare is a popular meat dish in Belgium. It is prepared with raw minced beef and is eaten with sauce, vegetables, and spices. Because it contains raw meat, steak tartare is highly prone to bacterial spoilage. The objective of this study was to explore the diversity of bacterial flora in steak tartare in Belgium according to the source and to determine which bacteria are able to grow during shelf life. A total of 58 samples from butchers' shops, restaurants, sandwich shops, and supermarkets were collected. These samples were analyzed using 16S rDNA metagenetics, a classical microbiological technique, and quantitative real-time PCR (qPCR) targeting the Lactobacillus genus. Samples were analyzed at the beginning and at the end of their shelf life, except for those from restaurants and sandwich shops, which were analyzed only on the purchase date. Metagenetic analysis identified up to 180 bacterial species and 90 genera in some samples. But only seven bacterial species were predominant in the samples, depending on the source: Brochothrix thermosphacta, Lactobacillus algidus, Lactococcus piscium, Leuconostoc gelidum, Photobacterium kishitani, Pseudomonas spp., and Xanthomonas oryzae. With this work, an alternative method is proposed to evaluate the total flora in food samples based on the number of reads from metagenetic analysis and the results of qPCR. The degree of underestimation of aerobic plate counts at 30°C estimated with the classical microbiology method was demonstrated in comparison with the proposed culture-independent method. Compared with culture-based methods, metagenetic analysis combined with qPCR targeting Lactobacillus provides valuable information for characterizing the bacterial flora of raw meat. PMID:26818982

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

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    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.

  6. Detection and quantification of viable Bacillus cereus group species in milk by propidium monoazide quantitative real-time PCR.

    Science.gov (United States)

    Cattani, Fernanda; Barth, Valdir C; Nasário, Jéssica S R; Ferreira, Carlos A S; Oliveira, Sílvia D

    2016-04-01

    The Bacillus cereus group includes important spore-forming bacteria that present spoilage capability and may cause foodborne diseases. These microorganisms are traditionally evaluated in food using culturing methods, which can be laborious and time-consuming, and may also fail to detect bacteria in a viable but nonculturable state. The purpose of this study was to develop a quantitative real-time PCR (qPCR) combined with a propidium monoazide (PMA) treatment to analyze the contamination of UHT milk by B. cereus group species viable cells. Thirty micrograms per milliliter of PMA was shown to be the most effective concentration for reducing the PCR amplification of extracellular DNA and DNA from dead cells. The quantification limit of the PMA-qPCR assay was 7.5×10(2)cfu/mL of milk. One hundred thirty-five UHT milk samples were analyzed to evaluate the association of PMA to qPCR to selectively detect viable cells. The PMA-qPCR was able to detect B. cereus group species in 44 samples (32.6%), whereas qPCR without PMA detected 78 positive samples (57.8%). Therefore, the PMA probably inhibited the amplification of DNA from cells that were killed during UHT processing, which avoided an overestimation of bacterial cells when using qPCR and, thus, did not overvalue potential health risks. A culture-based method was also used to detect and quantify B. cereus sensu stricto in the same samples and showed positive results in 15 (11.1%) samples. The culture method and PMA-qPCR allowed the detection of B. cereus sensu stricto in quantities compatible with the infective dose required to cause foodborne disease in 3 samples, indicating that, depending on the storage conditions, even after UHT treatment, infective doses may be reached in ready-to-consume products. PMID:26830746

  7. 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

  8. Validation of reference genes for quantitative real-time PCR in valproic acid rat models of autism.

    Science.gov (United States)

    Zhou, Jinlong; Zhang, Xiaozheng; Ren, Junrong; Wang, Ping; Zhang, Junfeng; Wei, Zhaoming; Tian, Yingfang

    2016-08-01

    Autism is a neurodevelopmental disorder, and embryonic exposure to valproic acid (VPA) in rodents is the most frequently studied environmentally triggered autism models. Valproic acid can affect gene transcription as a histone deacetylase inhibitor, and thus may alter the expression of the most genes including reference genes. The aim of the current study is to validate suitable reference genes for quantitative real-time PCR (qPCR) quantification in prefrontal cortex and hippocampus of VPA rat models of autism. Female rats received a single intraperitoneal injection of 400 mg/kg sodium VPA at day 12.5 post-conception and controls were injected with saline. Male offspring were used to observe the expression of nine commonly used reference genes by qPCR, and the data were analyzed by four commonly used reference selection program including geNorm, BestKeeper, NormFinder and RefFinder. The results showed that VPA affected the expression of these commonly used reference genes in prefrontal cortex and hippocampus on postnatal 3, 5 weeks and 80 days, Gapdh and Actin, two very frequently used reference genes, were identified as the least stable genes in VPA group. Hprt1 was selected as the most stable gene, and Hmbs and Tbp were the optimum gene pair in prefrontal cortex and hippocampus across all VPA and controls. Problematically, the use of unstable reference genes results in calculation of different PGRN mRNA expression levels. The results suggest that selection of suitable references is critical for accurate mRNA quantification, and specifically in VPA induced rat models of autism. PMID:27287459

  9. Fibroblast growth factor receptor 1 amplification in non-small cell lung cancer by quantitative real-time PCR.

    Directory of Open Access Journals (Sweden)

    Shirish M Gadgeel

    Full Text Available INTRODUCTION: Amplification of the fibroblast growth factor receptor 1 (FGFR1 gene has been described in tumors of non-small-cell lung cancer (NSCLC patients. Prior reports showed conflicting rates of amplification frequency and clinical relevance. MATERIALS AND METHODS: We developed a reliable real-time quantitative PCR assay to assess the frequency of FGFR1 amplification and assessed the optimal cutoff level of amplification for clinical application. RESULTS: In a training cohort of 203 NSCLCs, we established that a 3.5-fold amplification optimally divided patients into groups with different survival rates with a clear threshold level. Those with FGFR1 amplification levels above 3.5-fold had an inferior survival. These data were confirmed in a validation cohort of 142 NSCLC. After adjusting for age, sex, performance status, stage, and histology, patients with FGFR1 amplification levels above 3.5 fold had a hazard ratio of 2.91 (95% CI- 1.14, 7.41; pvalue-0.025 for death in the validation cohort. The rates of FGFR1 amplification using the cutoff level of 3.5 were 5.1% in squamous cell and 4.1% in adenocarcinomas. There was a non-significant trend towards higher amplifications rates in heavy smokers (> 15 pack-years of cigarette consumption as compared to light smokers. DISCUSSION: Our data suggest that a 3.5-fold amplification of FGFR1 is of clinical importance in NSCLC. Our cutpoint analysis showed a clear threshold effect for the impact of FGFR1 amplification on patients' survival, which can be used as an initial guide for patient selection in trials assessing efficacy of novel FGFR inhibitors.

  10. Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

    Institute of Scientific and Technical Information of China (English)

    Ming Shi; Yong Zhang; Ying-Hua Zhu; Jing Zhang; Wei-Jia Xu

    2008-01-01

    AIM: To compare the clinical performance of a real-time PCR assay with the COBAS Amplicor Hepatitis B Virus (HBV) Monitor test for quantitation of HBV DNA in serum samples. METHODS: The reference sera of the Chinese National Institute for the Control of Pharmaceutical and Biological Products and the National Center for Clinical Laboratories of China, and 158 clinical serum samples were used in this study. The linearity, accuracy, reproducibility, assay time, and costs of the real-time PCR were evaluated and compared with those of the Cobas Amplicor test. RESULTS: The intra-assay and inter-assay variations of the real-time PCR ranged from 0.3% to 3.8% and 1.4% to 8.1%, respectively. The HBV DNA levels measured by the real-time PCR correlated very well with those obtained with the COBAS Amplicor test (r = 0.948). The real-time PCR HBV DNA kit was much cheaper and had a wider dynamic range. CONCLUSION: The real-time PCR assay is an excellent tool for monitoring of HBV DNA levels in patients with chronic hepatitis B.

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

    Directory of Open Access Journals (Sweden)

    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

  12. 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.

  13. 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 Diges

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

    DEFF Research Database (Denmark)

    Nielsen, K. K.; Boye, Mette

    2005-01-01

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

  15. 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...

  16. Quantitative real-time RT-PCR in sentinel lymph nodes from melanoma patients. Detection of melanocytic mRNA predicts disease-free survival

    DEFF Research Database (Denmark)

    Riber-Hansen, Rikke; Abrahamsen, Helene Nortvig; Sorensen, Boe Sandahl;

    2008-01-01

    Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) for specific melanoma markers is more sensitive than histology for detecting cells of melanocytic origin in sentinel lymph nodes (SLNs) in cutaneous melanoma. The clinical significance of a positive qRT-PCR analysis ...

  17. Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR

    Science.gov (United States)

    Hu, Meizhen; Hu, Wenbin; Xia, Zhiqiang; Zhou, Xincheng; Wang, Wenquan

    2016-01-01

    Reverse transcription quantitative real-time polymerase chain reaction (real-time PCR, also referred to as quantitative RT-PCR or RT-qPCR) is a highly sensitive and high-throughput method used to study gene expression. Despite the numerous advantages of RT-qPCR, its accuracy is strongly influenced by the stability of internal reference genes used for normalizations. To date, few studies on the identification of reference genes have been performed on cassava (Manihot esculenta Crantz). Therefore, we selected 26 candidate reference genes mainly via the three following channels: reference genes used in previous studies on cassava, the orthologs of the most stable Arabidopsis genes, and the sequences obtained from 32 cassava transcriptome sequence data. Then, we employed ABI 7900 HT and SYBR Green PCR mix to assess the expression of these genes in 21 materials obtained from various cassava samples under different developmental and environmental conditions. The stability of gene expression was analyzed using two statistical algorithms, namely geNorm and NormFinder. geNorm software suggests the combination of cassava4.1_017977 and cassava4.1_006391 as sufficient reference genes for major cassava samples, the union of cassava4.1_014335 and cassava4.1_006884 as best choice for drought stressed samples, and the association of cassava4.1_012496 and cassava4.1_006391 as optimal choice for normally grown samples. NormFinder software recommends cassava4.1_006884 or cassava4.1_006776 as superior reference for qPCR analysis of different materials and organs of drought stressed or normally grown cassava, respectively. Results provide an important resource for cassava reference genes under specific conditions. The limitations of these findings were also discussed. Furthermore, we suggested some strategies that may be used to select candidate reference genes. PMID:27242878

  18. Quantitative Analysis of the Relative Transcript Levels of ABC Transporter Atr Genes in Aspergillus nidulans by Real-Time Reverse Transcription-PCR Assay

    OpenAIRE

    Pizeta Semighini, Camile; Marins, Mozart; Goldman, Maria Helena S.; Goldman, Gustavo Henrique

    2002-01-01

    The development of assays for quantitative analysis of the relative transcript levels of ABC transporter genes by real-time reverse transcription-PCR (RT-PCR) might provide important information about multidrug resistance in filamentous fungi. Here, we evaluate the potential of real-time RT-PCR to quantify the relative transcript levels of ABC transporter Atr genes from Aspergillus nidulans. The AtrA to AtrD genes showed different and higher levels in the presence of structurally unrelated dr...

  19. 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

  20. 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.

  1. 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

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Huang, S-H; Tsai, M-H; Lin, C-W [Department of Biotechnology, College of Health Science, Asia University, Wufeng, Taichung, Taiwan (China); Yang, T-C; Chuang, P-H [Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan (China); Tsai, I-S; Lu, H-C [Nanotechnology Research Center, Feng Chia University, Taichung, Taiwan (China); Wan Lei; Lin, Y-J [Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung, Taiwan (China); Lai, C-H [Department of Microbiology and Immunology, China Medical University, Taichung, Taiwan (China)], E-mail: cwlin@mail.cmu.edu.tw

    2008-10-08

    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.

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

    Science.gov (United States)

    Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

    2008-10-01

    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.

  5. 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.

  6. A frequency response analysis approach for quantitative assessment of actuator tracking for real-time hybrid simulation

    International Nuclear Information System (INIS)

    Real-time hybrid simulation is a viable and economical technique that allows researchers to observe the behavior of critical elements at full scale when an entire structure is subjected to dynamic loading. To ensure reliable experimental results, it is necessary to evaluate the actuator tracking after the test, even when sophisticated compensation methods are used to negate the detrimental effect of servo-hydraulic dynamics. Existing methods for assessment of actuator tracking are often based on time-domain analysis. This paper proposes a frequency-domain-based approach to the assessment of actuator tracking for real-time hybrid simulations. To ensure the accuracy of the proposed frequency response approach, the effects of spectrum leakage are investigated as well as the length and sampling frequency requirements of the signals. Two signal pre-processing techniques (data segmentation and window transform) are also discussed and compared to improve the accuracy of the proposed approach. Finally the effectiveness of the proposed frequency-domain-based approach is demonstrated through both computational analyses and laboratory tests, including real-time tests with predefined displacement and real-time hybrid simulation. (paper)

  7. A frequency response analysis approach for quantitative assessment of actuator tracking for real-time hybrid simulation

    Science.gov (United States)

    Guo, Tong; Chen, Cheng; Xu, WeiJie; Sanchez, Frank

    2014-04-01

    Real-time hybrid simulation is a viable and economical technique that allows researchers to observe the behavior of critical elements at full scale when an entire structure is subjected to dynamic loading. To ensure reliable experimental results, it is necessary to evaluate the actuator tracking after the test, even when sophisticated compensation methods are used to negate the detrimental effect of servo-hydraulic dynamics. Existing methods for assessment of actuator tracking are often based on time-domain analysis. This paper proposes a frequency-domain-based approach to the assessment of actuator tracking for real-time hybrid simulations. To ensure the accuracy of the proposed frequency response approach, the effects of spectrum leakage are investigated as well as the length and sampling frequency requirements of the signals. Two signal pre-processing techniques (data segmentation and window transform) are also discussed and compared to improve the accuracy of the proposed approach. Finally the effectiveness of the proposed frequency-domain-based approach is demonstrated through both computational analyses and laboratory tests, including real-time tests with predefined displacement and real-time hybrid simulation.

  8. Real-time systems

    OpenAIRE

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

    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...

  9. Quantitative detection of pork in commercial meat products by TaqMan® real-time PCR assay targeting the mitochondrial D-loop region.

    Science.gov (United States)

    Kim, Miju; Yoo, Insuk; Lee, Shin-Young; Hong, Yeun; Kim, Hae-Yeong

    2016-11-01

    The TaqMan® real-time PCR assay using the mitochondrial D-loop region was developed for the quantitative detection of pork in processed meat products. The newly designed primers and probe specifically amplified pork without any cross-reactivity with non-target animal species. The limit of detection of the real-time PCR assay was 0.1pg of heat-treated pork meat and 0.1% (w/w) pork meat in beef and chicken meat mixtures. The quantitative real-time PCR assay was applied to analyze the pork meat content in 22 commercial processed meat products including jerkies, press hams, sausages, hamburger patties and steaks, grilled short rib patties, and nuggets. The developed real-time PCR method was able to detect pork meat in various types of processed meat products that declared the use of pork meat on their label. All processed meat products that declared no use of pork meat showed a negative result in the assay. The method developed in this study showed sensitivity and specificity in the quantification of pork meat in commercial processed meat products. PMID:27211626

  10. Analysis of maternal microchimerism in rhesus monkeys (Macaca mulatta) using real-time quantitative PCR amplification of MHC polymorphisms

    OpenAIRE

    Bakkour, Sonia; Baker, Chris AR; Tarantal, Alice F; Wen, Li; Busch, Michael P; Lee, Tzong-Hae; McCune, Joseph M

    2014-01-01

    Although pregnancy-associated microchimerism is known to exist in humans, its clinical significance remains unclear. Fetal microchimerism has been documented in rhesus monkeys, but the trafficking and persistence of maternal cells in the monkey fetus and infant have not been fully explored. To investigate the frequency of maternal microchimerism in the rhesus monkey (Macaca mulatta), a real-time polymerase chain reaction (PCR) strategy was developed and validated to target polymorphic major h...

  11. Quantification of viable Giardia cysts and Cryptosporidium oocysts in wastewater using propidium monoazide quantitative real-time PCR

    OpenAIRE

    Alonso Molina, José Luís; Amoros-Muñoz, Inmaculada; Guy, Rebecca A.

    2014-01-01

    Real-time PCR (qPCR) is a rapid tool to quantify pathogens in the aquatic environment; however, it quantifies all pathogens, including both viable and nonviable. Propidium monoazide (PMA) is a membrane-impairment dye that penetrates only membrane-damaged cells. Once inside the cell, PMA is covalently cross-linked to DNA through light photoactivation, and PCR amplification is strongly inhibited. The goal of this study was to evaluate PMA-qPCR assays for rapid quantifica...

  12. Quantitative Real-Time PCR Detection of Rift Valley Fever Virus and Its Application to Evaluation of Antiviral Compounds

    OpenAIRE

    Garcia, Stephan; Crance, Jean Marc; Billecocq, Agnes; Peinnequin, Andre; Jouan, Alain; Bouloy, Michele; Garin, Daniel

    2001-01-01

    The Rift Valley fever virus (RVFV), a member of the genus Phlebovirus (family Bunyaviridae) is an enveloped negative-strand RNA virus with a tripartite genome. Until 2000, RVFV circulation was limited to the African continent, but the recent deadly outbreak in the Arabian Peninsula dramatically illustrated the need for rapid diagnostic methods, effective treatments, and prophylaxis. A method for quantifying the small RNA segment by a real-time detection reverse transcription (RT)-PCR using Ta...

  13. 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.

  14. 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

    2015-01-01

    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. PMID:26492259

  15. 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. PMID:27157323

  16. LEMming: A Linear Error Model to Normalize Parallel Quantitative Real-Time PCR (qPCR) Data as an Alternative to Reference Gene Based Methods

    OpenAIRE

    Feuer, Ronny; Vlaic, Sebastian; Arlt, Janine; Sawodny, Oliver; Dahmen, Uta; Zanger, Ulrich M; Thomas, Maria

    2015-01-01

    Background Gene expression analysis is an essential part of biological and medical investigations. Quantitative real-time PCR (qPCR) is characterized with excellent sensitivity, dynamic range, reproducibility and is still regarded to be the gold standard for quantifying transcripts abundance. Parallelization of qPCR such as by microfluidic Taqman Fluidigm Biomark Platform enables evaluation of multiple transcripts in samples treated under various conditions. Despite advanced technologies, cor...

  17. [The development of a test-system for the quantitative and qualitative evaluation of DNA content in criminalistic objects by the real-time polymerase chain reaction].

    Science.gov (United States)

    Lapenkov, M I; Plakhina, N V; Alekseev, Ia I; Varlamov, D A

    2011-01-01

    An original test-system for the preliminary quantitative and qualitative evaluation of isolated DNA is proposed by the polymerase chain reaction in real time (PCR-RT) based on the TaqMan technology. This test-system permits to simultaneously measure the amount of DNA in the sample, identify the genetic gender, and detect PCR inhibitors. The method has been approbated in the practical work of forensic medical experts. PMID:21735715

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

    OpenAIRE

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

    2014-01-01

    Background 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 mR...

  19. Relationship between pp65 antigenemia levels and real-time quantitative DNA PCR for Human Cytomegalovirus (HCMV) management in immunocompromised patients

    OpenAIRE

    Bonfanti Carlo; Perandin Francesca; Valloncini Barbara; Pollara Caterina P; Cariani Elisabetta; Manca Nino

    2007-01-01

    Abstract Background Quantitative real-time PCR assays, which are more rapid and practical than pp65 antigenemia determination, are progressively becoming the preferred method for monitoring Human Cytomegalovirus (HCMV) reactivation. However, the relationship between HCMV DNA and antigenemia levels is still under investigation. The aim of this study was to analyse the relationship between HCMV DNA and pp65 antigenemia levels in order to identify clinically useful threshold values for the manag...

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

    OpenAIRE

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

    2010-01-01

    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 i...

  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

    OpenAIRE

    Ralevski Filip; Lau Rachel; Martin Donald; Khairnar Krishna; Pillai Dylan R

    2009-01-01

    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 polymera...

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

    OpenAIRE

    Pascal E. Saikaly; 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...

  3. A new immunoprecipitation-real time quantitative PCR assay for anti-Th/To and anti-U3RNP antibody detection in systemic sclerosis

    OpenAIRE

    Ceribelli, A; Satoh, M; E.K.L. Chan

    2012-01-01

    Introduction Classic anti-nucleolar antibodies anti-Th/To and U3 ribonucleoprotein (-U3RNP) can help in the diagnosis, prediction of organ involvement and prognosis in systemic sclerosis (SSc); however, no validated commercial assay is available. We aimed at establishing a novel quantitative real time PCR (qPCR) method to detect these antibodies. Methods Standard immunoprecipitation (IP) was performed using K562 cell extract and RNA components were extracted. cDNA was reverse transcribed from...

  4. Real-Time PCR

    Science.gov (United States)

    Evrard, A.; Boulle, N.; Lutfalla, G. S.

    Over the past few years there has been a considerable development of DNA amplification by polymerase chain reaction (PCR), and real-time PCR has now superseded conventional PCR techniques in many areas, e.g., the quantification of nucleic acids and genotyping. This new approach is based on the detection and quantification of a fluorescent signal proportional to the amount of amplicons generated by PCR. Real-time detection is achieved by coupling a thermocycler with a fluorimeter. This chapter discusses the general principles of quantitative real-time PCR, the different steps involved in implementing the technique, and some examples of applications in medicine. The polymerase chain reaction (PCR) provides a way of obtaining a large number of copies of a double-stranded DNA fragment of known sequence. This DNA amplification technique, developed in 1985 by K. Mullis (Cetus Corporation), saw a spectacular development over the space of a few years, revolutionising the methods used up to then in molecular biology. Indeed, PCR has many applications, such as the detection of small amounts of DNA, cloning, and quantitative analysis (assaying), each of which will be discussed further below.

  5. Quantitative TaqMan® real-time PCR assays for gene expression normalisation in feline tissues

    Directory of Open Access Journals (Sweden)

    Riond Barbara

    2009-12-01

    Full Text Available Abstract Background Gene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels. Co-analysis of suitable reference genes is crucial for accurate expression normalisation. Reference gene expression may vary, e.g., among species or tissues; thus, candidate genes must be tested prior to use in expression studies. The domestic cat is an important study subject in both medical research and veterinary medicine. The aim of the present study was to develop TaqMan® real-time PCR assays for eight potential reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from healthy cats, and neoplastic tissues from FeLV-infected cats. Results RNA extraction from tissues was optimised for minimal genomic DNA (gDNA contamination without use of a DNase treatment. Real-time PCR assays were established and optimised for v-abl Abelson murine leukaemia viral oncogene homolog (ABL, β-actin (ACTB, β-2-microglobulin (B2M, β-glucuronidase (GUSB, hydroxymethyl-bilane synthase (HMBS, hypoxanthine phosphoribosyltransferase (HPRT, ribosomal protein S7 (RPS7, and tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ. The presence of pseudogenes was confirmed for four of the eight investigated genes (ACTB, HPRT, RPS7, and YWHAZ. The assays were tested together with previously developed TaqMan® assays for feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH and the universal 18S rRNA gene. Significant differences were found among the expression levels of the ten candidate reference genes, with a ~106-fold expression difference between the most abundant (18S rRNA and the least abundant genes (ABL, GUSB, and HMBS. The expression stability determined by the geNorm and NormFinder programs differed significantly. Using the ANOVA-based NormFinder program, RPS7 was the most stable gene in the

  6. Development of TaqMan real-time reverse transcription-polymerase chain reaction for the detection and quantitation of porcine kobuvirus.

    Science.gov (United States)

    Zhu, Xiangdong; Wang, Yufei; Chen, Jianfei; Zhang, Xin; Shi, Hongyan; Shi, Da; Gao, Jing; Feng, Li

    2016-08-01

    Porcine kobuvirus (PKV) is a newly emerging virus that has been detected in diarrheic pigs. Presently, reverse transcription-polymerase chain reaction (RT-PCR) and RT-loop-mediated amplification are the only methods that can be used to detect PKV. To develop a TaqMan real-time RT-PCR for the rapid detection and quantitation of PKV nucleic acid in fecal samples, a pair of primers and a probe were designed to amplify the conserved 3D region of the PKV genome. After optimization, the TaqMan real-time RT-PCR was highly specific and ∼1000 times more sensitive than conventional RT-PCR, and the detection limit was as low as 30 DNA copies. Among the 148 intestinal samples from piglets with diarrhea, 136 and 118 were positive based on the TaqMan and conventional RT-PCR methods, respectively, indicating that the TaqMan RT-PCR was more sensitive than conventional RT-PCR, and the total concordance of the two methods was approximately 87.84%. Thus, the TaqMan real-time RT-PCR should be a useful tool for the early detection and quantitation of PKV. PMID:26912233

  7. Quantitative detection of Aeromonas salmonicida in fish tissue by real-time PCR using self-quenched, fluorogenic primers.

    Science.gov (United States)

    Balcázar, José Luis; Vendrell, Daniel; de Blas, Ignacio; Ruiz-Zarzuela, Imanol; Gironés, Olivia; Múzquiz, José Luis

    2007-03-01

    In this study a real-time PCR assay using self-quenched primers labelled with a single fluorophore for the detection of Aeromonas salmonicida was developed. Probe specificity was confirmed by amplification of 16 A. salmonicida strain templates and by the lack of a PCR product with 26 non-A. salmonicida strains. With a pure culture of A. salmonicida, the assay was linear over a range of 0.5 pg to 50 ng and was able to detect 16 c.f.u. per reaction. A similar sensitivity was observed in DNA extracted from a mixture of A. salmonicida and fish tissue. Results using artificially inoculated tissues and diseased fish from outbreaks indicated that the assay can provide sensitive species-specific detection and quantification of A. salmonicida in fish tissue. PMID:17314361

  8. Validation and standardization of IS900 and F57 real-time quantitative PCR assays for the specific detection and quantification of Mycobacterium avium subsp. paratuberculosis.

    Science.gov (United States)

    Sidoti, Francesca; Banche, Giuliana; Astegiano, Sara; Allizond, Valeria; Cuffini, Anna Maria; Bergallo, Massimiliano

    2011-05-01

    Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease and may contribute to the onset and development of Crohn's disease in humans. Rapid detection of Map is fundamental because of its reported isolation from pasteurized milk and its potential for transmission through environmental sources. In this study, we developed two independent real-time quantitative PCR assays targeting the IS900 genetic insertion sequence and the F57 sequence, which proved capable of detecting and quantifying Map DNA. Validation and standardization of the developed methods were performed by evaluating diagnostic trueness, precision, and accuracy of the techniques. Specificity of the IS900 and F57 methods was verified in both in silico and experimental studies. The assays were found to be very accurate and precise with high repeatability and reproducibility. Moreover, the two real-time assays were very specific for Map, discriminating most of mycobacterial and nonmycobacterial species. PMID:21510779

  9. Absolute High-Precision Localisation of an Unmanned Ground Vehicle by Using Real-Time Aerial Video Imagery for Geo-referenced Orthophoto Registration

    Science.gov (United States)

    Kuhnert, Lars; Ax, Markus; Langer, Matthias; Nguyen van, Duong; Kuhnert, Klaus-Dieter

    This paper describes an absolute localisation method for an unmanned ground vehicle (UGV) if GPS is unavailable for the vehicle. The basic idea is to combine an unmanned aerial vehicle (UAV) to the ground vehicle and use it as an external sensor platform to achieve an absolute localisation of the robotic team. Beside the discussion of the rather naive method directly using the GPS position of the aerial robot to deduce the ground robot's position the main focus of this paper lies on the indirect usage of the telemetry data of the aerial robot combined with live video images of an onboard camera to realise a registration of local video images with apriori registered orthophotos. This yields to a precise driftless absolute localisation of the unmanned ground vehicle. Experiments with our robotic team (AMOR and PSYCHE) successfully verify this approach.

  10. Quantitative detection of Legionella pneumophila in water samples by immunomagnetic purification and real-time PCR amplification of the dotA gene.

    Science.gov (United States)

    Yáñez, M A; Carrasco-Serrano, C; Barberá, V M; Catalán, V

    2005-07-01

    A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples. PMID:16000746

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

    International Nuclear Information System (INIS)

    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)

  12. Comparison of Six Methods of Extracting Mycobacterium tuberculosis DNA from Processed Sputum for Testing by Quantitative Real-Time PCR

    OpenAIRE

    Aldous, Wade K.; Pounder, June I.; Cloud, Joann L.; Woods, Gail L.

    2005-01-01

    Six methods of extracting Mycobacterium tuberculosis DNA from sputum for testing by quantitative PCR were compared: Tris-EDTA (TE) buffer, PrepMan Ultra, 2% sodium dodecyl sulfate (SDS)-10% Triton X with and without sonication, Infectio Diagnostics, Inc. (IDI) lysing tubes, and QIAGEN QIAamp DNA mini kit; all included a 15-min boiling step. Pooled digested and decontaminated sputum was spiked with M. tuberculosis ATCC 27294. Each extraction method was repeated eight times. Quantitative PCR wa...

  13. Selection of reference genes for gene expression studies in Siberian Apricot (Prunus sibirica L. Germplasm using quantitative real-time PCR.

    Directory of Open Access Journals (Sweden)

    Jun Niu

    Full Text Available Quantitative real time reverse transcription polymerase chain reaction has been applied in a vast range of studies of gene expression analysis. However, real-time PCR data must be normalized with one or more reference genes. In this study, eleven putative consistently expressed genes (ACT, TUA, TUB, CYP, DNAj, ELFA, F-box27, RPL12, GAPDH, UBC and UBQ in nine Siberian Apricot Germplasms (including much variability were evaluated for their potential as references for the normalization of gene expression by NormFinder and geNorm programs. From our studies, ACT, UBC, CYP, UBQ and RPL12 as suitable for normalization were identified by geNorm, while UBC and CYP as the best pair by NormFinder. Moreover, UBC was selected as the most stably expressed gene by both algorithms in different Siberian Apricot seed samples. We also detected that a set of three genes (ACT, CYP and UBC by geNorm as control for normalization could lead to accurate results. Furthermore, the expression levels of oleosin gene were analyzed to validate the suitability of the selected reference genes. These obtained experimental results could make an important contribution to normalize real-time PCR data for gene expression analysis in Siberian Apricot Germplasm.

  14. A field-portable membrane introduction mass spectrometer for real-time quantitation and spatial mapping of atmospheric and aqueous contaminants.

    Science.gov (United States)

    Bell, Ryan J; Davey, Nicholas G; Martinsen, Morten; Collin-Hansen, Christian; Krogh, Erik T; Gill, Christopher G

    2015-02-01

    Environmental concentrations of volatile and semivolatile organic compounds (VOC/SVOCs) can vary dramatically in time and space under the influence of environmental conditions. In an industrial setting, multiple point and diffuse sources can contribute to fugitive emissions. Assessments and monitoring programs using periodic grab sampling provide limited information, often with delay times of days or weeks. We report the development and use of a novel, portable membrane introduction mass spectrometry (MIMS) system capable of resolving and quantifying VOC and SVOCs with high spatial and temporal resolution, in the field, in real-time. An electron impact ionization cylindrical ion trap mass spectrometer modified with a capillary hollow fiber polydimethylsiloxane membrane interface was used for continuous air and water sampling. Tandem mass spectrometry and selected ion monitoring scans performed in series allowed for the quantitation of target analytes, and full scan mode was used to survey for unexpected analytes. Predeployment and in-field external calibrations were combined with a continuously infused internal standard to enable real-time quantitation and monitor instrument performance. The system was operated in a moving vehicle with internet-linked data processing and storage. Software development to integrate MIMS and relevant meta-data for visualization and geospatial presentation in Google Earth is presented. Continuous quantitation enables the capture of transient events that may be missed or under-represented by traditional grab sampling strategies. Real-time geospatial maps of chemical concentration enable adaptive sampling and in-field decision support. Sample datasets presented in this work were collected in Northern Alberta in 2010-2012. PMID:25477082

  15. Improvement of Radar Quantitative Precipitation Estimation Based on Real-Time Adjustments to Z-R Relationships and Inverse Distance Weighting Correction Schemes

    Institute of Scientific and Technical Information of China (English)

    WANG Gaili; LIU Liping; DING Yuanyuan

    2012-01-01

    The errors in radar quantitative precipitation estimations consist not only of systematic biases caused by random noises but also spatially nonuniform biases in radar rainfall at individual rain-gauge stations.In this study,a real-time adjustment to the radar reflectivity-rainfall rates (Z R) relationship scheme and the gauge-corrected,radar-based,estimation scheme with inverse distance weighting interpolation was developed.Based on the characteristics of the two schemes,the two-step correction technique of radar quantitative precipitation estimation is proposed.To minimize the errors between radar quantitative precipitation estimations and rain gauge observations,a real-time adjustnent to the Z-R relationship scheme is used to remove systematic bias on the time-domain.The gauge-corrected,radar-based,estination scheme is then used to eliminate non-uniform errors in space.Based on radar data and rain gauge observations near the Huaihe River,the two-step correction technique was evaluated using two heavy-precipitation events.The results show that the proposed scheme improved not only in the underestination of rainfall but also reduced the root-mean-square error and the mean relative error of radar-rain gauge pairs.

  16. Identification of Suitable Reference Genes for Gene Expression Normalization in the Quantitative Real-Time PCR Analysis of Sweet Osmanthus (Osmanthus fragrans Lour.)

    OpenAIRE

    Zhang, Chao; Fu, Jianxin; Wang, Yiguang; Bao, Zhiyi; Zhao, Hongbo

    2015-01-01

    Quantitative real-time PCR (RT-qPCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. Several studies examining the selection of reference genes have been performed in ornamental plants but none in sweet osmanthus (Osmanthus fragrans Lour.). Based on transcriptomic sequencing data from O. fragrans buds at four developmental stages, six reference genes (OfACT, OfEF1α, OfIDH, OfRAN1, OfTUB, and OfUBC2) with st...

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

    OpenAIRE

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

    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, usi...

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

    OpenAIRE

    M.V. Resende; A.C. Lucio; A.P. Perini; L.Z Oliveira; A.O. Almeida; B.C.A Alves; Moreira-Filho, C. A.; I. W. Santos; V.F.M. Hossepian de Lima

    2011-01-01

    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 ...

  19. New In Situ Capture Quantitative (Real-Time) Reverse Transcription-PCR Method as an Alternative Approach for Determining Inactivation of Tulane Virus

    OpenAIRE

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

    2014-01-01

    Human noroviruses (HuNoVs) are the major cause of epidemic nonbacterial gastroenteritis. Although quantitative (real-time) reverse transcription-PCR (qRT-PCR) is widely used for detecting HuNoVs, it only detects the presence of viral RNA and does not indicate viral infectivity. Human blood group antigens (HBGAs) have been identified as receptors/co-receptors for both HuNoVs and Tulane virus (TV) and are crucial for viral infection. We propose that viral infectivity can be evaluated with a mol...

  20. Use of Quantitative Real-Time PCR To Study the Kinetics of Extracellular DNA Released from Candida albicans, with Implications for Diagnosis of Invasive Candidiasis

    OpenAIRE

    Kasai, Miki; Francesconi, Andrea; Petraitiene, Ruta; Petraitis, Vidmantas; Kelaher, Amy M.; Kim, Hee-sup; Meletiadis, Joseph; Sein, Tin; Bacher, John; Walsh, Thomas J.

    2006-01-01

    Quantitative real-time PCR (qPCR) is considered one of the most sensitive methods to detect low levels of DNA from pathogens in clinical samples. To improve the design of qPCR for the detection of deeply invasive candidiasis, we sought to develop a more comprehensive understanding of the kinetics of DNA released from Candida albicans in vitro and in vivo. We developed a C. albicans-specific assay targeting the rRNA gene complex and studied the kinetics of DNA released from C. albicans alone, ...

  1. 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

    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...... 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...

  2. The combination of hypoxia-response enhancers and an oxygen-dependent proteolytic motif enables real-time imaging of absolute HIF-1 activity in tumor xenografts

    International Nuclear Information System (INIS)

    The transcriptional activity of hypoxia-inducible factor-1 (HIF-1) is associated with tumor malignancies; therefore, it is important to comprehend its dynamism in solid tumors. However, a molecular imaging strategy to accurately access it remains to be developed. We constructed here a novel HIF-1-dependent reporter gene, 5HREp-ODD-luc, in which 5 copies of the hypoxia-response element (5HRE) enhance expression of the oxygen-dependent degradation (ODD) domain and luciferase (luc) fusion under hypoxia. Because the ODD domain caused the oxygen-dependent degradation of the ODD-Luc protein, the novel reporter gene showed little leak of luminescence under normoxia. Such a property caused an increase of the hypoxia-responsiveness up to about 4.7 x 104-fold. Moreover, the ODD domain caused rapid degradation of the ODD-Luc protein under normoxia, the luminescence reflected the dynamism of HIF-1 activity in real-time. The superiority of the novel reporter gene will surely accelerate analysis of the intratumoral HIF-1 activity during tumor progression and cancer treatments

  3. 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......, such as in vitro antimicrobic susceptibility testing or in vivo immunopathological experiments....

  4. 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; Vestereng, Vibeke H; Lundgren, Bettina; Fischer, Steven H; Gill, Vee J

    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 <5 copies of a plasmid standard per tube. It was reproducibly quantitative (r = 0.99) over...... 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. 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...

  5. Evaluation of a Rapid, Quantitative Real-Time PCR Method for Enumeration of Pathogenic Candida Cells in Water

    OpenAIRE

    Brinkman, Nichole E.; Haugland, Richard A.; Wymer, Larry J.; Byappanahalli, Muruleedhara; Whitman, Richard L.; Vesper, Stephen J.

    2003-01-01

    Quantitative PCR (QPCR) technology, incorporating fluorigenic 5′ nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one ...

  6. Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid `real-time' polymerase chain reaction

    OpenAIRE

    Pirnay, Jean-Paul; Vos, Daniel De; Duinslaeger, Luc; Reper, Pascal; Vandenvelde, Christian; Cornelis, Pierre; Vanderkelen, Alain

    2000-01-01

    Introduction: Early diagnosis of wound colonisation or prediction of wound sepsis provides an opportunity for therapeutic intervention. There is need for qualitative and quantitative tests that are more rapid than bacterial culture. Pseudomonas aeruginosa results in high morbidity and mortality rates, is inherently resistant to common antibiotics, and is increasingly being isolated as a nosocomial pathogen. We developed three PCR-based methods to detect and quantify P aeruginosa in wound biop...

  7. 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.

  8. Rapid quantification of viable Campylobacter on chicken carcasses by real-time PCR and propidium monoazide as a tool for quantitative risk assessment

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Löfström, Charlotta; Hansen, Tina Beck; Christensen, Laurids Siig; Olsen, John Elmerdahl; Hoorfar, Jeffrey

    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...... of foodborne Campylobacter, combining real-time PCR (Q-PCR) with a simple propidium monoazide (PMA) sample treatment. In less than 3 hours, this method generates a signal from only viable and viable but non-culturable (VBNC) Campylobacter with an intact membrane. The method performance was evaluated...... correlated with Ct-values (R2 = 0.993), with a quantification range from 1×1021×107 CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R2 = 0.844). The amplification efficiency of the Q-PCR method was not affected by chicken...

  9. Evaluation of a rapid, quantitative real-time PCR method for enumeration of pathogenic Candida cells in water

    Science.gov (United States)

    Brinkman, Nichole E.; Haugland, Richard A.; Wymer, Larry J.; Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Vesper, Stephen J.

    2003-01-01

    Quantitative PCR (QPCR) technology, incorporating fluorigenic 5′ nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

  10. Selection of Reference Genes for Quantitative Real-Time PCR during Flower Development in Tree Peony (Paeonia suffruticosa Andr.)

    Science.gov (United States)

    Li, Jian; Han, Jigang; Hu, Yonghong; Yang, Ji

    2016-01-01

    Tree peony (Paeonia suffruticosa) is a perennial plant indigenous to China known for its elegant and vibrantly colorful flowers. A few genes involved in petal pigmentation have been cloned in tree peony. However, to date, there have been few studies on the comparison and selection of stable reference genes for gene expression analysis by quantitative reverse-transcription PCR (qRT-PCR) in this species. In this study, 10 candidate reference genes were evaluated for the normalization of qRT-PCR in three tree peony cultivars. GAPDH and UBC were identified as the top two most stable reference genes in ‘Feng Dan’ and ‘Xi Shi,’ and EF-1α/UBC was recommended to be the best combination for ‘Que Hao.’ The expression stability of various reference genes differed across cultivars, suggesting that selection and validation of reliable reference genes for quantitative gene expression analysis was necessary not only for different species but also for different cultivars. The results provided a list of reference genes for further study on gene expression in P. suffruticosa. However, in any case, a preliminary check on the accuracy of the best performing reference genes is requested for each qRT-PCR experiment. PMID:27148337

  11. 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.

  12. Development and validation of a harmonized TaqMan-based triplex real-time RT-PCR protocol for the quantitative detection of normalized gene expression profiles of seven porcine cytokines.

    Directory of Open Access Journals (Sweden)

    Anja Petrov

    Full Text Available Dysregulation of cytokine responses plays a major role in the pathogenesis of severe and life-threatening infectious diseases like septicemia or viral hemorrhagic fevers. In pigs, diseases like African and classical swine fever are known to show exaggerated cytokine releases. To study these responses and their impact on disease severity and outcome in detail, reliable, highly specific and sensitive methods are needed. For cytokine research on the molecular level, real-time RT-PCRs have been proven to be suitable. Yet, the currently available and most commonly used SYBR Green I assays or heterogeneous gel-based RT-PCRs for swine show a significant lack of specificity and sensitivity. The latter is however absolutely essential for an accurate quantification of rare cytokine transcripts as well as for detection of small changes in gene expressions. For this reason, a harmonized TaqMan-based triplex real-time RT-PCR protocol for the quantitative detection of normalized gene expression profiles of seven porcine cytokines was designed and validated within the presented study. Cytokines were chosen to represent different immunological pathways and targets known to be involved in the pathogenesis of the above mentioned porcine diseases, namely interleukin (IL-1β, IL-2, IL-4, IL-6, IL-8, tumor necrosis factor (TNF-α and interferon (IFN-α. Beta-Actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH served as reference genes for normalization. For absolute quantification a synthetic standard plasmid was constructed comprising all target cytokines and reference genes within a single molecule allowing the generation of positive control RNA. The standard as well as positive RNAs from samples, and additionally more than 400 clinical samples, which were collected from animal trials, were included in the validation process to assess analytical sensitivity and applicability under routine conditions. The resulting assay allows the reliable assessment of gene

  13. Establishment of TaqMan Real-time Quantitative PCR Assay for Foreign Gene Copy Numbers in Transgenic Soybean

    Institute of Scientific and Technical Information of China (English)

    Qiu You-wen; Gao Xue-jun; Qi Bang-ruo; Li Lu; Zhen Zhen

    2012-01-01

    TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.

  14. In-depth analysis of internal control genes for quantitative real-time PCR in Brassica oleracea var. botrytis.

    Science.gov (United States)

    Sheng, X G; Zhao, Z Q; Yu, H F; Wang, J S; Zheng, C F; Gu, H H

    2016-01-01

    Quantitative reverse-transcription PCR (qRT-PCR) is a versatile technique for the analysis of gene expression. The selection of stable reference genes is essential for the application of this technique. Cauliflower (Brassica oleracea L. var. botrytis) is a commonly consumed vegetable that is rich in vitamin, calcium, and iron. Thus far, to our knowledge, there have been no reports on the validation of suitable reference genes for the data normalization of qRT-PCR in cauliflower. In the present study, we analyzed 12 candidate housekeeping genes in cauliflower subjected to different abiotic stresses, hormone treatment conditions, and accessions. geNorm and NormFinder algorithms were used to assess the expression stability of these genes. ACT2 and TIP41 were selected as suitable reference genes across all experimental samples in this study. When different accessions were compared, ACT2 and UNK3 were found to be the most suitable reference genes. In the hormone and abiotic stress treatments, ACT2, TIP41, and UNK2 were the most stably expressed. Our study also provided guidelines for selecting the best reference genes under various experimental conditions. PMID:27525844

  15. Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR.

    Science.gov (United States)

    Zampieri, Denise; Nora, Luísa C; Basso, Vanessa; Camassola, Marli; Dillon, Aldo J P

    2014-08-01

    Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a methodology that facilitates the quantification of mRNA expression in a given sample. Analysis of relative gene expression by qRT-PCR requires normalization of the data using a reference gene that is expressed at a similar level in all evaluated conditions. Determining an internal control gene is essential for gene expression studies. Gene expression studies in filamentous fungi frequently use the β-actin gene (actb), β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase as reference genes because they are known to have consistent expression levels. Until now, no study has been performed to select an internal control gene for the filamentous fungal species Penicillium echinulatum. The aim of this study was to evaluate and validate internal control genes to enable the study of gene expression in P. echinulatum using qRT-PCR. P. echinulatum strain S1M29 was grown in conditions to either induce (cellulose and sugar cane bagasse) or repress (glucose) gene expression to analyze 23 candidate normalization genes for stable expression. Two software programs, BestKeeper and geNorm, were used to assess the expression of the candidate normalization genes. The results indicate that the actb reference gene is more stably expressed in P. echinulatum. This is the first report in the literature that determines a normalization gene for this fungus. From the results obtained, we recommend the use of the P. echinulatum actb gene as an endogenous control for gene expression studies of cellulases and hemicellulases by qRT-PCR. PMID:24509829

  16. Quantitative Analysis of the Relative Transcript Levels of ABC Transporter Atr Genes in Aspergillus nidulans by Real-Time Reverse Transcription-PCR Assay

    Science.gov (United States)

    Pizeta Semighini, Camile; Marins, Mozart; Goldman, Maria Helena S.; Goldman, Gustavo Henrique

    2002-01-01

    The development of assays for quantitative analysis of the relative transcript levels of ABC transporter genes by real-time reverse transcription-PCR (RT-PCR) might provide important information about multidrug resistance in filamentous fungi. Here, we evaluate the potential of real-time RT-PCR to quantify the relative transcript levels of ABC transporter Atr genes from Aspergillus nidulans. The AtrA to AtrD genes showed different and higher levels in the presence of structurally unrelated drugs, such as camptothecin, imazalil, itraconazole, hygromycin, and 4-nitroquinoline oxide. We also verified the relative transcript levels of the Atr genes in the A. nidulans imazalil-resistant mutants. These genes displayed a very complex pattern in different ima genetic backgrounds. The imaB mutant has higher basal transcript levels of AtrB and -D than those of the wild-type strain. The levels of these two genes are comparable when the imaB mutant is grown in the presence and absence of imazalil. The imaC, -D, and -H mutants have higher basal levels of AtrA than that of the wild type. The same behavior is observed for the relative transcript levels of AtrB in the imaG mutant background. PMID:11872487

  17. Real-Time Monitoring and Quantitative Evaluation of Cavitation Bubbles Induced by High Intensity Focused Ultrasound Using B-Mode Imaging

    Science.gov (United States)

    Yu, Jie; Chen, Chu-Yi; Chen, Gong; Guo, Xia-Sheng; Ma, Yong; Tu, Juan; Zhang, Dong

    2014-03-01

    A software-based method is proposed to eliminate the flooding interference strips in B-mode images, and to evaluate the cavitation bubbles generated during high intensity focused ultrasound (HIFU) exposures. In vitro tissue phantoms are exposed to 1.12 MHz HIFU pulses with a fixed 100 Hz pulse repetition frequency. HIFU-induced cavitation bubbles are detected as hyperechoic regions in B-mode images. The temporal evolution of cavitation bubbles, generated by HIFU pulses with varying driving amplitude and pulse length, is analyzed by measuring the time-varying area of the hyperechoic region. The results show that: first, it is feasible to monitor HIFU-induced cavitation bubble activity in real-time using B-mode imaging; second, more cavitation bubbles can be generated with higher acoustic energy delivered; third, the hyperechoic region is observed to shrink gradually after ceasing the HIFU pulses, which indicates the dissolution of cavitation bubbles. This work will be helpful for developing an effective tool to realize real-time monitoring and quantitative evaluation of HIFU-induced cavitation bubble activity using a current commercialized B-mode machine.

  18. A new molecular breast cancer subclass defined from a large scale real-time quantitative RT-PCR study

    International Nuclear Information System (INIS)

    Current histo-pathological prognostic factors are not very helpful in predicting the clinical outcome of breast cancer due to the disease's heterogeneity. Molecular profiling using a large panel of genes could help to classify breast tumours and to define signatures which are predictive of their clinical behaviour. To this aim, quantitative RT-PCR amplification was used to study the RNA expression levels of 47 genes in 199 primary breast tumours and 6 normal breast tissues. Genes were selected on the basis of their potential implication in hormonal sensitivity of breast tumours. Normalized RT-PCR data were analysed in an unsupervised manner by pairwise hierarchical clustering, and the statistical relevance of the defined subclasses was assessed by Chi2 analysis. The robustness of the selected subgroups was evaluated by classifying an external and independent set of tumours using these Chi2-defined molecular signatures. Hierarchical clustering of gene expression data allowed us to define a series of tumour subgroups that were either reminiscent of previously reported classifications, or represented putative new subtypes. The Chi2 analysis of these subgroups allowed us to define specific molecular signatures for some of them whose reliability was further demonstrated by using the validation data set. A new breast cancer subclass, called subgroup 7, that we defined in that way, was particularly interesting as it gathered tumours with specific bioclinical features including a low rate of recurrence during a 5 year follow-up. The analysis of the expression of 47 genes in 199 primary breast tumours allowed classifying them into a series of molecular subgroups. The subgroup 7, which has been highlighted by our study, was remarkable as it gathered tumours with specific bioclinical features including a low rate of recurrence. Although this finding should be confirmed by using a larger tumour cohort, it suggests that gene expression profiling using a minimal set of

  19. Comprehensive selection of reference genes for expression studies in meniscus injury using quantitative real-time PCR.

    Science.gov (United States)

    Leal, Mariana Ferreira; Arliani, Gustavo Gonçalves; Astur, Diego Costa; Franciozi, Carlos Eduardo; Debieux, Pedro; Andreoli, Carlos Vicente; Smith, Marília Cardoso; Pochini, Alberto de Castro; Ejnisman, Benno; Cohen, Moises

    2016-06-10

    The meniscus plays critical roles in the knee function. Meniscal tears can lead to knee osteoarthritis. Gene expression analysis may be a useful tool for understanding meniscus tears, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. We evaluated the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1 and TBP) using meniscus samples of (1) 19 patients with isolated meniscal tears, (2) 20 patients with meniscal tears and combined anterior cruciate ligament injury (ACL), and (3) 11 controls without meniscal tears. The stability of the candidate reference genes was determined using the NormFinder, geNorm, BestKeeper DataAssist and RefFinder software packages and comparative ΔCt method. Overall, HPRT1 was the best single reference gene. However, GenEx software demonstrated that two or more reference genes should be used for gene expression normalization, which was confirmed when we evaluated TGFβR1 expression using several reference gene combinations. HPRT1+TBP was the most frequently identified pair from the analysis of samples of (1) meniscal tear samples of patients with a concomitant ACL tears, (2) all meniscal tears, and (3) all samples. HPRT1+GAPDH was the most frequently identified pair from the analysis of samples of isolated meniscal tear samples and controls. In the analysis involving only controls, GAPDH+18S was the most frequently identified pair. In the analysis of only isolated meniscal tear samples and in the analysis of meniscal tear samples of patients with concomitant ACL tears and controls, both HPRT1+TBP and HPRT1+GAPDH were identified as suitable pairs. If the gene expression study aims to compare non-injured meniscus, isolated meniscal tears and meniscal tears of patients with ACL tears as three independent groups, the trio of HPRT1+TBP+GAPDH is the most suitable

  20. Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing.

    Science.gov (United States)

    Li, Wenbin; Hartung, John S; Levy, Laurene

    2006-07-01

    Citrus huanglongbing (HLB, ex greening) is one of the most serious diseases of citrus. Different forms of the disease are caused by different Candidatus Liberobacter species, Candidatus Liberibacter asiaticus (Las), Ca. L. africanus (Laf) and Ca. L. americanus (Lam). The pathogen is transmitted by psyllid insects and by budding with contaminated plant materials. The vector psyllid Diaphorina citri can transmit both Las and Lam. Establishment of this vector into Florida, reports of Lam and Las in Brazil in 2004, and recent confirmation of HLB in Florida in September 2005 is of great concern to the citrus industry. Research on HLB has been hampered by the unculturable nature of the causal bacterium in artificial media. It has also been difficult to detect and identify the pathogens, possibly because of low concentration and uneven distribution in host plants and vector psyllids. In this study, we developed quantitative TaqMan PCR using 16S rDNA-based TaqMan primer-probe sets specific to the different Ca. Liberobacter spp. An additional primer-probe set based on plant cytochrome oxidase (COX) was used as a positive internal control to assess the quality of the DNA extracts. The assays do not cross-react with other pathogens or endophytes commonly resident in citrus plants, and are very sensitive. HLB pathogen DNA was successfully amplified from the equivalent of 20 ng of midrib tissue from symptomatic leaves. The consistent results of the assays with DNA extracted from plants infected by various Ca. Liberibacter species grown in greenhouses and in the field demonstrated a degree of reproducibility for these TaqMan assays. Inhibitors of the PCR that are frequently present in plant extracts did not affect the assay results. The population of the pathogens was estimated to be 5 x 10(7) and 2 x 10(6) cells/g of fresh midribs of symptomatic sweet orange leaves infected by Las and Lam, respectively. The ratio of pathogen DNA to host plant DNA was estimated by to be 1

  1. Real-time quantitative PCR assay with Taqman(®) probe for rapid detection of MCR-1 plasmid-mediated colistin resistance.

    Science.gov (United States)

    Chabou, S; Leangapichart, T; Okdah, L; Le Page, S; Hadjadj, L; Rolain, J-M

    2016-09-01

    Here we report the development of two rapid real-time quantitative PCR assays with TaqMan(®) probes to detect the MCR-1 plasmid-mediated colistin resistance gene from bacterial isolates and faecal samples from chickens. Specificity and sensitivity of the assay were 100% on bacterial isolates including 18 colistin-resistant isolates carrying the mcr-1 gene (six Klebsiella pneumoniae and 12 Escherichia coli) with a calibration curve that was linear from 10(1) to 10(8) DNA copies. Five out of 833 faecal samples from chickens from Algeria were positive, from which three E. coli strains were isolated and confirmed to harbour the mcr-1 gene by standard PCR and sequencing. PMID:27489722

  2. Rapid detection of Streptococcus pneumoniae by real-time fluorescence quantitation PCR%实时荧光定量PCR快速诊断肺炎链球菌

    Institute of Scientific and Technical Information of China (English)

    颜善活; 孙雷; 符可鹏; 卓永光; 杨善业; 樊祖茜; 黄永霞

    2013-01-01

    目的 建立实时荧光定量聚合酶链反应(PCR),用于肺炎链球菌检测和流行病学调查.方法 以肺炎链球菌自溶素(lyt)和溶血素(ply)的基因为目的序列分别设计引物和探针,将其分别与10株肺炎链球菌、13株非肺炎链球菌DNA以及不同浓度梯度的肺炎链球菌DNA进行荧光定量PCR,并将引物和探针应用于200例临床标本检测,同时通过传统的微生物培养鉴定的方法来验证荧光定量PCR的特异性和敏感性.结果 10株肺炎链球菌均获得了明显的扩增产物,13株非肺炎链球菌DNA无明显的扩增信号,其检测敏感性可达100 fg;200例临床标本,实时荧光定量PCR检测出42例肺炎链球菌阳性(阳性率为21%),而培养法阳性16例(阳性率为8%).结论 实时荧光定量PCR是一种敏感、特异、快速的检测肺炎链球菌方法,可用于肺炎链球菌的诊断和流行病学调查.%Objective To establish real-time fluorescence quantitation polymerase chain reaction ( PCR) for the detection and epidemiological studies of Streptococcus pneumoniae. Methods The autolysin (lyt) gene and hemolysin (ply) gene sequences were selected to develop primers and probe. A total of 10 strains of Streptococcus pneumoniae,13 strains of non-Streptococcus pneumoniae DISA and the different concentration strains of Streptococcus pneumoniae DNA were detected by real-time fluorescence quantitation PCR, and 200 clinical specimens were detected by primers and probe. The specificity and sensitivity were also analyzed. Results The 10 strains of Streptococcus pneumoniae were measured to obtain amplification products. However, 13 strains of non-Streptococcus pneumoniae DNA had no obvious amplification, and the sensitivity was 100fg. Among the 200 clinical specimens, real-time fluorescence quantitation PCR detected 42 cases of Streptococcus pneumoniae-positive (the positive rate was 21% ), while the culture method detected 16 cases of Streptococcus pneumoniae

  3. 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. PMID:21234520

  4. 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. 

  5. Distribution and persistence of probiotic Streptococcus salivarius K12 in the human oral cavity as determined by real-time quantitative polymerase chain reaction.

    Science.gov (United States)

    Horz, H-P; Meinelt, A; Houben, B; Conrads, G

    2007-04-01

    The bacteriocin producer Streptococcus salivarius K12 is used as a probiotic targeting the oral cavity, so our study aimed to assess whether its dispersal and persistence could be monitored using real-time quantitative polymerase chain reaction. To this end, we designed polymerase chain reaction primers and a hybridization probe specifically targeting salA, which encodes for the prepropeptide of salivaricin A. Using a single individual as our subject, we administered four lozenges of K12 Throat Guard per day over 3 days, then measured salA gene levels for 16 different oral sites at six different intervals over 35 days. Four samples each from gingival sulci and from teeth all remained negative. In contrast, in saliva and at all mucosal membranes K12 was detected, but with varying amounts and time profiles. Relatively high salA gene copy numbers, calibrated on the basis of colony-forming units, were seen on the tongue (maximum 4.6 x 10(4)/swab at day 4), in stimulated saliva (2.4 x 10(4)/ml, day 4) and on buccal membranes (1.3 x 10(4)/swab, day 8). K12 was present on both sides of the pharynx but asymmetrically in both quantity and duration. In conclusion, we have developed a real-time quantitative-polymerase chain reaction for counting S. salivarius K12 at various sites in the oral cavity. In the individual studied, K12 could be detected at the mucosal membranes for as long as 3 weeks, but with steadily decreasing numbers after day 8. Thus, K12 may have the potential to control oral bacterial infections only when the uptake is repeated frequently. PMID:17311636

  6. Development and optimization of a sensitive TaqMan® real-time PCR with synthetic homologous extrinsic control for quantitation of Human cytomegalovirus viral load.

    Science.gov (United States)

    Slavov, Svetoslav Nanev; Otaguiri, Katia Kaori; de Figueiredo, Glauciane Garcia; Yamamoto, Aparecida Yulie; Mussi-Pinhata, Marisa Marcia; Kashima, Simone; Covas, Dimas Tadeu

    2016-09-01

    Human cytomegalovirus (Human herpesvirus 5, HCMV) causes frequent asymptomatic infections in the general population. However, in immunosuppressed patients or congenitally infected infants, HCMV is related to high morbidity and mortality. In such cases, a rapid viral detection is crucial for monitoring the clinical outcome and the antiviral treatment. In this study, we optimized a sensitive biplex TaqMan® real-time PCR for the simultaneous detection and differentiation of a partial HCMV UL97 sequence and homologous extrinsic control (HEC) in the same tube. HEC was represented by a plasmid containing a modified HCMV sequence retaining the original primer binding sites, while the probe sequence was substituted by a phylogenetically divergent one (chloroplast CF0 subunit plant gene). It was estimated that the optimal HEC concentration, which did not influence the HCMV amplification is 1,000 copies/reaction. The optimized TaqMan® PCR demonstrated high analytical sensitivity (6.97 copies/reaction, CI = 95%) and specificity (100%). Moreover, the reaction showed adequate precision (repeatability, CV = 0.03; reproducibility, CV = 0.0027) and robustness (no carry-over or cross-contamination). The diagnostic sensitivity (100%) and specificity (97.8%) were adequate for the clinical application of the molecular platform. The optimized TaqMan® real-time PCR is suitable for HCMV detection and quantitation in predisposed patients and monitoring of the applied antiviral therapy. J. Med. Virol. 88:1604-1612, 2016. © 2016 Wiley Periodicals, Inc. PMID:26890091

  7. Determination of Cytochrome P450 2D6 (CYP2D6 Gene Copy Number by Real-Time Quantitative PCR

    Directory of Open Access Journals (Sweden)

    Laurent Bodin

    2005-01-01

    Full Text Available Gene dosage by real-time quantitative PCR has proved to be accurate for measuring gene copy number. The aim of this study was to apply this approach to the CYP2D6 gene to allow for rapid identification of poor and ultrarapid metabolizers (0, 1, or more than 2 gene copy number. Using the 2−ΔΔCt calculation method and a duplex reaction, the number of CYP2D6 gene copies was determined. Quantitative PCR was performed on 43 samples previously analyzed by Southern blotting and long PCR including 20 samples with a heterozygous deletion, 11 with normal copy number (2 copies, and 12 samples with duplicated genes. The average ratio ranged from 1.02 to 1.28, 1.85 to 2.21, and 2.55 to 3.30, respectively, for the samples with 1 copy, 2 copies, and 3 copies. This study shows that this method is sensitive enough to detect either a heterozygous gene deletion or duplication.

  8. 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. PMID:27041341

  9. HHV-6 encephalitis may complicate the early phase after allogeneic hematopoietic stem cell transplantation: Detection by qualitative multiplex PCR and subsequent quantitative real-time PCR.

    Science.gov (United States)

    Inazawa, Natsuko; Hori, Tsukasa; Yamamoto, Masaki; Hatakeyama, Naoki; Yoto, Yuko; Nojima, Masanori; Yasui, Hiroshi; Suzuki, Nobuhiro; Shimizu, Norio; Tsutsumi, Hiroyuki

    2016-02-01

    Viral reactivation following hematopoietic stem cell transplantation (HSCT) can cause various complications especially viral encephalitis. In this prospective study, we investigated the correlation of post-HSCT viral reactivation in blood with CNS dysfunction. We employed a multiplex PCR that detects 13 kinds of viruses as a first-line screening test and real-time PCR for subsequent quantitative evaluation. Five hundred ninety-one whole blood samples were collected from 105 patients from before until 42 days after HSCT. Seven patients developed CNS dysfunction such as altered consciousness. In six of the seven, the multiplex PCR test detected HHV-6 DNA in at least one sample. In contrast, DNA from other viruses, such as CMV, EBV, HHV-7, adenovirus, and HBV was never detected in any of the seven patients throughout the study period. Quantitative measurement of whole blood HHV-6 DNA levels demonstrated four of the six HHV-6 DNA loads were elevated at successive time points during the CNS dysfunction. In addition, the virus DNA peaks were temporally associated with the development of CNS dysfunction. CSF was tested in two of the four patients and high HHV-6 DNA levels comparable to those in whole blood were confirmed in both. These four patients were, thus, suspected to have developed HHV-6 encephalitis, a rate of 3.8% in the study population. Our results suggest that early diagnosis of probable HHV-6 encephalitis can be improved by confirming high HHV-6 DNA load in blood. PMID:26241219

  10. 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....

  11. 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

  12. B1 Sequence-Based Real-Time Quantitative PCR: A Sensitive Method for Direct Measurement of Mouse Plasma DNA Levels After Gamma Irradiation

    International Nuclear Information System (INIS)

    Purpose: Current biodosimetric techniques for determining radiation exposure have inherent delays, as well as quantitation and interpretation limitations. We have identified a new technique with the advantage of directly measuring circulating DNA by amplifying inter-B1 regions in the mouse genome, providing a sensitive method for quantitating plasma DNA. Methods and Materials: Real-time quantitative polymerase chain reaction (PCR) was used to detect levels of DNA by amplifying inter-B1 genomic DNA in plasma samples collected at 0-48 h from mice receiving 0-10 Gy total- or partial-body irradiation (137Cs γ-ray source at ∼1.86 Gy/min; homogeneity: ± 6.5%). Results: The correlation coefficient between DNA levels and the threshold cycle value (CT) was 0.996, and the average recoveries of DNA in the assay were 87%. This assay revealed that when BALB/c mice were exposed to 10 Gy total-body irradiation (TBI), plasma DNA levels gradually increased beginning at 3 h after irradiation, peaked at 9 h, and returned to baseline within 48 h. Increased plasma DNA levels were also detected following upper-torso or lower-torso partial-body irradiation; however, TBI approximately doubled those plasma DNA levels at the same radiation dose. This technique therefore reflects total body cell damage. The advantages of this assay are that DNA extraction is not required, the assay is highly sensitive (0.002 ng), and results can be obtained within 2.5 h after collection of plasma samples. Conclusions: A radiation dose-dependent increase of plasma DNA was observed in the dose range from 2 to 10 Gy, suggesting that plasma DNA may be a useful radiation biomarker and adjunct to existing cell-based assays.

  13. Detection of anatid herpesvirus 1 gC gene by TaqMan™ fluorescent quantitative real-time PCR with specific primers and probe

    Directory of Open Access Journals (Sweden)

    Jia Renyong

    2010-02-01

    Full Text Available Abstract Background Anatid herpesvirus 1 (AHV-1 is known for the difficulty of monitoring and controlling, because it has a long period of asymptomatic carrier state in waterfowls. Furthermore, as a significant essential agent for viral attachment, release, stability and virulence, gC (UL44 gene and its protein product (glycoprotein C may play a key role in the epidemiological screening. The objectives of this study were to rapidly, sensitively, quantitatively detect gC gene of AHV-1 and provide the underlying basis for further investigating pcDNA3.1-gC DNA vaccine in infected ducks by TaqMan™ fluorescent quantitative real-time PCR assay (FQ-PCR with pcDNA3.1-gC plasmid. Results The repeatable and reproducible quantitative assay was established by the standard curve with a wide dynamic range (eight logarithmic units of concentration and very good correlation values (1.000. This protocol was able to detect as little as 1.0 × 101 DNA copies per reaction and it was highly specific to AHV-1. The TaqMan™ FQ-PCR assay successfully detected the gC gene in tissue samples from pcDNA3.1-gC and AHV-1 attenuated vaccine (AHV-1 Cha strain inoculated ducks respectively. Conclusions The assay offers an attractive method for the detection of AHV-1, the investigation of distribution pattern of AHV-1 in vivo and molecular epidemiological screening. Meanwhile, this method could expedite related AHV-1 and gC DNA vaccine research.

  14. 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

  15. QUANTITATIVE REAL-TIME PCR (Q-PCR FOR SPUTUM SMEAR DIAGNOSIS OF PULMONARY TUBERCULOSIS AMONG PEOPLE WITH HIV/AIDS

    Directory of Open Access Journals (Sweden)

    Yvana Maria Maia de Albuquerque

    2014-04-01

    Full Text Available Objective: To assess quantitative real-time polymerase chain reaction (q-PCR for the sputum smear diagnosis of pulmonary tuberculosis (PTB in patients living with HIV/AIDS with a clinical suspicion of PTB. Method: This is a prospective study to assess the accuracy of a diagnostic test, conducted on 140 sputum specimens from 140 patients living with HIV/AIDS with a clinical suspicion of PTB, attended at two referral hospitals for people living with HIV/AIDS in the city of Recife, Pernambuco, Brazil. A Löwenstein-Jensen medium culture and 7H9 broth were used as gold standard. Results: Of the 140 sputum samples, 47 (33.6% were positive with the gold standard. q-PCR was positive in 42 (30% of the 140 patients. Only one (0.71% did not correspond to the culture. The sensitivity, specificity and accuracy of the q-PCR were 87.2%, 98.9% and 95% respectively. In 39 (93% of the 42 q-PCR positive cases, the CT (threshold cycle was equal to or less than 37. Conclusion: q-PCR performed on sputum smears from patients living with HIV/AIDS demonstrated satisfactory sensitivity, specificity and accuracy, and may therefore be recommended as a method for diagnosing PTB.

  16. Verification of suitable and reliable reference genes for quantitative real-time PCR during adipogenic differentiation in porcine intramuscular stromal-vascular cells.

    Science.gov (United States)

    Li, X; Huang, K; Chen, F; Li, W; Sun, S; Shi, X-E; Yang, G

    2016-06-01

    Intramuscular fat (IMF) is an important trait influencing meat quality, and intramuscular stromal-vascular cell (MSVC) differentiation is a key factor affecting IMF deposition. Quantitative real-time PCR (qPCR) is often used to screen the differentially expressed genes during differentiation of MSVCs, where proper reference genes are essential. In this study, we assessed 31 of previously reported reference genes for their expression suitability in porcine MSVCs derived form longissimus dorsi with qPCR. The expression stability of these genes was evaluated using NormFinder, geNorm and BestKeeper algorithms. NormFinder and geNorm uncovered ACTB, ALDOA and RPS18 as the most three stable genes. BestKeeper identified RPL13A, SSU72 and DAK as the most three stable genes. GAPDH was found to be the least stable gene by all of the three software packages, indicating it is not an appropriate reference gene in qPCR assay. These results might be helpful for further studies in pigs that explore the molecular mechanism underlying IMF deposition. PMID:26781521

  17. Short communication: Improved method for centrifugal recovery of bacteria from raw milk applied to sensitive real-time quantitative PCR detection of Salmonella spp.

    Science.gov (United States)

    Brewster, Jeffrey D; Paul, Moushumi

    2016-05-01

    Centrifugation is widely used to isolate and concentrate bacteria from dairy products before assay. We found that more than 98% of common pathogenic bacteria added to pasteurized, homogenized, or pasteurized homogenized milk were recovered in the pellet after centrifugation, whereas less than 7% were recovered from raw milk. The remaining bacteria partitioned into the cream layer of raw milk within 5 min, and half-saturation of the cream layer required a bacterial load of approximately 5×10(8) cfu/mL. Known treatments (e.g., heat, enzymes or solvents) can disrupt cream layer binding and improve recovery from raw milk, but can also damage bacteria and compromise detection. We developed a simple, rapid agitation treatment that disrupted bacteria binding to the cream layer and provided more than 95% recovery without affecting bacteria viability. Combining this simple agitation treatment with a previously developed real-time quantitative PCR assay allowed the detection of Salmonella spp. in raw milk at 4 cfu/mL within 3 h. To our knowledge, this is the first report of an effective method for achieving high centrifugal recovery of bacteria from raw milk without impairing bacterial viability. PMID:26971150

  18. Evaluation of Sensitivities and Specificities of SARS-CoV Detection by Real-time Quantitative Reverse Transcription-PCR Assays

    Institute of Scientific and Technical Information of China (English)

    Li-li XU; Zhi-hong HU; Hua-lin WANG; Xiao HAN; Fei DENG

    2009-01-01

    The etiological agent of severe acute respiratory syndrome (SARS) was identified as a new coronavirus, termed SARS-CoV. Establishment of an efficient and sensitive diagnostic system of SARS-CoV genetic materials is crucial for SARS control. In this study, we quantified SARS-CoV mRNAs in both infected cell culture lysate and in supernatant by using Real-time quantitative revere transcription-PCR based on EvaGreen鈩?dye and Taqman-MGB probes. For extensive evaluation of sensitivities and specificities, 13 pairs of primers and 4 probes were designed based on different genes of SARS-CoV. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was selected as the internal control gene. Results showed that S-gene-specific PCR was the most sensitive for detection, but because of its sequence variability in the different viral strains, primers and a probe based on the N gene were suitable substitutions. Meanwhile, we found the mRNA concentrations in cell culture lysates were much higher than in cell supernatant and facilited more sensitive detection of the SARS-CoV.

  19. Comparison of quantitative real-time polymerase chain reaction with NanoString(®) methodology using adipose and liver tissues from rats fed seaweed.

    Science.gov (United States)

    Bentley-Hewitt, Kerry L; Hedderley, Duncan I; Monro, John; Martell, Sheridan; Smith, Hannah; Mishra, Suman

    2016-05-25

    Experimental methods are constantly being improved by new technology. Recently a new technology, NanoString(®), has been introduced to the market for the analysis of gene expression. Our experiments used adipose and liver samples collected from a rat feeding trial to explore gene expression changes resulting from a diet of 7.5% seaweed. Both quantitative real-time polymerase chain reaction (qPCR) and NanoString methods were employed to look at expression of genes related to fat and glucose metabolism and this paper compares results from both methods. We conclude that NanoString offers a valuable alternative to qPCR and our data suggest that results are more accurate because of the reduced sample handling and direct quantification of gene copy number without the need for enzymatic amplification. However, we have highlighted a potential challenge for both methods, which needs to be addressed when designing primers or probes. We suggest a literature search for known splice variants of a particular gene to be completed so that primers or probes can be designed that do not span exons which may be affected by alternative gene sequences. PMID:26820124

  20. Validation of Reference Genes for Gene Expression by Quantitative Real-Time RT-PCR in Stem Segments Spanning Primary to Secondary Growth in Populus tomentosa

    Science.gov (United States)

    Wang, Ying; Chen, Yajuan; Ding, Liping; Zhang, Jiewei; Wei, Jianhua

    2016-01-01

    The vertical segments of Populus stems are an ideal experimental system for analyzing the gene expression patterns involved in primary and secondary growth during wood formation. Suitable internal control genes are indispensable to quantitative real time PCR (qRT-PCR) assays of gene expression. In this study, the expression stability of eight candidate reference genes was evaluated in a series of vertical stem segments of Populus tomentosa. Analysis through software packages geNorm, NormFinder and BestKeeper showed that genes ribosomal protein (RP) and tubulin beta (TUBB) were the most unstable across the developmental stages of P. tomentosa stems, and the combination of the three reference genes, eukaryotic translation initiation factor 5A (eIF5A), Actin (ACT6) and elongation factor 1-beta (EF1-beta) can provide accurate and reliable normalization of qRT–PCR analysis for target gene expression in stem segments undergoing primary and secondary growth in P. tomentosa. These results provide crucial information for transcriptional analysis in the P. tomentosa stem, which may help to improve the quality of gene expression data in these vertical stem segments, which constitute an excellent plant system for the study of wood formation. PMID:27300480

  1. 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.

  2. Real-time polymerase chain reaction (PCR) quantitative detection of Brassica napus using a locked nucleic acid TaqMan probe.

    Science.gov (United States)

    Schmidt, Anna-Mary; Rott, Michael E

    2006-02-22

    Several countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms. Real-time quantitative Polymerase Chain Reaction (PCR) has quickly become the method of choice in support of these regulations and requires the development of separate PCR assays targeting the transgenic sequence as well as a specific endogenous gene sequence. To develop a Brassica napus-specific PCR assay, partial sequences of the acetyl-CoA carboxylase BnACCg8 gene from B. napus and the closely related Brassica rapa were determined and compared, and a region of unique nucleotide sequence was identified. Universal amplification primers were designed to either side of this region, and a locked nucleic acid TaqMan probe was designed to the B. napus-specific sequence. Evaluation of this primer/probe combination indicated a high level of specificity to B. napus: no amplification signal was observed with any other species tested, including five closely related Brassica species. The method was assayed with 14 different B. napus cultivars, and comparable amplification curves were consistently obtained for all. The assay was highly sensitive, with a limit of detection between 1 and 10 haploid copies. Practically, the method was demonstrated to be effective for the detection of processed food samples and for the quantification of Roundup Ready canola content in mixed samples. PMID:16478231

  3. Influence of storage time on DNA of Chlamydia trachomatis, Ureaplasma urealyticum, and Neisseria gonorrhoeae for accurate detection by quantitative real-time polymerase chain reaction.

    Science.gov (United States)

    Lu, Y; Rong, C Z; Zhao, J Y; Lao, X J; Xie, L; Li, S; Qin, X

    2016-01-01

    The shipment and storage conditions of clinical samples pose a major challenge to the detection accuracy of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Ureaplasma urealyticum (UU) when using quantitative real-time polymerase chain reaction (qRT-PCR). The aim of the present study was to explore the influence of storage time at 4°C on the DNA of these pathogens and its effect on their detection by qRT-PCR. CT, NG, and UU positive genital swabs from 70 patients were collected, and DNA of all samples were extracted and divided into eight aliquots. One aliquot was immediately analyzed with qRT-PCR to assess the initial pathogen load, whereas the remaining samples were stored at 4°C and analyzed after 1, 2, 3, 7, 14, 21, and 28 days. No significant differences in CT, NG, and UU DNA loads were observed between baseline (day 0) and the subsequent time points (days 1, 2, 3, 7, 14, 21, and 28) in any of the 70 samples. Although a slight increase in DNA levels was observed at day 28 compared to day 0, paired sample t-test results revealed no significant differences between the mean DNA levels at different time points following storage at 4°C (all P>0.05). Overall, the CT, UU, and NG DNA loads from all genital swab samples were stable at 4°C over a 28-day period. PMID:27580005

  4. Evaluation of reference genes for quantitative real-time PCR analysis of gene expression during early development processes of the tongue sole (Cynoglossus semilaevis)

    Institute of Scientific and Technical Information of China (English)

    MA Qian; ZHUANG Zhimeng; FENG Wenrong; LIU Shufang; TANG Qisheng

    2015-01-01

    Differential expression of genes is crucial to growth and development of fish. To select the appropriate genes for gene normalization duringCynoglossus semilaevis early developmental process, eight candidate reference genes (ACTB, B2M, EF1A, GADPH, RPL7, TUBA, UBCE and 18S) were tested for their adequacy by using quantitative real-time PCR. The results showed that the expression of all the examined genes exhibited tissue dependent variations in the matureC.semilaevis. EF1A was listed as the most stable reference among the 14 tissues by RefFinder. Furthermore, the recommended comprehensive ranking of the stability determined by RefFinder showed that 18S was the most stable gene during the early developmental stages (from oosphere to 90 days old) in this study. However, when divided theCt value data of the above mentioned early developmental stages into two separate periods (embryo and post-hatching periods), TUBA and 18S represented the most stable references of these two developmental periods, respectively. Consequently, the reference gene should be carefully and accurately chosen even for studies of the same species at various developmental processes. The relevant data may help in selecting appropriate reference genes for mRNA expression analysis, and is of great value in the studies of fish growth and development.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. 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. PMID:27052702

  7. 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

    2016-07-01

    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. PMID:27154315

  8. Enumeration of salmonellae in table eggs, pasteurized egg products, and egg-containing dishes by using quantitative real-time PCR.

    Science.gov (United States)

    Jakociune, Dziuginta; Pasquali, Frédérique; da Silva, Cristiana Soares; Löfström, Charlotta; Hoorfar, Jeffrey; Klein, Günter; Manfreda, Gerardo; Olsen, John Elmerdahl

    2014-03-01

    Salmonellae are a major cause of food-borne outbreaks in Europe, with eggs and egg products being identified as major sources. Due to the low levels of salmonellae in eggs and egg products, direct quantification is difficult. In the present study, enrichment quantitative real-time PCR (qPCR) was employed for enumeration of salmonellae in different matrices: table eggs, pasteurized egg products, and egg-containing dishes. Salmonella enterica serovar Enteritidis and S. enterica serovar Tennessee were used to artificially contaminate these matrices. The results showed a linear regression between the numbers of salmonellae and the quantification cycle (Cq) values for all matrices used, with the exception of pasteurized egg white. Standard curves were constructed by using both stationary-phase cells and heat-stressed cells, with similar results. Finally, this method was used to evaluate the fate of salmonellae in two egg-containing dishes, long egg and tiramisu, at abused refrigeration temperatures, and results indicated the growth of bacteria over a 1-week period. In conclusion, enrichment qPCR was shown to be reliable for enumeration of salmonellae in different egg products. PMID:24362433

  9. Validation of housekeeping genes as an internal control for gene expression studies in Giardia lamblia using quantitative real-time PCR.

    Science.gov (United States)

    Marcial-Quino, Jaime; Fierro, Francisco; De la Mora-De la Mora, Ignacio; Enríquez-Flores, Sergio; Gómez-Manzo, Saúl; Vanoye-Carlo, America; Garcia-Torres, Itzhel; Sierra-Palacios, Edgar; Reyes-Vivas, Horacio

    2016-04-25

    The analysis of transcript levels of specific genes is important for understanding transcriptional regulation and for the characterization of gene function. Real-time quantitative reverse transcriptase PCR (RT-qPCR) has become a powerful tool to quantify gene expression. The objective of this study was to identify reliable housekeeping genes in Giardia lamblia. Twelve genes were selected for this purpose, and their expression was analyzed in the wild type WB strain and in two strains with resistance to nitazoxanide (NTZ) and metronidazole (MTZ), respectively. RefFinder software analysis showed that the expression of the genes is different in the three strains. The integrated data from the four analyses showed that the NADH oxidase (NADH) and aldolase (ALD) genes were the most steadily expressed genes, whereas the glyceraldehyde-3-phosphate dehydrogenase gene was the most unstable. Additionally, the relative expression of seven genes were quantified in the NTZ- and MTZ-resistant strains by RT-qPCR, using the aldolase gene as the internal control, and the results showed a consistent differential pattern of expression in both strains. The housekeeping genes found in this work will facilitate the analysis of mRNA expression levels of other genes of interest in G. lamblia. PMID:26778241

  10. Use of quantitative real-time PCR for studying the dissemination of Leptospira interrogans in the guinea pig infection model of leptospirosis.

    Science.gov (United States)

    Lourdault, Kristel; Aviat, Florence; Picardeau, Mathieu

    2009-05-01

    The dynamics of leptospirosis infection have been poorly studied. The purpose of this study was to determine the LD(50), rate of bacterial dissemination, histopathology and antibody responses against leptospira following inoculation with the highly virulent Leptospira interrogans Fiocruz L1-130 strain in a guinea pig model of leptospirosis. Three routes of infection (intraperitoneal, conjunctival and subcutaneous inoculation) were used to establish disease in guinea pigs. The size and kinetics of leptospiral burdens in the blood and tissues of infected animals were determined over a 1 week course of infection using quantitative real-time PCR (qPCR). Bacteraemia peaked at day 5 post-infection reaching more than 5x10(4) leptospires ml(-1). The highest spirochaetal load was found in the liver and kidneys, and was associated with alterations in organ tissues and a decline in liver and kidney functions. In contrast, lesions and bacteria were not detected in guinea pigs infected with an avirulent strain derived from a high-passage-number in vitro-passaged variant of the Fiocruz L1-130 strain. The use of qPCR supports the findings of earlier studies and provides an easy and reliable method for the quantification of L. interrogans in the tissues of infected animals. qPCR will be used in future studies to evaluate the efficacy of vaccine candidates against leptospirosis and the virulence of selected L. interrogans mutants relative to the parental strain. PMID:19369528

  11. 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. PMID:24934261

  12. 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.

  13. Development of a new real-time TaqMan PCR assay for quantitative analyses of Candida albicans resistance genes expression.

    Science.gov (United States)

    Kofla, Grzegorz; Ruhnke, Markus

    2007-01-01

    Candida albicans is an important opportunistic pathogen that can cause serious fungal diseases in immunocompromised patients including cancer patients, transplant patients, and patients receiving immunosuppressive therapy in general, those with human immunodeficiency virus infections and undergoing major surgery. Its emergence spectrum varies from mucosal to systemic infections and the first line treatment is still based on fluconazole, a triazole derivate with a potent antifungal activity against most of C. albicans strains. Nevertheless the emergence of fluconazole-resistant C. albicans strains can lead to treatment failures and thus become a clinical problem in the management of such infections. For that reason we consider it important to study mechanisms inducing azole resistance and the possibilities to influence this process. In this work we give a short report on a real-time PCR (TaqMan) assay, which can be used for quantitative analyses of gene expression levels of MDR1, CDR1 and ERG11, genes supposed to contribute to development of the resistance mechanisms. We show some results achieved with that assay in fluconazole susceptible and resistant strains that confirm results seen earlier in experiments using Northern blot hybridisation and prove that the comparative DeltaCt method is valid for our system. PMID:16945439

  14. Identification of Valid Housekeeping Genes for Real-Time Quantitative PCR Analysis of Collapsed Lung Tissues of Neonatal Somatic Cell Nuclear Transfer-Derived Cattle.

    Science.gov (United States)

    Liu, Yan; Zhang, Yunhai; Jiang, Qiuling; Rao, Man; Sheng, Zheya; Zhang, Yu; Du, Weihua; Hao, Haisheng; Zhao, Xueming; Xu, Zhe; Liu, Jianning; Zhu, Huabin

    2015-10-01

    Cloned calves produced by somatic cell nuclear transfer frequently suffer alveolar collapse as newborns. To study the underlying pathophysiological mechanisms responsible for this phenomenon, the expression profiles of numerous genes involved in lung development need to be investigated. Quantitative real-time PCR is commonly adopted in gene expression analysis. However, selection of an appropriate reference gene for normalization is critical for obtaining reliable and accurate results. Seven housekeeping genes-β-glucuronidase (GUSB), phosphoglycerate kinase 1 (PGK1), β-2-microglobolin (B2M), peptidylprolyl isomerase A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA-box binding protein (TBP), and 5.8S ribosomal RNA (5.8S rRNA)-were selected and evaluated as candidates. Their gene expression levels in the collapsed lungs of deceased neonate cloned calves and normal lung derived from normal calves were assessed. The ranking of gene expression stability was estimated by the geNorm, NormFinder, and BestKeeper programs. 5.8S rRNA and PPIA were determined to be the most stable reference genes by geNorm and BestKeeper, whereas the combination of GAPDH and TBP was suggested as reference genes by NormFinder. Taking these results into account, we conclude that 5.8S rRNA and PPIA could be the most reliable reference genes for studying the genes involved in alveolar collapse. Moreover, 5.8S rRNA could be represented as a uniform reference gene in similar cases. PMID:26393896

  15. 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)

    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.

  16. 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)

    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

  17. Detection of live Salmonella sp. cells in produce by a TaqMan-based quantitative reverse transcriptase real-time PCR targeting invA mRNA.

    Science.gov (United States)

    González-Escalona, Narjol; Hammack, Thomas S; Russell, Mindi; Jacobson, Andrew P; De Jesús, Antonio J; Brown, Eric W; Lampel, Keith A

    2009-06-01

    Salmonella enterica contamination in foods is a significant concern for public health. When DNA detection methods are used for analysis of foods, one of the major concerns is false-positive results from the detection of dead cells. To circumvent this crucial issue, a TaqMan quantitative real-time RT-PCR (qRT-PCR) assay with an RNA internal control was developed. invA RNA standards were used to determine the detection limit of this assay as well as to determine invA mRNA levels in mid-exponential-, late-exponential-, and stationary-phase cells. This assay has a detection limit of 40 copies of invA mRNA per reaction. The levels of invA mRNA in mid-exponential-, late-exponential-, and stationary-phase S. enterica cells was approximately 1 copy per 3 CFU, 1 copy per CFU, and 4 copies per 10(3) CFU, respectively. Spinach, tomatoes, jalapeno peppers, and serrano peppers were artificially contaminated with four different Salmonella serovars at levels of 10(5) and less than 10 CFU. These foods were analyzed with qRT-PCR and with the FDA's Bacteriological Analytical Manual Salmonella culture method (W. A. Andrews and T. S. Hammack, in G. J. Jackson et al., ed., Bacteriological analytical manual online, http://www.cfsan.fda.gov/ approximately ebam/bam-5.html, 2007). Comparable results were obtained by both methods. Only live Salmonella cells could be detected by this qRT-PCR assay, thus avoiding the dangers of false-positive results from nonviable cells. False negatives (inhibition of the PCR) were also ruled out through the use of an RNA internal control. This assay allows for the fast and accurate detection of viable Salmonella spp. in spinach, tomatoes, and in both jalapeno and serrano peppers. PMID:19376910

  18. Typing of human adenoviruses in specimens from immunosuppressed patients by PCR-fragment length analysis and real-time quantitative PCR.

    Science.gov (United States)

    Ebner, Karin; Rauch, Margit; Preuner, Sandra; Lion, Thomas

    2006-08-01

    Currently, 51 human adenovirus (AdV) serotypes, which are divided into six species (A to F), are known. AdV infections are a major cause of morbidity and mortality in immunosuppressed individuals, particularly in allogeneic stem cell transplant (SCT) recipients. Any AdV species may cause life-threatening disease, but little information is available on the clinical relevance of individual serotypes. The use of serological testing for serotype identification is limited due to the impaired immune response during the posttransplant period. A new molecular approach to serotype identification is presented here that exploits variable regions within the hexon gene. All serotypes belonging to the species A, B, C, E, and F can be determined by fragment length analysis of a single PCR product. For species C, which is the most prevalent in many geographic regions, an alternative technique based on serotype-specific real-time quantitative PCR was established. Of 135 consecutive pediatric patients screened for AdV infections after allogeneic SCT, 40 tested positive. Detailed analysis revealed the presence of 10 different serotypes; serotypes 1 and 2 from species C (C01 and C02) showed the highest prevalence, accounting for 77% of the AdV-positive cases. Representatives of other species were observed less commonly: serotype A12 in 6.5%; serotype A31 in 4.5%; and B03, B16, C05, C06, D19, and F41 in 2%. The approach to rapid molecular serotype analysis presented here provides a basis for detailed studies on adenovirus epidemiology and on the transmission of nosocomial infections. Moreover, in view of the increasing importance of tailored therapy approaches, serotype identification may in the future have implications for the selection of the most appropriate antiviral treatment. PMID:16891496

  19. 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

  20. Quantitative identification of fecal water pollution sources by TaqMan real-time PCR assays using Bacteroidales 16S rRNA genetic markers.

    Science.gov (United States)

    Lee, Dae-Young; Weir, Susan C; Lee, Hung; Trevors, Jack T

    2010-12-01

    PCR-based analysis of Bacteroidales 16S rRNA genes has emerged as a promising tool to identify sources of fecal water pollution. In this study, three TaqMan real-time PCR assays (BacGeneral, BacHuman, and BacBovine) were developed and evaluated for their ability to quantitatively detect general (total), human-specific, and bovine-specific Bacteroidales 16S rRNA genetic markers. The detection sensitivity was determined to be 6.5 copies of 16S rRNA gene for the BacGeneral and BacHuman assays and 10 copies for the BacBovine assay. The assays were capable of detecting approximately one to two cells per PCR. When tested with 70 fecal samples from various sources (human, cattle, pig, deer, dog, cat, goose, gull, horse, and raccoon), the three assays positively identified the target markers in all samples without any false-negative results. The BacHuman and BacBovine assays exhibited false-positive reactions with non-target samples in a few cases. However, the level of the false-positive reactions was about 50 times smaller than that of the true-positive ones, and therefore, these cross-reactions were unlikely to cause misidentifications of the fecal pollution sources. Microbial source-tracking capability was tested at two freshwater streams of which water quality was influenced by human and cattle feces, respectively. The assays accurately detected the presence of the corresponding host-specific markers upon fecal pollution and the persistence of the markers in downstream areas. The assays are expected to reliably determine human and bovine fecal pollution sources in environmental water samples. PMID:20871990

  1. 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

    Directory of Open Access Journals (Sweden)

    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.

  2. Quantitative determination, by real-time reverse transcription polymerase chain reaction, of aromatase mRNA in invasive ductal carcinoma of the breast

    International Nuclear Information System (INIS)

    Estrogen is a mitogenic factor that is implicated in the genesis and progression of breast cancer via its binding to estrogen receptor (ER)-α. Synthesis of estrogen in situ is believed to be catalyzed mainly by aromatase. Previous studies comparing the relative contributions from tumor cells and stromal cells to local estrogen synthesis, as assessed by immunohistochemical analysis, were quite controversial and no consistent relationship was found between the presence of aromatase and any clinicopathologic factor. In addition, previous studies into aromatase gene expression and clinicopathologic factors are limited. We assessed the level of expression of aromatase mRNA, using quantitative real-time RT-PCR, in 162 cases of invasive ductal carcinoma of the breast. Associations between aromatase expression and different clinicopathologic factors were sought. It was found that aromatase mRNA was expressed at significantly higher levels in patients older than 50 years, in those without axillary lymph node involvement, in those with tumor size less than 2 cm, and in ER-α positive tumors. However, no relationship was found between aromatase mRNA expression and any other clinicopathologic factor, including histologic grade and progesterone receptor status. Patients with high levels of expression of aromatase mRNA tended to have a better prognosis than did those patients with low expression. These findings imply that ER-α and aromatase may be coexpressed in endocrine responsive patients. They may also indicate that aromatase expression could be a marker of endocrine responsiveness, and it may have prognostic implications for breast cancer progression

  3. 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. PMID:26320872

  4. Detection and quantification of parapoxvirus DNA by use of a quantitative real-time polymerase chain reaction assay in calves without clinical signs of parapoxvirus infection.

    Science.gov (United States)

    Yaegashi, Gakuji; Fukunari, Kazuhiro; Oyama, Takayuki; Murakami, Ryu-Koh; Inoshima, Yasuo

    2016-04-01

    OBJECTIVE To investigate the presence of parapoxvirus (PPV) in cattle without clinical signs of infection and in farm environments of PPV-infected cattle. ANIMALS 28 calves without clinical signs of PPV infection on 2 farms and 11 clinically affected calves on 6 farms. PROCEDURES 164 oral swab samples were collected at regular intervals from 28 calves without clinical signs of PPV infection, and 11 swab samples were collected from 11 clinically affected calves. Viral DNA load was quantified by use of a PPV-specific quantitative real-time PCR (qRT-PCR) assay. RESULTS Of 28 calves without clinical signs of PPV infection, 12 had positive results for PPV DNA by use of the qRT-PCR assay. Viral DNA was detected continuously over a period of 2 to 5 months from 9 of these 12 calves, particularly from calves with dermatomycosis or respiratory tract disease. The PPV DNA loads in 32 oral swab samples from these 12 calves were significantly lower (median, 3.2 copies/mg) than those in samples collected from the 11 clinically affected calves (median, 3.2 × 10(4) copies/mg). Moreover, PPV DNA was detected in the residual feed and drinking water on both farms that housed the calves without clinical signs of PPV infection. CONCLUSIONS AND CLINICAL RELEVANCE PPV in cattle without clinical signs of infection and in the environments of these cattle may represent sources of PPV transmission to susceptible cattle. IMPACT FOR HUMAN MEDICINE Humans should wear gloves to prevent zoonotic disease transmission when handling cattle with or without clinical signs of PPV infection. PMID:27027837

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

    Energy Technology Data Exchange (ETDEWEB)

    Malik, Afshan N., E-mail: afshan.malik@kcl.ac.uk [King' s College London, Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Medicine (United Kingdom); Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil [King' s College London, Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Medicine (United Kingdom)

    2011-08-19

    Highlights: {yields} Mitochondrial dysfunction is central to many diseases of oxidative stress. {yields} 95% of the mitochondrial genome is duplicated in the nuclear genome. {yields} Dilution of untreated genomic DNA leads to dilution bias. {yields} 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 {beta}-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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Dunn Sade N

    2008-06-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  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; Moreira, Otacilio da Cruz; 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; Guedes, Paulo Marcos da Matta; 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; Galvão, Lúcia Maria da Cunha; da Câmara, Antonia Cláudia Jácome; Espinoza, Bertha; de Noya, Belkisyole Alarcón; Puerta, Concepción; Riarte, Adelina; Diosque, Patricio; Sosa-Estani, Sergio; Guhl, Felipe; Ribeiro, Isabela; Aznar, Christine; Britto, Constança; Yadón, Zaida Estela; Schijman, Alejandro G.

    2015-01-01

    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. PMID:26320872

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

    Science.gov (United States)

    Guil-Luna, S; Stenvang, J; Brünner, N; Sánchez-Céspedes, R; Millán, Y; Gómez-Laguna, J; de las Mulas, J Martín

    2014-09-01

    Cloning and sequencing of the progesterone receptor gene in dogs have revealed 2 isoforms, A and B, transcribed from a single gene. Distribution of isoforms A and B in canine mammary lesions has hitherto been investigated only by Western blot analysis. This study analyzed progesterone receptor 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 tumors, and 59% of carcinomas. Carcinomas, and particularly simple epithelial-type carcinomas, displayed the lowest levels of expression. A high rate of agreement was recorded between RT-qPCR and immunohistochemical labeling. Isoforms A and B were successfully amplified, with correlation coefficients of 0.99 and amplification efficiencies close to 2, and were expressed in all lesion types analyzed. Predominance of A over B expression was observed in carcinomas and complex adenomas. Low-grade tumors exhibited higher progesterone receptor messenger RNA (mRNA) levels, but no difference was observed 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 of the canine mammary gland. These findings will facilitate future research into the role of progesterone receptor isoforms in the progression of canine mammary tumors. PMID:24249219

  11. Expression of steroidogenic enzymes and their transcription factors in cortisol-producing adrenocortical adenomas: immunohistochemical analysis and quantitative real-time polymerase chain reaction studies.

    Science.gov (United States)

    Kubota-Nakayama, Fumie; Nakamura, Yasuhiro; Konosu-Fukaya, Sachiko; Azmahani, Abdullah; Ise, Kazue; Yamazaki, Yuto; Kitawaki, Yuko; Felizola, Saulo J A; Ono, Yoshikiyo; Omata, Kei; Morimoto, Ryo; Iwama, Noriyuki; Satoh, Fumitoshi; Sasano, Hironobu

    2016-08-01

    Adrenal Cushing syndrome (CS) is caused by the overproduction of cortisol in adrenocortical tumors including adrenal cortisol-producing adenoma (CPA). In CS, steroidogenic enzymes such as 17α-hydroxylase/17, 20-lase (CYP17A1), 3β-hydroxysteroid dehydrogenase (HSD3B), and 11β-hydroxylase (CYP11B1) are abundantly expressed in tumor cells. In addition, several transcriptional factors have been reported to play pivotal roles in the regulation of these enzymes in CPA, but their correlations with those enzymes above have still remained largely unknown. Therefore, in this study, we examined the status of steroidogenic enzymes and their transcriptional factors in 78 and 15 CPA cases by using immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR), respectively. Immunoreactivity of HSD3B2, CYP11B1, CYP17A1, steroidogenic factor-1 (SF1[NR5A1]), GATA6, and nerve growth factor induced-B (NGFIB[NR4A1]) was detected in tumor cells. Results of qPCR analysis revealed that expression of HSD3B2 mRNA was significantly higher than that of HSD3B1, and CYP11B1 mRNA was significantly higher than CYP11B2. In addition, the expression of CYP11B1 mRNA was positively correlated with those of NR5A1, GATA6, and NR4A1. These results all indicated that HSD3B2 but not HSD3B1 was mainly involved in cortisol overproduction in CPA. In addition, NR5A1, GATA6, and NR4A1 were all considered to play important roles in cortisol overproduction through regulating CYP11B1 gene transcription. PMID:27085553

  12. Time profile of viral DNA in aqueous humor samples of patients treated for varicella-zoster virus acute retinal necrosis by use of quantitative real-time PCR.

    Science.gov (United States)

    Bernheim, D; Germi, R; Labetoulle, M; Romanet, J P; Morand, P; Chiquet, C

    2013-07-01

    The objective of this study was to evaluate the kinetics of varicella-zoster virus (VZV) loads using quantitative PCR (qPCR) in patients treated for acute retinal necrosis (ARN). Six patients (52 ± 13 years old) with ARN syndrome were consecutively studied. Aqueous humor (AH) was sampled from both eyes of all patients for qPCR evaluation. The patients were treated with intravenous acyclovir and intravitreal injections of antiviral drugs. The mean follow-up time was 17.6 ± 16.4 months. Main outcome measures were the numbers of viral genome copies in the AH, assessed using real-time qPCR with hydrolysis probe technology with a threshold of detection of 200 copies/ml. Two main portions of the viral load curves were observed for each patient: a plateau phase (27.8 ± 24.9 days) and a decrease in the number of viral genome copies. The mean baseline viral load was 3.4 × 10(7) ± 4.45 × 10(7) copies/ml (6 × 10(6) to 1.2 × 10(8) copies/ml). The viral load decreased according to a logarithmic model, with a 50% reduction obtained in 3 ± 0.7 days. There was a significant viral load (>102 copies/ml) at 50 days after the onset of treatment, despite antiviral drugs. qPCR use demonstrated reproducible VZV DNA kinetics with a two-phase evolution: a plateau followed by a logarithmic decrease. These data suggest that high-dosage antiviral therapy administered for the conventional 10-day duration is insufficient for most patients. This series of patients responded with a similar decrease in viral load once treatment was initiated, and the data from these patients may be used to predict the responses of future patients. PMID:23637296

  13. 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.

    Directory of Open Access Journals (Sweden)

    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.

  14. Quantitative Real-Time PCR Analysis of YKL-40 and Its Comparison with Mammalian Chitinase mRNAs in Normal Human Tissues Using a Single Standard DNA

    Directory of Open Access Journals (Sweden)

    Misa Ohno

    2015-04-01

    Full Text Available YKL-40 (YKL for the first three N-terminal residues of a 40 kDa protein belongs to a group of human chitinase-like proteins (CLPs, which are similar to chitinases but lack chitinolytic activity. YKL-40 mRNA and its protein levels have been reported elevated in multiple disorders including asthma, cystic fibrosis, rheumatoid arthritis and malignant tumors. Here, we quantified the YKL-40 mRNA levels and compared them with chitinases and housekeeping genes in normal human tissues. To establish the quantitative real-time PCR (qPCR system for evaluation of relative YKL-40 mRNA levels, we constructed a human standard DNA molecule by ligating cDNAs of YKL-40, two mammalian chitinases and two housekeeping genes in a one-to-one ratio. We generated cDNAs from various normal human tissues and analyzed the YKL-40 mRNA expression levels using a qPCR system with the standard DNA. We found that YKL-40 mRNA is present widely in human tissues while its expression patterns exhibit clear tissue specificity. Highest YKL-40 mRNA levels were detected in the liver, followed by kidney, trachea and lung. The levels of YKL-40 mRNA in the kidney and liver were more than 100-times higher than those of chitotriosidase mRNA. Our study provides for the first time a comprehensive analysis of the relative expression levels of YKL-40 mRNA versus mammalian chitinases in normal human tissues.

  15. 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.

  16. Development of quantitative real-time PCR assays for fathead minnow (Pimephales promelas) gonadotropin ß subunit mRNAs to support endocrine disruptor research.

    Energy Technology Data Exchange (ETDEWEB)

    Villeneuve, Daniel L.; Miracle, Ann L.; Jensen, Kathleen M.; Degitz, Sigmund J.; Kahl, Michael D.; Korte, Joseph J.; Greene, Katie J.; Blake, Lindsey S.; Linnum, Ann; Ankley, Gerald T.

    2007-03-01

    Fathead minnows (Pimephales promelas) are one of the most widely-used small fish models for regulatory ecotoxicology testing and research related to endocrine disrupting chemicals (EDCs). In this study, we isolated and sequenced cDNAs for fathead minnow follicle-stimulating hormone-like and luteinizing hormone-like β (FSHβ and LHβ) and glycoprotein α (GPα) subunits. Quantitative real-time PCR assays for measuring gonadotropin (GtH) β subunit transcripts were developed and used to examine “baseline” transcript levels over a range of age classes and reproductive states encompassed in EDC testing. In females, FSHβ and LHβ transcripts were greater in 4-5 month old than in younger fish and were significantly correlated with one another across all age classes examined. In males, FSHβ transcripts were greatest in 2-3 month old fish and were inversely correlated with various measures of testis development including, gonadal-somatic index (GSI), and histological stage. Overall, the pattern of GtHβ expression over age classes associated with gonad development was similar to that reported for other asynchronous-spawning fish. Despite significant changes in female GSI, gonad stage, and plasma vitellogenin within 24 h of spawning, GtHβ transcript levels in fish that had spawned within the preceding 24 h were not significantly different from those in fish that were 2-3 days post-spawn and expected to spawn within the next 24 h based on spawning history. Results of this study provide insights related to the role of GtHs in fathead minnow reproductive development and function. Additionally they provide useful “baseline” data needed to design and interpret effective experiments for studying direct and indirect effects of EDCs on GtH subunit mRNA expression, which will facilitate a greater understanding of integrated system-wide responses of the fathead minnow brain-pituitary-gonadal axis to stressors including EDCs.

  17. Real-time radiography

    International Nuclear Information System (INIS)

    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

  18. Mass Spectrometry-Based Approaches Toward Absolute Quantitative Proteomics

    OpenAIRE

    Kito, Keiji; Ito, Takashi

    2008-01-01

    Mass spectrometry has served as a major tool for the discipline of proteomics to catalogue proteins in an unprecedented scale. With chemical and metabolic techniques for stable isotope labeling developed over the past decade, it is now routinely used as a method for relative quantification to provide valuable information on alteration of protein abundance in a proteome-wide scale. More recently, absolute or stoichiometric quantification of proteome is becoming feasible, in particular, with th...

  19. Real-Time Shading

    CERN Document Server

    Olano, Marc

    2002-01-01

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

  20. Application of quantitative real-time reverse transcription-PCR in assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum in vitro.

    Science.gov (United States)

    Cai, Xiaomin; Woods, Keith M; Upton, Steve J; Zhu, Guan

    2005-11-01

    We report here on a quantitative real-time reverse transcription-PCR (qRT-PCR) assay for assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum. The qRT-PCR assay detects 18S rRNA transcripts from both parasites, that is, the cycle threshold for 18S rRNA from parasites (C(T)([P18S])) and host cells (C(T)([H18S])), and evaluates the relative expression between parasite and host rRNA levels (i.e., deltaC(T) = C(T)([P18S]) - C(T)([H18S])) to minimize experimental and operational errors. The choice of qRT-PCR over quantitative PCR (qPCR) in this study is based on the observations that (i) the relationship between the logarithm of infected parasites (log[P]) and the normalized relative level of rRNA (deltadeltaC(T)) is linear, with a fourfold dynamic range, by qRT-PCR but sigmoidal (nonlinear) by qPCR; and (ii) the level of RNA represents that of live parasites better than that of DNA, because the decay of RNA (99% in approximately 3 h) in dead parasites is faster than that of DNA (99% in approximately 24 to 48 h) under in vitro conditions. The reliability of the qRT-PCR method was validated by testing the efficacies of nitazoxanide and paromomycin on the development of two strains of C. parvum (IOWA and KSU-1) in HCT-8 cells in vitro. Both compounds displayed dose-dependent inhibitions. The observed MIC50 values for nitazoxanide and paromomycin were 0.30 to 0.45 micro/ml and 89.7 to 119.0 microg/ml, respectively, comparable to the values reported previously. Using the qRT-PCR assay, we have also observed that pyrazole could inhibit C. parvum development in vitro (MIC50 = 15.8 mM), suggesting that the recently discovered Cryptosporidium alcohol dehydrogenases may be explored as new drug targets. PMID:16251280

  1. Human parvovirus B19, varicella zoster virus, and human herpes virus 6 in temporal artery biopsy specimens of patients with giant cell arteritis: analysis with quantitative real time polymerase chain reaction

    OpenAIRE

    Alvarez-Lafuente, R.; Fernandez-Gutierr..., B; Jover, J; Judez, E; Loza, E; Clemente, D; Garcia-Asenjo, J; Lamas, J

    2005-01-01

    Methods: Temporal artery biopsy specimens from 57 patients with GCA and 56 controls were investigated. DNA was obtained by biopsy, and quantitative real time polymerase chain reaction assay performed to establish the prevalence and viral load of B19, VZV, and HHV-6. Amplification of the human ß-globin gene was used as internal positive control.

  2. TRI12 based quantitative real-time PCR assays reveal the distribution of trichothecene genotype of F. graminearum and F. culmorum isolates in Danish small grain cereals

    DEFF Research Database (Denmark)

    Nielsen, L. K.; Jensen, J. D.; Rodríguez, A.;

    2012-01-01

    Quantitative real-time PCR assays, based on polymorphisms in the TRI12 gene of the trichothecene pathway, were developed to identify and quantify the trichothecene genotypes producing 3-acetyl-deoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON) or nivalenol (NIV) in the Fusarium graminearum...

  3. Understanding the Mechanisms Enabling an Ultra-high Efficiency Moving Wire Interface for Real-time Carbon 14 Accelerator Mass Spectrometry Quantitation of Samples Suspended in Solvent

    Science.gov (United States)

    Thomas, Avraham Thaler

    Carbon 14 (14C) quantitation by accelerator mass spectrometry (AMS) is a powerfully sensitive and uniquely quantitative tool for tracking labeled carbonaceous molecules in biological systems. This is due to 14C's low natural abundance of 1 ppt, the nominal difference in biological activity between an unlabeled and a 14C-labeled molecule, and the ability of AMS to measure isotopic ratios independently of a sample's other characteristics. To make AMS more broadly accessible, a moving wire interface for real-time coupling of high pressure liquid chromatography (HPLC) to AMS and high throughput AMS quantitation of minute single samples has been developed. Prior to this work, samples needed to be converted to solid carbon before measurement. This conversion process has many steps and requires that the sample size be large enough to allow precise handling of the resulting graphite. These factors make the process susceptible to error and time consuming, as well as requiring 0.5 ug of carbon. Samples which do not contain enough carbon, such as HPLC fractions, must be bulked up. This adds background and increases effort. The moving wire interface overcomes these limitations by automating sample processing. Samples placed on the wire are transported through a solvent removal stage followed by a combustion stage after which the combustion products are directed to a gas accepting ion source. The ion source converts the carbon from the CO2 combustion product into C ions, from which an isotopic ratio can be determined by AMS. Although moving wire interfaces have been implemented for various tasks since 1964, the efficiency of these systems at transferring fluid from an HPLC to the wire was only 3%, the efficiency of transferring combustion products from the combustion oven to ion source was only 30%, the flow and composition of the carrier gas from the combustion oven to the ion source needed to be optimized for coupling to an AMS gas accepting ion source and the drying ovens

  4. LEMming: A Linear Error Model to Normalize Parallel Quantitative Real-Time PCR (qPCR Data as an Alternative to Reference Gene Based Methods.

    Directory of Open Access Journals (Sweden)

    Ronny Feuer

    Full Text Available Gene expression analysis is an essential part of biological and medical investigations. Quantitative real-time PCR (qPCR is characterized with excellent sensitivity, dynamic range, reproducibility and is still regarded to be the gold standard for quantifying transcripts abundance. Parallelization of qPCR such as by microfluidic Taqman Fluidigm Biomark Platform enables evaluation of multiple transcripts in samples treated under various conditions. Despite advanced technologies, correct evaluation of the measurements remains challenging. Most widely used methods for evaluating or calculating gene expression data include geNorm and ΔΔCt, respectively. They rely on one or several stable reference genes (RGs for normalization, thus potentially causing biased results. We therefore applied multivariable regression with a tailored error model to overcome the necessity of stable RGs.We developed a RG independent data normalization approach based on a tailored linear error model for parallel qPCR data, called LEMming. It uses the assumption that the mean Ct values within samples of similarly treated groups are equal. Performance of LEMming was evaluated in three data sets with different stability patterns of RGs and compared to the results of geNorm normalization. Data set 1 showed that both methods gave similar results if stable RGs are available. Data set 2 included RGs which are stable according to geNorm criteria, but became differentially expressed in normalized data evaluated by a t-test. geNorm-normalized data showed an effect of a shifted mean per gene per condition whereas LEMming-normalized data did not. Comparing the decrease of standard deviation from raw data to geNorm and to LEMming, the latter was superior. In data set 3 according to geNorm calculated average expression stability and pairwise variation, stable RGs were available, but t-tests of raw data contradicted this. Normalization with RGs resulted in distorted data contradicting

  5. Screening Brucella spp. in bovine raw milk by real-time quantitative PCR and conventional methods in a pilot region of vaccination, Edirne, Turkey.

    Science.gov (United States)

    Kaynak-Onurdag, F; Okten, S; Sen, B

    2016-05-01

    Brucellosis is a worldwide zoonotic disease transmitted to humans by consumption of contaminated milk and milk products. Brucellosis is endemic in Turkey, and Edirne has a high Brucella prevalence. Brucellosis is prevented by live-attenuated vaccines for animals and the vaccination program has been in place since 1984 in Turkey. Thrace is the pilot region for this vaccination program. The gold standard diagnostic technique for brucellosis is still the isolation of suspicious bacterial colonies followed by bacteriological identification, but it is very time consuming and laborious. In many studies, Brucella has been investigated by PCR techniques. However, PCR-based methods cannot differentiate between the vaccine strain and the virulent strain; thus, the vaccine strain may interfere with the virulent strain and causes false-positive reactions. To monitor brucellosis control programs effectively, it is important to distinguish vaccine and field strains of Brucella spp. In this study, raw milk samples were collected from 99 cows at 12 different barns in 5 villages of Edirne (Turkey). Bacteriological analyses and real-time quantitative (q)PCR experiments were applied to all samples. The DNA was isolated using Biospeedy DNA-Tricky Purification Kit (Bioeksen, Istanbul, Turkey). For all reactions, Roche Light Cycler Nano (Roche Diagnostics, Mannheim, Germany) instrument and Biospeedy EvaGreen qPCR Pre-Mix (Bioeksen) were used. The data were analyzed using Roche LightCycler NanoSoftware 1.0. For samples that were negative by bacteriological analyses and positive by qPCR, we developed a novel qPCR-based method to differentiate the virulent B. abortus strains and B. abortus S19 vaccine strain. We designed qPCR primers targeting the outer membrane protein of B. abortus. The qPCR products were sequenced using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit on an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA). In total, 2.02% of the

  6. 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

  7. Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa.

    Science.gov (United States)

    Bhoora, Raksha; Quan, Melvyn; Franssen, Linda; Butler, Catherine M; van der Kolk, Johannes H; Guthrie, Alan J; Zweygarth, Erich; Jongejan, Frans; Collins, Nicola E

    2010-03-25

    A quantitative real-time polymerase chain reaction (qPCR) assay using a TaqMan minor groove binder (MGB) probe was developed for the detection of Babesia caballi infection in equids from South Africa. Nine previously published sequences of the V4 hypervariable region of the B. caballi 18S rRNA gene were used to design primers and probes to target unique, conserved regions. The B. caballi TaqMan MGB qPCR assay was shown to be efficient and specific. The detection limit, defined as the concentration at which 95% of positive samples can be detected, was determined to be 0.000114% parasitized erythrocytes (PE). We further evaluated a previously reported Theileria equi-specific qPCR assay and showed that it was able to detect the 12 T. equi 18S rRNA sequence variants previously identified in South Africa. Both qPCR assays were tested on samples from two ponies experimentally infected with either T. equi or B. caballi. The qPCR assays were more sensitive than the indirect fluorescent antibody test (IFAT) and the reverse-line blot (RLB) during the early onset of the disease. The assays were subsequently tested on field samples collected from 41 horses, resident on three stud farms in the Northern Cape Province, South Africa. The IFAT detected circulating T. equi and B. caballi antibody in, respectively, 83% and 70% of the samples. The RLB detected T. equi parasite DNA in 73% of the samples, but none of the samples were positive for B. caballi, although 19 T. equi-positive samples also hybridized to the Babesia genus-specific probe. This could indicate a mixed T. equi and B. caballi infection in these samples, with either the B. caballi parasitaemia at a level below the detection limit of the B. caballi RLB probe, or the occurrence of a novel Babesia genotype or species. In contrast, the qPCR assays correlated fairly well with the IFAT. The B. caballi TaqMan MGB qPCR assay was able to detect B. caballi parasite DNA in 78% of the samples. The T. equi-specific qPCR assay

  8. Route around real time

    International Nuclear Information System (INIS)

    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)

  9. 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

    by antibiotics (ionophores) presently used to prevent parasitic coccidiosis. From 2012 the European Union has banned these anticoccidials as feed additives, wherefore alternatives are needed to suppress C. perfringens and/or α-toxin production. A real-time PCR primer-probe set targeting the α-toxin gene...

  10. Rapid and Quantitative Detection of Hepatitis A Virus from Green Onion and Strawberry Rinses by Use of Real-Time Reverse Transcription-PCR

    OpenAIRE

    Shan, X. C.; Wolffs, P; Griffiths, M. W.

    2005-01-01

    In this study, an immunomagnetic capture method and a real-time reverse transcription-PCR assay were used to quantify hepatitis A virus (HAV) in green onion and strawberry rinses. This combined protocol detected as low as 0.5 PFU HAV in produce rinses and concentrated HAV levels up to 20-fold.

  11. 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 TaqMa

  12. DEVELOPMENT AND VALIDATION OF A REAL-TIME TAQMAN(R) PCR ASSAY FOR THE DETECTION AND QUANTITATION OF INFECTIOUS LARYNGOTRACHEITIS VIRUS IN POULTRY

    Science.gov (United States)

    In this study, we report the development and validation of a real-time PCR assay using a Taqman(R) labeled probe (ILTV assay) for the detection and quantification of infectious larygotracheitis virus (ILTV). The ILTV assay was highly specific with a detection limit of 25 viral template copies/amplif...

  13. 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

  14. 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…

  15. Real Time Processing

    CERN Document Server

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

    2015-01-01

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

  16. 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...

  17. [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.

  18. Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses.

    Science.gov (United States)

    Simmons, Monika; Myers, Todd; Guevara, Carolina; Jungkind, Donald; Williams, Maya; Houng, Huo-Shu

    2016-07-01

    Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping. PMID:27098955

  19. 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...... the 3D gene of FMDV. The assay was validated for the efficacy to detect all known FMDV serotypes. The test method was linear over a range of at least 7 orders of magnitude and the detection limit was below the equivalent of 10 genomic copies. Analysing recent African probang samples the method was able...... to detect FMDV in materials from both cattle and buffalo. When compared to traditional virus cultivation the virus detection sensitivity was similar but the RT-PCR method can provide a laboratory result much faster than virus cultivation. The real-time PCR method confirms the identity of the amplicon...

  20. DETECTION SENSITIVITY AND QUANTITATION OF PLASMODIUM FALCIPARUM VAR GENE TRANSCRIPTS BY REAL-TIME RT-PCR IN COMPARISON WITH CONVENTIONAL RT-PCR

    OpenAIRE

    Gatton, Michelle L; Peters, Jennifer M.; GRESTY, KARRYN; Fowler, Elizabeth V.; Chen, Nanhua; Cheng, Qin

    2006-01-01

    Antigenic variation in Plasmodium falciparum erythrocyte membrane protein 1, caused by a switch in transcription of the encoding var gene, is an important feature of malaria. In this study, we quantified the relative abundance of var gene transcripts present in P. falciparum parasite clones using real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional RT-PCR combined with cloning and sequencing, with the aim of directly comparing the results obtained. When there wa...

  1. 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

  2. Real- Time Simulation Applications

    Directory of Open Access Journals (Sweden)

    V. Srinivas

    1987-10-01

    Full Text Available This paper deals with development of the real-time simulation systems. Two systems, one for supporting the research and development programmes of the aircraft industry and the other to provide pilot training of modem aircraft are described. The presentation brings out the effectiveness of such systems in their respective roles and the techniques developed in the design and realisation of hardware and software.This also gives a overview of various activities which have culminated in the establishment of research simulation facility and training simulators. Also plans of actions and techniques proposed to be employed in terms of future programmes are discussed.

  3. Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS

    OpenAIRE

    Steen, Hanno; Jebanathirajah, Judith A.; Springer, Michael; Kirschner, Marc W.

    2005-01-01

    Qualitative and quantitative information are crucial to a detailed understanding of the function of protein phosphorylation. MS is now becoming a quantitative approach to analyze protein phosphorylation. All methods that have been described either require the elaborate/expensive use of stable isotopes to compare a limited number of samples or do not provide phosphorylation stoichiometries. Here, we present stable isotope-free MS strategies that allow relative and absolute quantitation of phos...

  4. Evaluation of the Aptima(®) HIV-1 Quant Dx assay for HIV-1 RNA viral load detection and quantitation in plasma of HIV-1-infected individuals: A comparison with Abbott RealTime HIV-1 assay.

    Science.gov (United States)

    Amendola, Alessandra; Pisciotta, Maria; Aleo, Loredana; Ferraioli, Valeria; Angeletti, Claudio; Capobianchi, Maria Rosaria

    2016-09-01

    The Hologic Aptima(®) HIV-1 Quant Dx assay (Aptima HIV) is a real-time transcription-mediated amplification method CE-approved for use in diagnosis and monitoring of HIV-1 infection. The analytical performance of this new assay was compared to the FDA-approved Abbott RealTime HIV-1 (RealTime). The evaluation was performed using 220 clinical plasma samples, the WHO 3rd HIV-1 International Standard, and the QCMD HIV-1 RNA EQA. Concordance on qualitative results, correlation between quantitative results, accuracy, and reproducibility of viral load data were analyzed. The ability to measure HIV-1 subtypes was assessed on the second WHO International Reference Preparation Panel for HIV-1 Subtypes. With clinical samples, inter-assay agreement for qualitative results was high (91.8%) with Cohen's kappa statistic equal to 0.836. For samples with quantitative results in both assays (n = 93), Lin's concordance correlation coefficient was 0.980 (P R(2)  > 0.970) and showed higher sensitivity compared to RealTime being able to detect HIV-1 RNA in 10 out of 10 replicates containing down to 7 cp/ml (20 IU/ml). Reproducibility was very high, even at low HIV-1 RNA values. The Aptima HIV was able to detect and accurately quantify all the main HIV-1 subtypes in both reference panels and clinical samples. Besides excellent performance, Aptima HIV shows full automation, ease of use, and improved workflow compared to RealTime. J. Med. Virol. 88:1535-1544, 2016. © 2016 Wiley Periodicals, Inc. PMID:26864171

  5. Real-time interferometric synthetic aperture microscopy

    OpenAIRE

    Ralston, Tyler S.; Marks, Daniel L.; Carney, P. Scott; Boppart, Stephen A.

    2008-01-01

    An interferometric synthetic aperture microscopy (ISAM) system design with real-time 2D cross-sectional processing is described in detail. The system can acquire, process, and display the ISAM reconstructed images at frame rates of 2.25 frames per second for 512 × 1024 pixel images. This system provides quantitatively meaningful structural information from previously indistinguishable scattering intensities and provides proof of feasibility for future real-time ISAM systems.

  6. Real-time interferometric synthetic aperture microscopy.

    Science.gov (United States)

    Ralston, Tyler S; Marks, Daniel L; Carney, P Scott; Boppart, Stephen A

    2008-02-18

    An interferometric synthetic aperture microscopy (ISAM) system design with real-time 2D cross-sectional processing is described in detail. The system can acquire, process, and display the ISAM reconstructed images at frame rates of 2.25 frames per second for 512 X 1024 pixel images. This system provides quantitatively meaningful structural information from previously indistinguishable scattering intensities and provides proof of feasibility for future real-time ISAM systems. PMID:18542337

  7. Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosa in sputum of cystic fibrosis patients

    Directory of Open Access Journals (Sweden)

    De Vos Daniel

    2009-11-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is the major pathogen involved in the decline of lung function in cystic fibrosis (CF patients. Early aggressive antibiotic therapy has been shown to be effective in preventing chronic colonization. Therefore, early detection is important and sensitive detection methods are warranted. In this study, we used a dilution series of P. aeruginosa positive sputa, diluted in a pool of P. aeruginosa negative sputa, all from CF patients - to mimick as closely as possible the sputa sent to routine laboratories - to compare the sensitivity of three culture techniques versus that of two conventional PCR formats and four real-time PCR formats, each targeting the P. aeruginosa oprL gene. In addition, we compared five DNA-extraction protocols. Results In our hands, all three culture methods and the bioMérieux easyMAG Nuclisens protocol Generic 2.0.1, preceded by proteinase K pretreatment and followed by any of the 3 real-time PCR formats with probes were most sensitive and able to detect P. aeruginosa up to 50 cfu/ml, i.e. the theoretical minimum of one cell per PCR mixture, when taking into account the volumes used in this study of sample for DNA-extraction, of DNA-elution and of DNA-extract in the PCR mixture. Conclusion In this study, no difference in sensitivity could be found for the detection of P. aeruginosa from sputum between microbiological culture and optimized DNA-extraction and real-time PCR. The results also indicate the importance of the optimization of the DNA-extraction protocol and the PCR format.

  8. Multicenter Evaluation of the New Abbott RealTime Assays for Quantitative Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA▿

    OpenAIRE

    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 TaqMan assays. Two different protocols used during the testing period with and without a pre-m1000 RNA isolation spin were compared. The difference proved to be nonsignificant. A uracil-N-glycosylase (U...

  9. 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)

  10. 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▿

    OpenAIRE

    Bravo, Dayana; Clari, María Ángeles; Costa, Elisa; Muñoz-Cobo Liria, Beatriz; Solano Vercet, Carlos; José Remigia, María; Navarro, David

    2011-01-01

    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 (Qia...

  11. 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...

  12. 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)

    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+0.0198X2). Conclusion: We find that γ ray has influence on the mtDNA4977bp deletion, so it may be an important biodosmeter in future. (authors)

  13. 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 the...... genetic potential for degrading PA in the soils. In 25 of the 45 mineralization scenarios, ∼60% mineralization was observed within 118 days. Elevated concentrations of tfdA in the range 1 × 105 to 5 × 107 gene copies g−1 of soil were observed in soils where mineralization could be described by using...... patterns which were best fitted by the exponential model yielded a higher tfdA gene potential after mineralization had occurred than the three mineralization patterns best fitted by the Lin model....

  14. 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.

  15. Detection of carcinoembryonic antigen messenger RNA in blood using quantitative real-time reverse transcriptase-polymerase chain reaction to predict recurrence of gastric adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Wang Zhi-qiang

    2010-10-01

    Full Text Available Abstract Background The existence of circulating tumor cells (CTCs in peripheral blood as an indicator of tumor recurrence has not been clearly established, particularly for gastric cancer patients. We conducted a retrospective analysis of the relationship between CTCs in peripheral blood at initial diagnosis and clinicopathologic findings in patients with gastric carcinoma. Methods Blood samples were obtained from 123 gastric carcinoma patients at initial diagnosis. mRNA was extracted and amplified for carcinoembryonic antigen (CEA mRNA detection using real-time RT-PCR. Periodic 3-month follow-up examinations included serum CEA measurements and imaging. Results The minimum threshold for corrected CEA mRNA score [(CEA mRNA/GAPDH mRNA × 106] was set at 100. Forty-five of 123 patients (36.6% were positive for CEA mRNA expression. CEA mRNA expression significantly correlated with T stage and postoperative recurrence status (P = 0.001. Recurrent disease was found in 44 of 123 cases (35.8%, and 25 of these (56.8% were positive for CEA mRNA. Of these patients, CEA mRNA was more sensitive than serum CEA in indicating recurrence. Three-year disease-free survival of patients positive for CEA mRNA was significantly poorer than of patients negative for CEA mRNA (P Conclusions CEA mRNA copy number in peripheral blood at initial diagnosis was significantly associated with disease recurrence in gastric adenocarcinoma patients. Real-time RT-PCR detection of CEA mRNA levels at initial diagnosis appears to be a promising predictor for disease recurrence in gastric adenocarcinoma patients.

  16. Designing Real Time Assistive Technologies

    DEFF Research Database (Denmark)

    Sonne, Tobias; Obel, Carsten; Grønbæk, Kaj

    2015-01-01

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

  17. Fluorescent quantitative real-time PCR for quantitative detection of Schistosoma japonicum cercariae in water%荧光实时定量PCR定量水体中日本血吸虫尾蚴方法的建立

    Institute of Scientific and Technical Information of China (English)

    王本敬; 王文波; 周霞; 陈艳勤; 张静; 刘晨晨; 梁幼生; 诸葛洪祥

    2011-01-01

    目的 建立快速、高效、特异的定量水体中尾蚴的数量和检测水体中日本血吸虫尾蚴残余基因组DNA的方法,来评估水体受日本血吸虫尾蚴污染的程度.方法 根据日本血吸虫基因组DNA中的3个多拷贝序列Sjrh1.0(序列号:U92488.1)、18S小亚基单位核楷体核酸基因(18SrRNA)序列(序列号:AY157226.1)和逆转录转座子SjR2的G55A序列(G55A)(序列号:AF412221.1),设计常规PCR引物和实时定量PCR引物,选取较好的靶序列建立SYBR GreenI实时定量PCR方法,绘制尾蚴数的对数与Ct值的标准曲线,并对疫水中日本血吸虫尾蚴残余的基因组DNA进行检测.结果 尾蚴数的对数与Ct值的标准曲线有良好的线性关系,相关系数r2为0.918 6,重复性良好.结论 本方法特异性高,灵敏,可定量水体中尾蚴数,对疫水检测有一定的预警作用.%The objective was to establish a rapid, sensitive and specific method to detect the number of cercariea in water and evaluate level of water stained by Schistosoma japonicum cercariea. Convenience PCR primer sequences were designed tar-geting pSjrH1.0(U92488. 1), Sjl8SrRNA( AY157226. 1) and the clone G55A of the highly repetitive retrotransposon SjR2 (AF412221. 1) in S. japonicum genome, and sequence the PCR product. Based on conserved sequence of pSjrHl. 0, Sjl8SrRNA and clone G55A of highly repetitive retrotransposon SjR2 (G55A), design primers and the quantitative real-time PCR essays were established, by which, the amplifying products were 150 to 170 bp. The sequence in S. japonicum genome and the best annealing temperature were selected by comparing their threshold cycle (Ct value). Then the quantitative real-time PCR essays were established under the better annealing temperature, generate standard curve between the logarithms of gradi-ent diluted DNA templates and Ct value. Five DNA samples extracted from 1, 5, 10, 20 and 80 cercariae were used as quanti-tative template to generate standard curve

  18. Real Time PCR: Principles and Application

    Directory of Open Access Journals (Sweden)

    Safie Amini

    2005-09-01

    absolute quantification or relative quantification. Absolute quantification determines the absolute amount of target (expressed as copy number or concentration, whereas relative quantification determines the ratio between the amount of target and an endogenous reference molecule, usually asuitable housekeeping gene. This normalized value can then be used to compare, for example, differential gene expression in different samples.Absolute QuantificationUse of external standards enables the expression level of a gene to be given as an absolute copy number. For gene expression analysis, the most accurate standards are RNA molecules of known copy number or concentration. Depending on the sequence and structure of the target and the efficiency of reverse transcription, only a proportion of the target RNA in the RNA sample will be reverse transcribed. The cDNA generated during reversetranscription then serves as template in the subsequent real-time PCR. The use of RNA standards takes into account the variable efficiency of reverse transcription as well as the possible presence of reverse-transcription inhibitors, which may be in the RNA sample(18, 19.Relative QuantificationAlternatively, gene expression levels can be calculated by determining the ratio between the amount of a target gene and an endogenous reference gene that is in all samples. This ratio is then compared between different samples. Usually, housekeeping or maintenance genes are chosen as an endogenous reference. The target and the reference genes are amplified from the same sample, either separately or in the same reaction (duplex real-time PCR. The normalized value is determined for each sample and can be used to compare, for example, differential expression of a gene in different tissues. However, the expression level of the endogenous reference gene must not vary under different experimental conditions or in different states of the tissue (e.g., "stimulated" versus "unstimulated" samples. When gene expression

  19. Evaluation of p16 hypermethylation in oral submucous fibrosis: A quantitative and comparative analysis in buccal cells and saliva using real-time methylation-specific polymerase chain reaction

    Science.gov (United States)

    Kaliyaperumal, Subadra; Sankarapandian, Sathasivasubramanian

    2016-01-01

    Aims: The aim of this study was to quantitatively investigate the hypermethylation of p16 gene in buccal cells and saliva of oral submucous fibrosis (OSMF) patients using real-time quantitative methylation-specific polymerase chain reaction (PCR) and to compare the values of two methods. Subjects and Methods: A total of 120 samples were taken from 60 subjects selected for this study, of which 30 were controls and 30 patients were clinically and histopathologically diagnosed with OSMF. In both groups, two sets of samples were collected, one directly from the buccal cells through cytobrush technique and the other through salivary rinse. We analyzed the samples for the presence of p16 hypermethylation using quantitative real-time PCR. Results: In OSMF, the hypermethylation status of p16 in buccal cells was very high (93.3%) and in salivary samples, it was partially methylated (50%). However, no hypermethylation was found in controls suggesting that significant quantity of p16 hypermethylation was present in buccal cells and saliva in OSMF. Conclusions: This study indicates that buccal cell sampling may be a better method for evaluation than the salivary samples. It signifies that hypermethylation of p16 is an important factor to be considered in epigenetic alterations of normal cells to oral precancer, i.e. OSMF.

  20. 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. PMID:27367320

  1. Detection of Live Salmonella sp. Cells in Produce by a TaqMan-Based Quantitative Reverse Transcriptase Real-Time PCR Targeting invA mRNA▿ †

    OpenAIRE

    González-Escalona, Narjol; Hammack, Thomas S.; Russell, Mindi; Jacobson, Andrew P.; De Jesús, Antonio J.; Brown, Eric W.; Lampel, Keith A.

    2009-01-01

    Salmonella enterica contamination in foods is a significant concern for public health. When DNA detection methods are used for analysis of foods, one of the major concerns is false-positive results from the detection of dead cells. To circumvent this crucial issue, a TaqMan quantitative real-time RT-PCR (qRT-PCR) assay with an RNA internal control was developed. invA RNA standards were used to determine the detection limit of this assay as well as to determine invA mRNA levels in mid-exponent...

  2. 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...

  3. 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...

  4. Quantitation of aflatoxins from corn and other food related materials by direct analysis in real time - mass spectrometry (DART-MS)

    Science.gov (United States)

    Ambient ionization coupled to mass spectrometry continues to be applied to new analytical problems, facilitating the rapid and convenient analysis of a variety of analytes. Recently, demonstrations of ambient ionization mass spectrometry applied to quantitative analysis of mycotoxins have been shown...

  5. Selection and Validation of Reference Genes for Quantitative Real-Time PCR in Buckwheat (Fagopyrum esculentum) Based on Transcriptome Sequence Data

    OpenAIRE

    Demidenko, Natalia V.; Logacheva, Maria D.; Aleksey A Penin

    2011-01-01

    Quantitative reverse transcription PCR (qRT-PCR) is one of the most precise and widely used methods of gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. We studied the expression stability of potential reference genes in common buckwheat (Fagopyrum esculentum) in order to find the optimal reference for gene expression analysis in this economically important crop. Recently sequenced buckwheat floral transcriptome was used a...

  6. Evaluation of an infectivity standard for real-time quality control of human immunodeficiency virus type 1 quantitative micrococulture assays. Participating Laboratories of The AIDS Clinical Trials Group.

    OpenAIRE

    Scott, W A; Brambilla, D; Siwak, E; Beatty, C.; Bremer, J.; Coombs, R W; Farzadegan, H; Fiscus, S A; Hammer, S M; Hollinger, F. B.; Khan, N.; Rasheed, S; Reichelderfer, P S

    1996-01-01

    Quantitative microculture assays of cryopreserved human immunodeficiency virus type 1-infected cell suspensions and culture supernatants were compared among seven assays sites. There was no significant change in titer during 1 year of storage. The overall standard deviation for infected cell suspensions was approximately 0.8 log10 virus titer. A method for detecting deviant assay results was developed and was used to identify two donor cell preparations (n = 54) that gave consistently low tit...

  7. Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR

    OpenAIRE

    Ray, Debashree L; Joshua C Johnson

    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 r...

  8. 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. PMID:25437743

  9. Comparison of DNA extraction kits and modification of DNA elution procedure for the quantitation of subdominant bacteria from piggery effluents with real-time PCR

    OpenAIRE

    Desneux, J.; Pourcher, A.M.

    2014-01-01

    Four commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. The PowerSoil®, PowerFecal®, NucleoSpin® Soil kits and QIAamp® DNA Stool Mini kit were tested on raw manure samples and on lagoon effluents for their ability to quantify total bacteria and a subdominant bacteria specific of pig manure contamination: Lactobacillus amylovorus. The NucleoSpin® Soil kit (NS kit), and to a lesser extent th...

  10. Dried blood spots versus plasma for the quantitation of HIV-1 RNA using a real-Time PCR, m2000rt assay.

    Science.gov (United States)

    Vidya, Madhavan; Saravanan, Shanmugam; Rifkin, Samara; Solomon, Sunil S; Waldrop, Greer; Mayer, Kenneth H; Solomon, Suniti; Balakrishnan, Pachamuthu

    2012-05-01

    High costs and stringent requirements for storage and transport of plasma, often prohibit the availability of HIV viral load quantification in resource-limited settings. Dried blood spots (DBS) represent a better method of specimen collection that removes many of these logistical and technical limitations. The present study aimed to assess the performance of the Abbott m2000rt assay for quantitation of HIV-1 RNA in DBS specimens using plasma as a "gold standard" for comparison. One hundred paired DBS and plasma specimens were collected from patients infected with HIV, who were 18 years and older during routine visits to a private tertiary-care clinic in Chennai, India. HIV-1 RNA was extracted manually and then detected using the m2000rt assay. The mean plasma and DBS viral loads were 4.27 (95% CI: 2.65, 5.88) and 4.14 (95% CI: 1.96, 6.32) log copies/mL, respectively. The overall sensitivity of DBS reached 95%; with sensitivities of 62%, 88% and 100% when stratified by viral load ranges of ≤1000, 1000-3000 and >3000 copies/mL, respectively. An over quantitation of the viral load with DBS was observed in pairs with plasma viral loadfailure in resource-limited settings. PMID:22401801

  11. Comparison of DNA extraction kits and modification of DNA elution procedure for the quantitation of subdominant bacteria from piggery effluents with real-time PCR.

    Science.gov (United States)

    Desneux, Jérémy; Pourcher, Anne-Marie

    2014-08-01

    Four commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. The PowerSoil(®), PowerFecal(®), NucleoSpin(®) Soil kits and QIAamp(®) DNA Stool Mini kit were tested on raw manure samples and on lagoon effluents for their ability to quantify total bacteria and a subdominant bacteria specific of pig manure contamination: Lactobacillus amylovorus. The NucleoSpin(®) Soil kit (NS kit), and to a lesser extent the PowerFecal(®) kit were the most efficient methods. Regardless of the kit utilized, the modified elution procedure increased DNA yield in the lagoon effluent by a factor of 1.4 to 1.8. When tested on 10 piggery effluent samples, compared to the QIAamp kit, the NS kit combined with the modified elution step, increased by a factor up to 1.7 log10 the values of the concentration of L. amylovorus. Regardless of the type of manure, the best DNA quality and the highest concentrations of bacteria were obtained using the NS kit combined with the modification of the elution procedure. The method recommended here significantly improved quantitation of subdominant bacteria in manure. PMID:24838631

  12. Real-time volume graphics

    CERN Document Server

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

    2006-01-01

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

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

    International Nuclear Information System (INIS)

    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

  14. How to develop an in-house real-time quantitative cytomegalovirus polymerase chain reaction: Insights from a cancer centre in Eastern India

    Directory of Open Access Journals (Sweden)

    Anusha Harishankar

    2015-01-01

    Full Text Available Development of a reliable, cost-effective cytomegalovirus quantitative polymerase chain reaction (QPCR is a priority for developing countries. Manufactured kits are expensive, and availability can be inconsistent. Development of an in-house QPCR kit that is reliable and quality assured requires significant effort and initial investment. However, the rewards of such an enterprise are manifold and include an in-depth understanding of molecular reactions, and expertise in the development of further low-cost molecular kits. The experience of an oncology centre in Eastern India has been shared. Hopefully, this would provide a brief roadmap for such an initiative. Staff with adequate understanding of molecular processes are essential along with vital infrastructure for molecular research and development.

  15. 探针荧光定量PCR在肺炎链球菌检测中的应用%Clinical application of real-time fluorescence quantitive PCR for detecting Streptococcus pneumoniae

    Institute of Scientific and Technical Information of China (English)

    曹东林; 胡亮彬; 林茂锐; 王婷; 黄基伟; 田军章

    2014-01-01

    目的:建立探针荧光定量PCR检测肺炎链球菌的方法。方法针对肺炎链球菌种属特异性基因lytA,设计合成了特异引物和探针,研究引物和探针的灵敏度和特异性,确定循环阈值(cycle threshold,ct)的临界值(cut-off value)。将荧光定量PCR和细菌培养法进行比较,同时检测158份肺炎患者痰液标本加以验证。结果针对LytA基因所设计的引物和探针能灵敏地检出常见致病的血清型肺炎链球菌株,检测灵敏度为每个反应100个基因组DNA拷贝。通过荧光量PCR方法,35株肺炎链球菌中检测结果为阳性有34株,检测结果为阴性的有1株;15株非肺炎链球菌全部为阴性。通过荧光定量PCR方法,158份痰液标本中共检测出34份肺炎链球菌阳性,其中10份培养出相应的病原菌。肺炎链球菌阳性患者的白细胞数量和住院时间均显著高于阴性患者(P<0.05)。肺炎链球菌阳性患者住院时间均显著长于阴性患者(P<0.05)。结论探针荧光定量PCR方法能特异地检测肺炎链球菌,具有很高的灵敏度,能提高临床肺炎链球菌感染患者标本的阳性检出率。%Objective To establish an assay for the detection of Streptococcus pneumoniae by real-time fluorescence quantititive polymerase chain reaction (PCR).Methods Special primers and probe for the autolysin A (lytA)gene were designed.The sensitivity and specificity of primers and probe were studied,and cut-off of cycle threshold was assayed.158 clinical specimens were confirmed by real-time fluorescence quantitative PCR and bacterial culture method.Results Primer and probe design for LytA gene could sensitively detect serotype Streptococcus pneumoniae strains of common pathogenic,and the sensitivity was 100 copies.Among 35 strains of Streptococcus pneumoniae,34 cases were detected to be positive for Streptococcus pneumoniae by real-time fluorescence quantitative PCR,while 1 case was

  16. Absolute Quantitative MALDI Imaging Mass Spectrometry: A Case of Rifampicin in Liver Tissues.

    Science.gov (United States)

    Chumbley, Chad W; Reyzer, Michelle L; Allen, Jamie L; Marriner, Gwendolyn A; Via, Laura E; Barry, Clifton E; Caprioli, Richard M

    2016-02-16

    Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) elucidates molecular distributions in thin tissue sections. Absolute pixel-to-pixel quantitation has remained a challenge, primarily lacking validation of the appropriate analytical methods. In the present work, isotopically labeled internal standards are applied to tissue sections to maximize quantitative reproducibility and yield accurate quantitative results. We have developed a tissue model for rifampicin (RIF), an antibiotic used to treat tuberculosis, and have tested different methods of applying an isotopically labeled internal standard for MALDI IMS analysis. The application of the standard and subsequently the matrix onto tissue sections resulted in quantitation that was not statistically significantly different from results obtained using HPLC-MS/MS of tissue extracts. Quantitative IMS experiments were performed on liver tissue from an animal dosed in vivo. Each microspot in the quantitative images measures the local concentration of RIF in the thin tissue section. Lower concentrations were detected from the blood vessels and around the portal tracts. The quantitative values obtained from these measurements were comparable (>90% similarity) to HPLC-MS/MS results obtained from extracts of the same tissue. PMID:26814665

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

    Science.gov (United States)

    Bao, Wenlong; Qu, Yanli; Shan, Xiaoyi; Wan, Yinglang

    2016-01-01

    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. PMID:27483238

  18. Selection and validation of reference genes for quantitative real-time PCR in buckwheat (Fagopyrum esculentum based on transcriptome sequence data.

    Directory of Open Access Journals (Sweden)

    Natalia V Demidenko

    Full Text Available Quantitative reverse transcription PCR (qRT-PCR is one of the most precise and widely used methods of gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. We studied the expression stability of potential reference genes in common buckwheat (Fagopyrum esculentum in order to find the optimal reference for gene expression analysis in this economically important crop. Recently sequenced buckwheat floral transcriptome was used as source of sequence information. Expression stability of eight candidate reference genes was assessed in different plant structures (leaves and inflorescences at two stages of development and fruits. These genes are the orthologs of Arabidopsis genes identified as stable in a genome-wide survey gene of expression stability and a traditionally used housekeeping gene GAPDH. Three software applications--geNorm, NormFinder and BestKeeper--were used to estimate expression stability and provided congruent results. The orthologs of AT4G33380 (expressed protein of unknown function, Expressed1, AT2G28390 (SAND family protein, SAND and AT5G46630 (clathrin adapter complex subunit family protein, CACS are revealed as the most stable. We recommend using the combination of Expressed1, SAND and CACS for the normalization of gene expression data in studies on buckwheat using qRT-PCR. These genes are listed among five the most stably expressed in Arabidopsis that emphasizes utility of the studies on model plants as a framework for other species.

  19. 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.

  20. 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.

  1. Selection and validation of reference house-keeping genes in the J774A1 macrophage cell line for quantitative real-time PCR.

    Science.gov (United States)

    Ferraz, F B; Fernandez, J H

    2016-01-01

    Macrophages are essential components of the innate and adaptive immune responses, playing a decisive role in atherosclerosis, asthma, obesity, and cancer. The differential gene expression resulting from adhesion of macrophages to the extra-cellular matrix (ECM) has been studied in the J774A1 murine macrophage cell line using quantitative polymerase chain reaction (qPCR). The goal of this study was to identify housekeeping genes (HKGs) that remain stable and unaltered under normal culture conditions and in the presence of laminin after a time lapse of 6 and 24 h. The expression stabilities of eight commonly used reference genes were analyzed by determining the comparative threshold cycle ((ΔΔ)Ct) values, and using the BestKeeper, NormFinder, and geNorm algorithms. BestKeeper analysis revealed that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), and ribosomal protein L13a (RPL13A) genes were highly stable, confirming the results of the (ΔΔ)Ct analysis. On the other hand, NormFinder proposed RPL13A and beta-glucuronidase (GUSB) to be the most suitable combination, and geNorm adjudged RPL13A, PPIA, and GUSB to be the most stable across all culture conditions. All programs discarded the use of actin beta and beta-2-microglobulin for normalization. The collected data indicated that RPL13A, PPIA, GAPDH, and GUSB as highly suitable as reference genes for qPCR analysis of murine macrophages under normal and ECM-simulated culture conditions. This study also emphasizes the importance of evaluating HKGs used for normalization to ensure the accuracy of qPCR data. PMID:26985962

  2. 应用实时荧光定量PCR快速分子诊断唐氏综合征%Application of real-time fluorescence quantitative PCR to rapid molecular detection of Down's syndrome

    Institute of Scientific and Technical Information of China (English)

    黄以宁; 廖灿; 涂新枝; 杨昕; 易翠兴; 黎丽仙

    2005-01-01

    目的探讨一种快速、准确诊断唐氏综合征的方法.方法采用实时荧光定量PCR技术,对25例唐氏患者、50名正常人外周血标本,扩增21号及1号、19号染色体上的多态位点,定量分析比较正常组及唐氏患者组的4对△Ct值.结果唐氏患者组△Ct值明显低于正常组,两组比较差异有统计学意义(P<0.001).初步建立了临床应用的参考值范围,可以有效区分出唐氏样本和正常样本.结论应用实时荧光定量PCR技术可快速、准确诊断唐氏综合征,为唐氏综合征的快速产前诊断开辟了新的途径.%Objective To develop a rapid and reliable technique for the detection of Down's syndrome. Methods The peripheral blood samples were collected from twenty-five Down's syndrome patients and fifty normal individuals. Four polymorphic loci on chromosomes 21, 1, 19 were anplified by real-time fluorescence quantitative PCR, and then four pairs of △Ct values were analytically compared between the two groups. Results The △Ct values of Down's syndrome patients were significantly lower than those of normal individuals, and the reference ranges for clinical applica tion were primarily established. The difference between the two groups was highly significant ( P < 0.001 ), and the ref erence ranges between the two groups were not overlapped. Real-time quantitative PCR technique can effectively differentiates Down's syndrome samples from the normal fetuses; furthermore, the results were consistent with those of the karyotype analysis. Conclusion Real-time quantitative PCR is a fast and reliable method that may provide a new approach for rapid detection of Down's syndrome.

  3. Development and Validation of LNA-Based Quantitative Real-Time PCR Assays for Detection and Identification of the Root-Knot Nematode Meloidogyne enterolobii in Complex DNA Backgrounds.

    Science.gov (United States)

    Kiewnick, Sebastian; Frey, Jürg E; Braun-Kiewnick, Andrea

    2015-09-01

    Meloidogyne enterolobii is a quarantine root-knot nematode posing a major threat to agricultural production systems worldwide. It attacks many host plants, including important agricultural crops, ornamentals, and trees. M. enterolobii is a highly virulent and pathogenic root-knot nematode species, able to reproduce on plants resistant to other Meloidogyne spp. Significant crop damage has been reported in Asia, South America, Africa, the United States, France, and greenhouses in Switzerland. To identify potential introduction pathways and ensure appropriate phytosanitary measures and management strategies, accurate detection and identification tools are needed. Therefore, two real-time quantitative polymerase chain reaction (PCR) assays based on the second intergenic spacer region of the ribosomal DNA cistron and the cytochrome oxidase c subunit I (COI) gene using locked nucleic acid probes were developed and validated for fast and reliable detection and identification of M. enterolobii. Analytical specificity was confirmed with 16 M. enterolobii populations, 16 populations of eight closely related Meloidogyne spp., and four species from other nematode genera. Optimizing and testing the assays on two real-time PCR platforms revealed an analytical sensitivity of one juvenile in a background of 1,000 nematodes and the intended limit of detection of one juvenile per 100 ml of soil. Both assays performed equally well, with the COI-based assay showing a slightly better performance concerning detection of M. enterolobii target DNA in complex DNA backgrounds. PMID:25775103

  4. Establishment and Application of Real-time Fluorogenetic Quantitative RT-PCR for Detection of Classical Swine Fever Virus%猪瘟病毒实时荧光定量RT-PCR

    Institute of Scientific and Technical Information of China (English)

    李军; 杨威; 潘艳; 禤雄标; 胡帅; 马春霞; 谢宇舟; 陈泽样; 许力干; 谢永平

    2011-01-01

    根据GenBank公布的猪瘟病毒基因组5'非编码区基因序列进行同源性比较分析,选择保守序列区作为扩增区域,设计1对特异性扩增引物,通过优化反应条件,建立了一个用于猪瘟病毒快速定量检测的SY13R GreenⅠ荧光定量RT-PC R方法.试验结果表明该方法重复性好,反应批内循环阈值差异不显著.与猪繁殖与呼吸综合征病毒、伪狂犬病病毒和猪圆环病毒2型等猪源病毒无交叉反应,具有高度的特异性,而且灵敏度高,最小检出量为2×10(2)病毒基因组拷贝数.利用此方法对15例临床样本进行检测,其结果与兔体交又免疫试验一致,表明此方法可作为猪瘟实验室快速诊断和疫情监测的一种快速、准确、简便的检测工具.%To establish a real-time fluorogenetic quantitative RT-PCR assay for detection of classical swine fever virus (CSFV), the 5' non-translated region sequences of CSFV in GenBank were aligned and a pair of specific primers was designed from the conserved sequence within 5' non-translated region.The reactive conditions were optimized to improve the sensitivity and specificity of the assay.The results of reproducibility of this assay were reliable and the intra assay variations were not significant.The specificity test proved that this assay had a high specificity which could not detected PRRSV,PRV and PCV2.The assay also proven to be specific,and the detection limit was up to 2 × 102 copies.The accuracy of real-time fluorogenetic quantitative RT-PCR was evaluated by testing 15 clinical samples.The results of real-time fluorogenetic quantitative RT-PCR were consistent with results of rabbit-cross reaction test,which suggested that real-time quantitative RT-PCR assay may be used as a powerful tool for repaid detection of CSFV.

  5. 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

    . 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......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...

  6. 实时荧光定量PCR对乙肝DNA检测的影响因素%Real-time Fluorescent Quantitative PCR for the Influence Factors of Hepatitis B DNA Testing

    Institute of Scientific and Technical Information of China (English)

    赖新华; 吕娜; 彭艳辉

    2014-01-01

    Objective:To explore effect of real-time fluorescent quantitative PCR detection of hepatitis B DNA,analyze the influence factors.Method:From January 2010 to January 2012 were between 300 patients with hepatitis b virus (HBV) infection,collect the blood specimens,by real-time fluorescent quantitative PCR detection method for hepatitis B DNA testing,the result of the inspection result and clinical diagnosis,this paper compares and analyzes the effect factors of test results is analyzed.Result:300 cases of hepatitis b infection in patients with blood specimens,using real-time fluorescent quantitative PCR of hepatitis B DNA test results and clinical diagnosis results of 276 cases of same (92.0%),misdiagnosis and misdiagnosed 24 cases,accounted for 8.0%,the main influence factors including laboratories,specimen and nucleic acid extraction method,etc.Conclusion:The real-time fluorescent quantitative PCR to detect HBV DNA,has higher diagnosis rate,but the influence on test results of many factors,the need to strengthen the attention,to decrease the rate of missed diagnosis and misdiagnosis,provide effective support for treatment.%目的:探讨实时荧光定量PCR对乙肝DNA检测的效果,对影响因素进行分析总结。方法:选择2010年1月-2012年1月笔者所在医院收治的300例乙肝病毒感染患者,收集其血标本,通过实时荧光定量PCR检测方法进行乙肝DNA检验,对检验结果与临床诊断结果进行对比分析,对影响检验结果的因素进行分析总结。结果:本组300例乙肝感染患者血标本中,采用实时荧光定量PCR对乙肝DNA检测结果与临床诊断结果相同276例(92.0%),漏诊误诊24例(8.0%),主要的影响因素包括实验室因素、标本因素和核酸提取方法因素等。结论:采用实时荧光定量PCR对乙肝DNA进行检测,具有较高的确诊率,但对检测结果产生影响的因素较多,需要加强注意,才能降低漏诊和误诊率,为治疗提供有效支持。

  7. 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.

  8. Standardization of real-time quantitative polymerase chain reaction for detection of the JAK2V617F mutation in BCR-ABL1 negative myeloproliferative neoplasms: A tertiary care centre experience

    Directory of Open Access Journals (Sweden)

    Tathagat Chatterjee

    2015-01-01

    Full Text Available Background: Identification of JAK2V617F mutations has led to a significant development in our understanding of the pathogenesis and therapy of BCR-ABL1 negative myeloproliferative neoplasms (MPNs. However, not all cases of BCR-ABL1 negative MPNs carry JAK2V617F mutations. The present study was undertaken with an aim to standardize the real-time quantitative polymerase chain reaction (PCR for the detection of JAK2V617F mutations and to find the prevalence of Janus kinase 2 (JAK2 mutations in MPNs in the Indian scenario. Materials and Methods: Real-time quantitative PCR was used to detect the JAK2V617F mutation. Standardization of the detection procedure was carried out using recommended guidelines to ensure the accuracy and reproducibility of results. Forty-nine patients of BCR-ABL1 negative MPNs were included in the study. Results: The JAK2V617F mutation was detected in 63.3% patients of BCR-ABL1 negative MPNs. On classification of these BCR-ABL1 negative MPNs, JAK2V617F mutation was detected in 78.3% patients with polycythemia vera (PV, 62.5% patients with essential thrombocythemia (ET, and 44.4% patients with Primary myelofibrosis (PMF. Conclusion: Role of detection of JAK2 mutations in BCR-ABL1 negative MPN has begun to be described and can be used in the diagnosis of PV, ET, and PMF along with other criterias. In future, it may be suitable for treatment monitoring and prognostication.

  9. 牛副结核分枝杆菌实时荧光定量PCR检测方法的建立%Development of Real-time Fluorescence Quantitative PCR Assay for Detection of Bovine Mycobacterium paratuberculosis

    Institute of Scientific and Technical Information of China (English)

    王素华; 王忠才; 李孝军; 杜爱芳

    2012-01-01

    根据GenBank上公布的牛副结核分枝杆菌C-2染色体的ISMav2基因保守区域序列设计合成1对特异性引物,建立了一套SYBR Green Ⅰ荧光定量PCR检测牛副结核分枝杆菌(Mycobacterium paratuberculosis)的方法.以实验室构建的牛副结核分枝杆菌pMD-ISMav2阳性重组质粒为标准品,通过优化反应条件,建立了标准曲线,其相关系数为0.999.以构建的标准品为模板,进行了特异性和敏感性试验.结果显示,该方法检测布氏杆菌、大肠杆菌、沙门氏菌、链球菌DNA均为阴性;最低可检测到相当于每微升1.96×101拷贝数的标准品阳性质粒.本研究建立的实时荧光定量PCR具有特异、敏感和快速等优点,可用于牛副结核杆菌病的监测.%According to the published sequences of bovine Mycobacterium paratuberculosis in GenBank, a pair of primers were designed and synthesized for the C-2 chromosome(ISMav2)gene of Mycobacterium paratuberculosis, and a SYBR-Green I fluorescent Real-time quantitative PCR assay was developed. The positive standard plasmid pMD-ISMav2 were used as quantitative template to make the standard curves by optimizing the reaction conditions, the correlation coefficient (R2) was 0. 999. By using the positive standard as template, specificity and sensitivity were tested. According to the experiment, all negative controls such as Brucella, Escherichia coli, Salmonella and Streptococcus showed negative detection. By sensitivity analysis, the Real-time PCR indicated that a minimum of 1. 96×101 copies of plasmid DNA was detected. As a result of the specificity and sensitivity of the assay with a relatively rapid and simple procedure, the Real-time PCR can be used as a routine assay for the diagnosis of Mycobacterium paratuberculosis.

  10. Quantification of Human Cytomegalovirus DNA by Real-Time PCR

    OpenAIRE

    Gault, Elyanne; Michel, Yanne; Dehée, Axelle; Belabani, Chahrazed; Nicolas, Jean-Claude; Garbarg-Chenon, Antoine

    2001-01-01

    A quantitative real-time PCR assay was developed to measure human cytomegalovirus (HCMV) DNA load in peripheral blood leukocytes (PBLs). The HCMV DNA load in PBLs was normalized by means of the quantification of a cellular gene (albumin). The results of the real-time PCR assay correlated with those of the HCMV pp65-antigenemia assay (P < 0.0001).

  11. HEVC real-time decoding

    Science.gov (United States)

    Bross, Benjamin; Alvarez-Mesa, Mauricio; George, Valeri; Chi, Chi Ching; Mayer, Tobias; Juurlink, Ben; Schierl, Thomas

    2013-09-01

    The new High Efficiency Video Coding Standard (HEVC) was finalized in January 2013. Compared to its predecessor H.264 / MPEG4-AVC, this new international standard is able to reduce the bitrate by 50% for the same subjective video quality. This paper investigates decoder optimizations that are needed to achieve HEVC real-time software decoding on a mobile processor. It is shown that HEVC real-time decoding up to high definition video is feasible using instruction extensions of the processor while decoding 4K ultra high definition video in real-time requires additional parallel processing. For parallel processing, a picture-level parallel approach has been chosen because it is generic and does not require bitstreams with special indication.

  12. Rapid diagnosis of Down's and Edward's syndrome by multiplex real-time quantitative PCR%多重实时定量PCR快速诊断唐氏及爱德华综合征

    Institute of Scientific and Technical Information of China (English)

    眭建忠; 张慧敏; 孙筱放

    2010-01-01

    Objective To establish a multiplex real-time quantitative PCR method for diagnosis of Down's and Edward's syndrome. Methods The sequences of the amyloid precursor protein gene (APP)in the Down's region of chromosome 21 and the thymidylate synthetase gene (TYMS) on chromosome 18 were co-amplified in the same tube. The relative quantitative index △CT value was used to differentiate Down's and Edward's syndrome patient from healthy individual. Four groups of samples, including 36 blood samples from normal controls (group A), 15 amniotic fluid samples from normal pregnancies (group B), 21 samples from patients with Down's syndrome (group C) and 6 samples from patients with Edward's syndrome (group D), were investigated in the study. Results The mean△CT values of the four groups were -0.48±0.15,-0.49±0.12,- 1.26±0.17 and 0.25±0.12 respectively. The △CT value from group B was not different from that from group A (P>0.05). However, the△CT values from group C and group D were significantly different from that from group A (P<0.01), and no overlapping was observed.Conclusion The △CT values from multiplex real-time quantitative PCR could be used to rapidly diagnose Down' s and Edward's syndrome simultaneously.%目的 建立多重实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RTqPCR)技术,快速分子诊断唐氏及爱德华综合征.方法 应用PCR方法同时扩增位于21号染色体上的淀粉样前蛋白基因(amyloid precursor protein gene,APP)和18号染色体上的胸苷酸合成酶基因(thymidylate synthetase gene,TYMS),以相对定量指标△CT值区分唐氏、爱德华综合征及正常人.用此方法检测4组样本:36名正常人外周血(A组)、15名正常核型的羊水细胞(B组)、21例唐氏综合征(C组)和6例爱德华综合征(D组).结果 A~D 4组样本△CT均值分别为-0.48±0.15、-0.49±0.12、-1.26±0.17和0.25±0.12.B组与A组间差异无统计学意义(P>0.05);C组与A组、D组

  13. 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

  14. ISTTOK real-time architecture

    International Nuclear Information System (INIS)

    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

  15. 促凝剂对乙型肝炎病毒实时荧光定量检测影响的评估%Assessment of coagulant on hepatitis B virus by real-time fluorescent quantitative detection*

    Institute of Scientific and Technical Information of China (English)

    姜立茹; 马莹

    2013-01-01

    目的评估促凝剂对乙型肝炎病毒实时荧光定量检测的影响。方法随机采集2011年10月至2012年3月到西安市中心医院门诊的41例患者每人2管血样,分别为无添加剂普通管和促凝剂管,检测分离血清时间及效果。采用实时荧光定量聚合酶链反应检测乙型肝炎病毒DNA ,结果对数值采用SPSS11.5统计软件进行处理,并对促凝剂管标本进行精密度和抗干扰性能评价。结果促凝剂管分离血清平均时间为(4.73±0.86)min ,普通管为(14.46±2.27)min ,两组数据经配对 t检验,差异有统计学意义(P<0.01);促凝剂管乙型肝炎病毒DNA结果对数平均值为(3.86±2.99),普通管组对数平均值为(3.85±2.96),两组经配对 t检验,差异无统计学意义(P>0.05),促凝剂管具有高精密度和抗干扰性能。结论促凝剂对乙型肝炎病毒实时荧光定量检测结果准确性没有影响,可减少报告周转时间,值得临床推广和应用。%Objective To evaluate effect of the coagulant on hepatitis B virus by real-time fluorescent quantita-tive detection .Methods From October 2011 to March 2012 were randomly collected in Xi'an city central hospital of 41 patients with each of 2 tubes of blood samples .which were without additives common tube and the coagulant tube . To detected the serum separation time and effect ,using real-time fluorescence quantitative PCR for detection of hepa-titis B virus DNA ,logarithmic mean was performed by SPSS11 .5 statistical analysis ,and the coagulant tube speci-mens for precision and anti-jamming performance evaluation .Results Coagulant group separation of serum average time was (4 .73 ± 0 .86)min ,common tube group was (14 .46 ± 2 .27)min ,data from the two groups were paired t test ,the difference was statistically significant (P0 .05) .coagulant tube had the advantages of high precision and anti-jamming performance .Conclusion

  16. 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

  17. A Real-time Data Model Based on Temporal Data

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiao-fang; LIU Yun-sheng

    2006-01-01

    Real-time database systems contain not only transaction timing constraints, but also data timing constraints. This paper discusses the temporal characteristics of data in real-time databases and offers a definition of absolute and relative temporal consistency. In real-time database systems, it is often the case that the policies of transaction schedules only consider the deadline of real-time transactions, making it insufficient to ensure temporal correctness of transactions. A policy is given by considering both the deadlines of transactions and the "data deadline" to schedule real-time transactions. A real-time relational data model and a real-time relational algebra based on the characteristics of temporal data are also proposed. In this model, the temporal data has not only corresponding values, but also validity intervals corresponding to the data values. At the same time, this model is able to keep historical data values. When validity interval of a relation is[NOW,NOW], real-time relational algebra will transform to traditional relational algebra.

  18. Real time psychrometric data collection

    International Nuclear Information System (INIS)

    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

  19. Quantitative detection of Egr-1 and Egr-4 gene expression in mice thymus after whole-body irradiation with X-rays by real-time RT-PCR

    International Nuclear Information System (INIS)

    Objective: To investigate the changes of Egr-1 and Egr-4 gene expression in mice thymus after whole-body irradiation (WBI) with X-rays by real-time quantitative reverse transcription-polymerase chain reaction (QRT-PCR). Methods: The mRNA was isolated from mice thymus 4 and 24 h after WBI with 0-6 Gy of X-ray irradiation. The changes of Egr-1 and Egr-4 gene expression 4 and 24 h post irradiation were examined with QRT-PCR. Paralleled counts of micronucleus rate in bone marrow polychromatic erythrocytes (PCE) was as a reference radiation biodosimetric control. Results: The relative expression of Egr-1 and Egr-4 genes at 4 and 24 h after WBI 0.5 to 6 Gy was changed with the dose (r=0.974, 0.987, 0.999, P<0.01). At all dose points the relative expression of Egr-1 and Egr-4 genes was highly correlated with the micronucleus rate of bone marrow PCE (r=0.866, 0.947, 0.983, 0.835, P<0.05). The dose-effect relationship could be fitted into linear-quadratic model. The expression of Egr-4 gene was significantly increased at 4 h post irradiation and best correlated with PCE micronucleus rate. Conclusions: QRT-PCR assay of early expression Egr-4 gene might be a candidate for fast, high-throughput radiation biodosimetry. (authors)

  20. Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Laticifers on the Basis of Latex Flow in Rubber Tree (Hevea brasiliensis Muell. Arg.)

    Science.gov (United States)

    Chao, Jinquan; Yang, Shuguang; Chen, Yueyi; Tian, Wei-Min

    2016-01-01

    Latex exploitation-caused latex flow is effective in enhancing latex regeneration in laticifer cells of rubber tree. It should be suitable for screening appropriate reference gene for analysis of the expression of latex regeneration-related genes by quantitative real-time PCR (qRT-PCR). In the present study, the expression stability of 23 candidate reference genes was evaluated on the basis of latex flow by using geNorm and NormFinder algorithms. Ubiquitin-protein ligase 2a (UBC2a) and ubiquitin-protein ligase 2b (UBC2b) were the two most stable genes among the selected candidate references in rubber tree clones with differential duration of latex flow. The two genes were also high-ranked in previous reference gene screening across different tissues and experimental conditions. By contrast, the transcripts of latex regeneration-related genes fluctuated significantly during latex flow. The results suggest that screening reference gene during latex flow should be an efficient and effective clue for selection of reference genes in qRT-PCR. PMID:27524995

  1. Quantification of mRNAs and housekeeping gene selection for quantitative real-time RT-PCR normalization in European beech (Fagus sylvatica L.) during abiotic and biotic stress.

    Science.gov (United States)

    Olbrich, Maren; Gerstner, Elke; Welzl, Gerhard; Fleischmann, Frank; Osswald, Wolfgang; Bahnweg, Günther; Ernst, Dieter

    2008-01-01

    Analyses of different plant stressors are often based on gene expression studies. Quantitative real-time RT-PCR (qRT-PCR) is the most sensitive method for the detection of low abundance transcripts. However, a critical point to note is the selection of housekeeping genes as an internal control. Many so-called 'housekeeping genes' are often affected by different stress factors and may not be suitable for use as an internal reference. We tested six housekeeping genes of European beech by qRT-PCR using the Sybr Green PCR kit. Specific primers were designed for 18S rRNA, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH1, GAPDH2), a-tubulin, and ubiquitin-like protein. Beech saplings were treated with increased concentrations of either ozone or CO2. In parallel, the expression of these genes was analyzed upon pathogen infection with Phytophthora citricola. To test the applicability of these genes as internal controls under realistic outdoor conditions, sun and shade leaves of 60-year-old trees were used for comparison. The regulation of all genes was tested using a linear mixed-effect model of the R-system. Results from independent experiments showed that the only gene not affected by any treatment was actin. The expression of the other housekeeping genes varied more or less with the degree of stress applied. These results highlight the importance of undergoing an individual selection of internal control genes for different experimental conditions. PMID:18811005

  2. Real Time Network Traffic Monitoring

    OpenAIRE

    Tripp, Gerald

    1999-01-01

    This paper looks at the problems of real time network traffic monitoring. Some of the existing approaches are reviewed, looking at both simple filtering systems and also systems based on the use of finite state machines that can report specific events or capture data only when in particular states. Finally, some existing implementation techniques are examined and an outline proposal made for the design of a network monitoring system that uses finite state machines implemented using associativ...

  3. 法医学组织病理切片DNA的实时荧光定量分析%Quantitative analysis of DNA by real-time PCR for forensic pathological section

    Institute of Scientific and Technical Information of China (English)

    张越; 陈阳; 杨元立; 李继周; 李朝品

    2012-01-01

    Objective:To investigate the effects of preserving period and tissue types of pathological sections on the quality of DNA genome for forensic science using real-time quantitative PCR detection with TaqMan probe-based gene analysis. Methods: The fresh tissue samples were obtained from 14 cases of autopsy by 7 death ages for preparing pathological sections of the brain, heart, liver, lung, spleen, kidney and ileum tissue, and randomly maintained these sections for 7,14,30,90,180,360 and 720 days, respectively, in accordance with Latin square by double 7-order design. Silicon bead method was used to extract the DNA template that was further quantitatively detected using the real-time fluorescent quanti-tative PCR. Results:The UNA quantitative results varied a lot in the same kind of tissue sections preserved in dissimilar phase (P0.6). Conclusion: Forensic pathological tissue sections commonly exhibit lower content of DNA, i. e. , low copy number DNA, suggesting that the DNA nature is associated with the intervals of sections kept and tissue types.%目的:运用TaqMan探针实时荧光定量扩增法,对法医学病理切片进行DNA定量分析,探讨切片组织类型、保存时间对DNA基因组的质量影响.方法:取14例尸检7个年龄段的新鲜组织样本,制作脑、心、肝、肾、脾、肺、肠等7类组织病理切片.以2个7阶拉丁方实验设计,将切片随机分组保存7d、14 d、30d、90d、180 d、360 d、720 d.用硅珠法提取切片DNA,实时荧光PCR进行DNA定量.结果:同一类型组织的不同保存时间的切片DNA含量存在差异(P<0.01),保存1月以内的切片DNA含量下降不显著,半年以上的样本DNA含量下降明显;同一保存时间的不同组织类型之间的DNA含量存在差异(P<0.01),肺、肝、肾三种组织切片DNA含量相对较高;身源年龄组之间差异不显著(P>0.6).结论:法医学病理组织切片DNA含量较低,为低拷贝模板,组织切片保存时间、组织类型是影响DNA质量的重要因素.

  4. Acute Aortic Dissection Biomarkers Identified Using Isobaric Tags for Relative and Absolute Quantitation.

    Science.gov (United States)

    Xiao, Ziya; Xue, Yuan; Yao, Chenling; Gu, Guorong; Zhang, Yaping; Zhang, Jin; Fan, Fan; Luan, Xiao; Deng, Zhi; Tao, Zhengang; Song, Zhen-Ju; Tong, Chaoyang; Wang, Haojun

    2016-01-01

    The purpose of this study was to evaluate the utility of potential serum biomarkers for acute aortic dissection (AAD) that were identified by isobaric Tags for Relative and Absolute Quantitation (iTRAQ) approaches. Serum samples from 20 AAD patients and 20 healthy volunteers were analyzed using iTRAQ technology. Protein validation was performed using samples from 120 patients with chest pain. A total of 355 proteins were identified with the iTRAQ approach; 164 proteins reached the strict quantitative standard, and 125 proteins were increased or decreased more than 1.2-fold (64 and 61 proteins were up- and downregulated, resp.). Lumican, C-reactive protein (CRP), thrombospondin-1 (TSP-1), and D-dimer were selected as candidate biomarkers for the validation tests. Receiver operating characteristic (ROC) curves show that Lumican and D-dimer have diagnostic value (area under the curves [AUCs] 0.895 and 0.891, P values for D-dimer were 93.33% and 68.33%. For Lumican and D-dimer AAD combined diagnosis, the sensitivity and specificity were 88.33% and 95%, respectively. In conclusion, Lumican has good specificity and D-dimer has good sensitivity for the diagnosis of AAD, while the combined detection of D-dimer and Lumican has better diagnostic value. PMID:27403433

  5. Real-Time Quantitative RT-PCR of Defense-Associated Gene Transcripts of Rhizoctonia solani-Infected Bean Seedlings in Response to Inoculation with a Nonpathogenic Binucleate Rhizoctonia Isolate.

    Science.gov (United States)

    Wen, Kui; Seguin, Philippe; St-Arnaud, Marc; Jabaji-Hare, Suha

    2005-04-01

    ABSTRACT Certain isolates of nonpathogenic binucleate Rhizoctonia spp. (np-BNR) are effective biocontrol agents against seedling root rot and damping-off. Inoculation of bean seed with np-BNR strain 232-CG at sowing reduced disease symptoms in bean (Phaseolus vulgaris) seedlings caused by R. solani. Molecular analyses of the spatial expression of three defense-associated genes were carried out using real-time quantitative reverse transcription-polymerase chain reaction (QRT-PCR) assays. This method allowed accurate quantitative evaluation of transcript levels of pG101 encoding for 1,3-beta-D-glucanase, gPAL1 encoding for phenylalanine ammonia lyase, and CHS17 encoding for chalcone synthase in 1- and 2-week-old bean seedlings that were inoculated simultaneously with np-BNR and infected with R. solani, and in seedlings that were singly inoculated with either fungi or not inoculated. In the seedlings that were infected with R. solani only, results revealed that, following infection, activation of all defense-associated gene transcripts was achieved with significant increases ranging from 7- to 40-fold greater than the control, depending on the defense gene and tissue analyzed. Seedlings that were treated with np-BNR and infected with R. solani had expression similar to those that were treated with np-BNR only, but the levels were significantly down-regulated compared with those that were infected with R. solani only. These findings indicate that disease suppression by np-BNR isolate is not correlated to pG101, gPAL1, and CHS17 gene activation. PMID:18943035

  6. 罗非鱼组织内无乳链球菌实时荧光定量 PCR 检测方法建立%Real-time quantitative PCR for detection of Streptococcus agalactiae from tilaPia tissue

    Institute of Scientific and Technical Information of China (English)

    王瑞; 李莉萍; 黄婷; 梁万文; 梁聪; 雷爱莹; 陈明

    2015-01-01

    . The results show that the real-time quantitative PCR assay which we developed was accurate,specific and sen-sitive,and could detect S. agalactiae from various tilapia tissues rapidly. The method can be used for surveillance and prevention of S. agalactiae disease of tilapia.

  7. Real-time PCR as a tool to study weed biology

    Science.gov (United States)

    Real-time polymerase chain reaction (Real-time PCR) also known as quantitative PCR is used to determine relative gene expression or to quantify exact levels of mRNA in cells or tissues. Before the advent of Real-time PCR, the major difficulty associated with traditional quantitative or semi-quantita...

  8. Real time automatic scene classification

    OpenAIRE

    Israël, Menno; Broek, van den, Wouter; Putten, van, M.J.A.M.; Uyl, den, T.M.; Verbrugge, R.; Taatgen, N.; Schomaker, L.

    2004-01-01

    This work has been done as part of the EU VICAR (IST) project and the EU SCOFI project (IAP). The aim of the first project was to develop a real time video indexing classification annotation and retrieval system. For our systems, we have adapted the approach of Picard and Minka [3], who categorized elements of a scene automatically with so-called ’stuff’ categories (e.g., grass, sky, sand, stone). Campbell et al. [1] use similar concepts to describe certain parts of an image, which they named...

  9. Development of Real-Time Fluorescent Quantitative PCR in the Field of Genetically Modified Food Detection%实时荧光定量PCR技术在转基因食品检测领域中的应用

    Institute of Scientific and Technical Information of China (English)

    吴永彬; 肖维威; 马文丽

    2011-01-01

    With wide application of genetic engineering technology in agricultural production, more and more improved characteristics of transgenic plants have been widely planted on a global scale, followed by genetically modified(GM) foods have to be developed rapidly. Because of security issues caused by large-scale commercialization of GM products, and ensuring GMO labeling system implement smoothly, a rapid, accurate, high-throughput quantitative assay is necessary. In this paper we reviewed the development of GM foods detection at home and abroad research progress, and focused on the application of real-time fluorescent quantitative PCR in the field of GM foods detection, and in its application prospects, constructing standard reference molecules were proposed to achieve more transgenic plant lines for quantitative detection.%随着基因工程技术在农业生产中应用的深入,越来越多具有改良特征的转基因植物在全球范围内得到广泛种植,随之而来的转基因食品也迅猛发展,转基因产品大规模商业化引起了对安全性问题的担忧.为保证转基因产品标签制度的顺利实施,建立快速、准确、高通量的定量检测方法十分必要.我们综述了国内外转基因食品检测技术的研究进展,重点阐述了实时荧光定量PCR技术在转基因食品检测领域中的应用,并展望了通过构建质粒标准分子的方法来实现对更多转基因植物品系的定量检测.

  10. Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae

    OpenAIRE

    Jensen Jørgen; Østergaard Lars; Birkebæk Niels H; Birkelund Svend; Mygind Tina; Christiansen Gunna

    2002-01-01

    Abstract Background Chlamydia pneumoniae infection has been detected by serological methods, but PCR is gaining more interest. A number of different PCR assays have been developed and some are used in combination with serology for diagnosis. Real-time PCR could be an attractive new PCR method; therefore it must be evaluated and compared to conventional PCR methods. Results We compared the performance of a newly developed real-time PCR with a conventional PCR method for detection of C. pneumon...

  11. Real-time detection of airborne chemicals

    Science.gov (United States)

    Hartenstein, Steven D.; Tremblay, Paul L. A.; Fryer, Michael O.; Kaser, Timothy

    1999-02-01

    Accurate, real time air quality measurements are difficult to make, because real time sensors for some gas species are not specific to a single gas. For example, some carbon dioxide sensors react to hydrogen sulfide. By combining the response of several types of real time gas sensors the Real-time Air Quality Monitoring System (RAQMS) accurately measures many different gases. The sensor suite for the INEEL's Real-time Air Quality Monitoring System (RAQMS) incudes seven, inexpensive, commercially-available chemical sensors for gases associated with air quality. These chemical sensors are marketed as devices to measure carbon dioxide, hydrogen sulfide, carbon monoxide, sulfur dioxide, nitrogen dioxide, water vapor and volatile organic compounds (VOC's). However, these chemical sensors respond to more than a single compound, e.g. both the VOC and the carbon dioxide sensors respond strongly to methane. This multiple sensor response to a given chemical is used to advantage in the RAQMS system, as patterns of responses by the sensors were found to be unique and distinguishable for several chemicals. Therefore, there is the potential that the seven sensors combined output can: (1) provide more accurate measurements of the advertized gases and (2) estimate the presence and quantity of additional gases. The patterns of sensor response can be thought of as clusters of data points in a seven dimensional space. One dimension for each sensor's output. For all of the gases tested, these clusters were separated enough that good quantitative results were obtained. As an example, the prototype RAQMS is able to distinguish methane from butane and predict accurate concentrations of both gases. A mathematical technique for estimating probability density functions from random samples is used to distinguish the data clusters from each other and to make gas concentration estimates. Bayes optimal estimates of gas concentration are calculated using the probability density function. The

  12. VEGF-A and its isoform VEGF121 mRNA expression measured by quantitative real-time RT-PCR. Correlation with F-18 FDG uptake and aggressiveness of lung adenocarcinoma. Preliminary study

    International Nuclear Information System (INIS)

    The objective of this study was to investigate the correlation of vascular endothelial growth factor (VEGF) A and its isoform VEGF121 mRNA expression with F-18 fluorodeoxyglucose (FDG) uptake and aggressiveness in lung adenocarcinoma. Twenty-three patients with lung adenocarcinoma underwent FDG positron emission tomography (PET) before surgery. As semi-quantitative analysis for FDG uptake, partial volume corrected standardized uptake value (PVC-SUV) of the tumor was calculated. Total RNA from lung adenocarcinoma tissue was prepared from the frozen specimens. Using the real-time reverse transcription polymerase chain reaction method, we analyzed the mRNA level of VEGF-A and VEGF-A isoform VEGF121 mRNA level. 18S ribosomal RNA was used as an endogenous control. VEGF-A and VEGF121 mRNA levels had significantly positive correlation with PVC-SUV in lung adenocarcinoma (r=0.477, p=0.021, r=0.539, p=0.008, respectively), while they were not correlated with tumor size (≤3 or >3 cm). VEGF-A and VEGF121 mRNA levels of the low FDG uptake group were significantly lower than those of the high FDG uptake group (p=0.005 and p=0.004, respectively). FDG uptake (PCV-SUV) of aggressive lung adenocarcinoma was higher than that of non-aggressive lung adenocarcinoma (p=0.01). VEGF-A and VEGF121 mRNA levels of aggressive lung adenocarcinoma were higher than those of non-aggressive lung adenocarcinoma (p=0.0001 and p=0.0001, respectively). VEGF-A and VEGF121 mRNA levels may correlate with FDG uptake and aggressiveness in lung adenocarcinoma. These findings support the hypothesis that VEGF-A and VEGF121 may help in predicting the outcome in patients with lung adenocarcinoma. (author)

  13. A Rapid and Sensitive One Step-SYBR Green Based Semi Quantitative Real Time RT-PCR for the Detection of peste des petits ruminants Virus in the Clinical Samples

    Institute of Scientific and Technical Information of China (English)

    Vinayagamurthy Balamurugan; Arnab Sen; Gnanavel Venkatesan; Vinita Yadav; Vandna Bhanot; Veerakyathappa Bhanuprakash; Raj Kumar Singh

    2012-01-01

    A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit(R) for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR.The assay amplifies a 124 bp fragment of the PPRV M gene with Tm of 78.28 to 78.50.The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg.Based on the serial dilution of the live-attenuated PPR vaccine virus,the detection limit was ~0.0001 cell culture infectious dose 50% units (TCID50).Additionally,swab materials spiked with known titre of vaccine virus were equally well detected in the assay.The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples.The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples.The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats.Therefore,the established,simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.

  14. Detection of Live Salmonella sp. Cells in Produce by a TaqMan-Based Quantitative Reverse Transcriptase Real-Time PCR Targeting invA mRNA▿ †

    Science.gov (United States)

    González-Escalona, Narjol; Hammack, Thomas S.; Russell, Mindi; Jacobson, Andrew P.; De Jesús, Antonio J.; Brown, Eric W.; Lampel, Keith A.

    2009-01-01

    Salmonella enterica contamination in foods is a significant concern for public health. When DNA detection methods are used for analysis of foods, one of the major concerns is false-positive results from the detection of dead cells. To circumvent this crucial issue, a TaqMan quantitative real-time RT-PCR (qRT-PCR) assay with an RNA internal control was developed. invA RNA standards were used to determine the detection limit of this assay as well as to determine invA mRNA levels in mid-exponential-, late-exponential-, and stationary-phase cells. This assay has a detection limit of 40 copies of invA mRNA per reaction. The levels of invA mRNA in mid-exponential-, late-exponential-, and stationary-phase S. enterica cells was approximately 1 copy per 3 CFU, 1 copy per CFU, and 4 copies per 103 CFU, respectively. Spinach, tomatoes, jalapeno peppers, and serrano peppers were artificially contaminated with four different Salmonella serovars at levels of 105 and less than 10 CFU. These foods were analyzed with qRT-PCR and with the FDA's Bacteriological Analytical Manual Salmonella culture method (W. A. Andrews and T. S. Hammack, in G. J. Jackson et al., ed., Bacteriological analytical manual online, http://www.cfsan.fda.gov/∼ebam/bam-5.html, 2007). Comparable results were obtained by both methods. Only live Salmonella cells could be detected by this qRT-PCR assay, thus avoiding the dangers of false-positive results from nonviable cells. False negatives (inhibition of the PCR) were also ruled out through the use of an RNA internal control. This assay allows for the fast and accurate detection of viable Salmonella spp. in spinach, tomatoes, and in both jalapeno and serrano peppers. PMID:19376910

  15. Development and Validation of TaqMan Real-Time Polymerase Chain Reaction Assays for the Quantitative and Differential Detection of Wild-Type Infectious Laryngotracheitis Viruses from a Glycoprotein G-Deficient Candidate Vaccine Strain.

    Science.gov (United States)

    Shil, Niraj K; Legione, Alistair R; Markham, Philip F; Noormohammadi, Amir H; Devlin, Joanne M

    2015-03-01

    Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens with a worldwide distribution. Differentiating between wild-type and vaccine strains of ILT virus (ILTV) would be useful for enhancing disease control, and in the early stages of a disease outbreak molecular diagnostic tools for the detection and differentiation of the circulating virus could be applied. This study developed TaqMan real-time PCR (qPCR) assays to detect and differentiate the glycoprotein G (gG)-deficient (ΔgG) ILTV candidate vaccine strain of ILTV from ILTV strains that contain the gG gene. The gG+ve and gG-ve ILTV TaqMan assays were used in individual and multiplex format to detect, differentiate, and quantitate ILTV DNA in laboratory and clinical samples. The assays were highly sensitive and highly specific, with a detection limit of 10 viral template copies for each assay. Low interassay coefficients of variation were recorded (0.021-0.042 and 0.013-0.039) for gG+ve and gG-ve TaqMan assays, respectively. The multiplex assay was successfully used to examine the replication kinetics of wild-type and ΔgG strains of ILTV in cultured leghorn male hepatoma cells and embryonated hen eggs under coinfection conditions. The results showed that the TaqMan qPCR assay, along with the ΔgG ILTV vaccine, has the potential to be used in a "Differentiating Infected from Vaccinated Animals" strategy for the control and eradication of ILT. PMID:26292527

  16. Research on Real-Time Quantitative PCR Technology of Common Aspergillus Species%常见曲霉菌的实时定量PCR诊断技术的研究

    Institute of Scientific and Technical Information of China (English)

    武学成; 龚文胜

    2012-01-01

    Objective The detected method of real-time quantitative PCR technology on common Aspergillus Species was developed. Methods The employing universal, fungus-specific primers and DNA probes, directed to the conservative internal transcribed spacer 2(ITS2) region of ribosomal DNA from A, terreus, A flavus, A. Fumigatus, A. Terreus, A. Nidulans and A niger were designed. Five kinds of common Aspergillus Species were detected by the quantitative real-time PCR and common PCR technology. Results DNA from each Aspergillus species for which DNA probes were designed produced am-plicons of approximately 350bp in size. Melting temperature of A, terreus, A. Flavus, A. Fumigatus,A terreus, A. Nidulajis and A. Niger was respectively 57℃ ± 0. 12℃ , 59℃ ± 0. 13℃ , 63℃ ± 0. 17℃ , 66°C ± 0. 15℃ , 66℃ ± 0. 14℃ and 68℃± 0. 12°C by melting peak analysis for five different Aspergillus spp. The minimum reaction concentration of DNA response template of A terreus, A flavus, A fumigatus, A nidulans and A niger was respectively 82. 4, 621. 3, 51. 2, 520. 8 and 19. 5 fg/μl by sensitivity analysis of common Aspergillus species. Conclusion The developed technology of real-time quantitative PCR is simple, rapid, and sensitive for the identification of medically important common Aspergillus species, which was a new option in invasive aspergillosis diagnosis.%目的 建立常见曲霉菌的实时定量PCR诊断技术.方法 针对黄曲霉菌、烟曲霉菌、构巢曲霉菌、黑曲霉菌和土曲霉菌内转录间隔区2(internal transcribed spacer 2,ITS2)保守基因设计引物和探针,通过普通PCR扩增其ITS2基因片段,作为实时定量PCR反应模板,通过实时定量扩增和分析其解链曲线来鉴定常见曲霉菌,同时分析该方法灵敏度.结果 真菌通用引物扩增五种常见曲霉菌ITS2基因DNA片段,大小为350 bp左右.实时定量PCR分析和探针解链曲线分析结果显示土曲霉解链温度(melting temperature

  17. Quantitative real-time RT-PCR validation of differential mRNA expression of SPARC, FADD, Fascin, COL7A1, CK4, TGM3, ECM1, PPL and EVPL in esophageal squamous cell carcinoma

    International Nuclear Information System (INIS)

    Esophageal squamous cell carcinoma (ESCC) is one of the most malignant tumors and typically presents at an advanced and rapidly fatal stage. To better understand the role of genetics in the etiology and prevention of ESCC and to identify potential susceptibility genes as well as early detection markers, we previously compared tumor and matched normal tissues from ESCC patients from a high-risk area of China using cDNA expression microarrays and identified 41 differentially-expressed genes (13 over-expressed and 28 under-expressed). In the current study, we validated and quantitated differential mRNA expression in a sample of nine of these 41 genes, including four that were over-expressed (SPARC, FADD, Fascin, COL7A1), and five that were under-expressed (CK4, TGM3, ECM1, PPL, EVPL), in 75 new ESCC patients using quantitative Real-time RT-PCR and the 2-ΔΔCT method to examine both tumor and matched normal tissue. In addition, we examined expression patterns for these genes by selected demographic and clinical characteristics. Four previously over-expressed (tumor ≥2-fold normal) genes were all increased in the majority of new ESCC patients: SPARC was increased in 71% of patients, Fascin in 70%, FADD in 63%, and COL7A1 in 57%. Five previously under-expressed (tumor ≤0.5-fold normal) genes similarly showed decreased mRNA expression in two-thirds or more of patients: CK4 was decreased in 83% of patients, TGM3 in 77%, ECM1 in 73%, and PPL and EVPL in 67% each. In subset analyses, associations with age (for COL7A1), family history (for PPL and ECM1), and alcohol use (for SPARC and Fascin) were also noted. These data indicate that these nine genes have consistent differential mRNA expression, validating results of our previous cDNA array results, and affirming their potential role in the early detection of ESCC

  18. Taqman实时定量PCR检测产毒艰难梭菌方法的建立%Detection of toxigenic genes Clostridium difficile by TaqMan real-time quantitative PCR

    Institute of Scientific and Technical Information of China (English)

    吴琳; 王毅谦; 邵景东; 吴福平; 傅春玲

    2011-01-01

    OBJECTIVE To develop a rapid real-time quantitative PCR assay targeting on toxin gene tcdA and tcdB of clostridium difficile. METHODS The special sequence of tcdA and tcdB gene of C. difficile was amplified with a pair primers and Taqman probe. The standard curves of the reaction for the detection of each gene were generated from the standard toxgenic clostridium difficile strain. RESULTS The specificity of each gene was demonstrated by the absence of amplification with DNA purified from bacterial species other than toxigenic C. difficile. Both amplification reactions showed a linear relationship between Ct and DNA amounts which yielded the R values of 0. 9975 and 0. 9984 for tcdA and tcdB gene respectively. And the detecting limit was 2. 5× 10-3. CONCLUSION It is a rapid, special, sensitive, method for quantitative detection of C. difficile and will allow the detection of toxigenic C. difficile in clinical specimens.%目的 建立以毒素基因A/B为靶基因的产毒艰难梭菌的快速定量检测方法.方法 通过设计艰难梭菌毒素A/B基因的特异引物及探针,建立标准产毒菌株DNA(ng)含量与Ct值的标准曲线.结果 该方法仅对产毒艰难梭菌进行特异性扩增,11种其他常见的致病菌及非产毒艰难梭菌均不能扩增; tcdA和tcdB基因扩增标准曲线线性关系R值分别为0.9975、0.9984,检测低限均为2.5×10-3ng.结论 该研究建立的方法具有快速、灵敏、特异性高等优点,可用于艰难梭菌毒素基因的定量检测.

  19. 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

  20. REAL TIME DATA PROCESSING FRAMEWORKS

    Directory of Open Access Journals (Sweden)

    Yash Sakaria

    2015-09-01

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

  1. 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. PMID:25728411

  2. Absolute quantitative analysis for sorbic acid in processed foods using proton nuclear magnetic resonance spectroscopy

    International Nuclear Information System (INIS)

    Highlights: ► A method using qHNMR was applied and validated to determine SA in processed foods. ► This method has good accuracy, precision, selectiveness, and linearity. ► The proposed method is more rapid and simple than the conventional method. ► We found that the proposed method is reliable for the accurate determination of SA. ► This method can be used for the monitoring of SA in processed foods. - Abstract: An analytical method using solvent extraction and quantitative proton nuclear magnetic resonance (qHNMR) spectroscopy was applied and validated for the absolute quantification of sorbic acid (SA) in processed foods. The proposed method showed good linearity. The recoveries for samples spiked at the maximum usage level specified for food in Japan and at 0.13 g kg−1 (beverage: 0.013 g kg−1) were larger than 80%, whereas those for samples spiked at 0.063 g kg−1 (beverage: 0.0063 g kg−1) were between 56.9 and 83.5%. The limit of quantification was 0.063 g kg−1 for foods (and 0.0063 g kg−1 for beverages containing Lactobacillus species). Analysis of the SA content of commercial processed foods revealed quantities equal to or greater than those measured using conventional steam-distillation extraction and high-performance liquid chromatography quantification. The proposed method was rapid, simple, accurate, and precise, and provided International System of Units traceability without the need for authentic analyte standards. It could therefore be used as an alternative to the quantification of SA in processed foods using conventional method.

  3. 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

  4. Comparative expression analysis of Crlea gene in Catharanthus roseus under drought stress by real-time quantitative PCR%荧光定量PCR检测干旱胁迫下长春花Crlea基因的相对表达

    Institute of Scientific and Technical Information of China (English)

    祖元刚; 聂明珠; 房思良; 于景华; 郭晓瑞

    2006-01-01

    A full-length Crlea(Crlea for Catharanthus roseus late embryogenesis abundant) gene was first isolated from Catharanthus roseus. Gene expression profiles of Crlea gene in leaves and roots under drought stress were monitored by real-time quantitative PCR. The results showed that a similar accumulation pattern of Crlea gene in leaves and roots over the observation period of 0.5 to 8 hours. The expression of Crlea mRNA was strengthened with the prolongation of stress time. In leaves, expression amounts of Crlea gene were 9.984 and 20.431 times higher than that of control respectively at 6 and 8 h. Similarly, the expression amounts of Crlea gene in root obviously increased (2.831 times higher than that of control) at 8 h. Primary results show the expression of Crlea gene is non-tissue-specific and up-regulated under drought stress.%首次从长春花中克隆了Crlea(Crlea for Catharanthus roseus late embryogenesis abundant)的全长基因,采用荧光定量PCR方法对干旱胁迫下长春花叶片和根部Crlea基因的表达模式进行监测,结果表明,在0.5~8h的胁迫时间中,叶片和根部的Crlea基因表现出相似的积累模式.长春花Crlea基因的表达随着胁迫时间的延长而表达增强.在叶片中,在6 h和8 h的干旱处理后,Crlea基因表达显著提高,分别是未处理材料的9.984和20.431倍.在根部, 在8 h的处理后,Crlea基因的表达量显著提高(2.831倍于对照).初步结果表明Crlea基因的表达没有组织特异性,并且为干旱胁迫正调控.

  5. 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)

    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

  6. 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.

  7. A real time monitoring system

    International Nuclear Information System (INIS)

    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)

  8. Mobile real time radiography system

    International Nuclear Information System (INIS)

    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

  9. Real-Time Imaging of Quantum Entanglement

    International Nuclear Information System (INIS)

    Full text: Photonic entanglement of spatial modes is routinely studied in many experiments and offers interesting features for quantum optical and quantum information experiments. To investigate the properties of these complex modes, it is crucial to gain information about the transversal structure with high precision and in an efficient way. 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 the spatial mode of its entangled partner photon. We determine from imaged intensity pattern the number of photons within a certain region, evaluate its error margin and thereby quantitatively verify the non-classicality of the measurements. In addition, the use of the ICCD camera allows us to demonstrate visually the enhanced remote angular sensing and the high flexibility of our setup in creating any desired spatial-mode entanglement. (author)

  10. Lentiviral vectors titration using real-time PCR

    OpenAIRE

    Amir Allahverdi; Fatemeh Eskandari; Mohammad Hossein Moghadasi; Mehdi Azad; Mehdi Goudarzi; Saied Abroun; Masoud Soleimani

    2015-01-01

    Lentiviral vectors (LVs) are useful vehicle for genetransfer to dividing and non-dividing cells and genetic manipulations. However, the use of lentiviruses in studies requires an accurate titration technique.Quantitative real-time PCR (qPCR) is a sensitive technique for the indication and quantitation of retrovirals particles. In this study, we used the qPCR for lentiviral vector titeration. The puromycin resistance gene as templates for an SYBR green-based real-time qPCR method and detect le...

  11. Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes

    OpenAIRE

    Deepak, Sa; Kottapalli, KR; Rakwal, R.; Oros, G; Rangappa, KS; H. Iwahashi; Masuo, Y; Agrawal, GK

    2007-01-01

    Invention of polymerase chain reaction (PCR) technology by Kary Mullis in 1984 gave birth to real-time PCR. Real-time PCR — detection and expression analysis of gene(s) in real-time — has revolutionized the 21st century biological science due to its tremendous application in quantitative genotyping, genetic variation of inter and intra organisms, early diagnosis of disease, forensic, to name a few. We comprehensively review various aspects of real-time PCR, including technological refinement ...

  12. 弯曲菌荧光定量PCR检测方法的建立及初步应用%Development and Application of Real-time PCR Assay for Quantitative Detection of Campylobacter spp

    Institute of Scientific and Technical Information of China (English)

    朱佳琪; 张小燕; 黄金林; 焦新安

    2014-01-01

    根据弯曲菌16S rRNA基因的靶序列设计特异性引物和探针,对反应条件和试剂浓度进行优化,建立了快速检测鸡肉中弯曲菌的荧光定量PCR方法,并对其特异性、敏感性和重复性进行评价。该方法除了对弯曲菌有扩增曲线外,对其他常见食源性病原菌均未有扩增曲线,具有较好的特异性;弯曲菌的最低检测限为10 CFU/mL;重复性实验获得标准曲线相关系数为R2=0.999,批内可重复性变异系数在0.12%-2.1%之间,批间可重复性变异系数为1.7%。应用该方法对68份鸡肉样品检测弯曲菌阳性率为95.6%(65/68),传统分离方法阳性率为89.5%(60/68),两种方法的阳性符合率为89.7%。本研究建立的荧光定量PCR方法灵敏度高、特异性好、操作简单,大大缩短检测周期,可作为检测鸡肉样品中弯曲菌的手段,为鸡肉样品弯曲菌的流行病学调查研究提供新的工具。%To establish a rapid assay for quantitative detection of Campylobacter spp.,a pair of primers and fluorescent probe were designed according to 16S rRNA sequence of Campylobacter spp.,and then a real-time PCR method was successfully established to detect Campylobacter spp. Results demonstrated that the method was speciifc for ampliifcation of Campylobacter spp.,but no ampliifcation for other related bacteria. The detection limit of the assay was 10CFU/mL and the correlation coefifcient of standard curve(R2)was 0.999. The detection of 68 chicken meat samples showed that the positive rate was 95.6%,while the positive rate was 89.5% by traditional culture method. The method was simple,rapid,speciifc and sensitive for rapid detection of Campylobacter spp. and a new tool for epidemiological investigation of Campylobacter spp. in chicken meats.

  13. Quantitative analysis of phosphoric acid esters in aqueous samples by isotope dilution stir-bar sorptive extraction combined with direct analysis in real time (DART)-Orbitrap mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Bridoux, Maxime C., E-mail: maxime.bridoux@cea.fr [CEA, DAM, DIF, F-91297 Arpajon (France); Malandain, Hélène [SANTEN SAS, 1 rue Pierre Fontaine, Bâtiment Genavenir IV, F-91058 Evry (France); Leprince, Françoise; Progent, Frédéric; Machuron-Mandard, Xavier [CEA, DAM, DIF, F-91297 Arpajon (France)

    2015-04-15

    Highlights: • Theory and modeling of organophosphorus SBSE extraction are presented. • Organophosphorus are quantitatively analyzed by isotopic dilution SBSE/DART/Orbitrap-MS. • PDMS coated Twisters™ extract phosphate acid esters in the neutral form from water samples. • Good linearity between concentrations and relative peak areas (0.1–750 ng mL{sup −1}). • This novel SBSE/DART/Orbitrap-MS method should be very valuable for on-site sampling/monitoring - Abstract: A novel hyphenated technique, namely the combination of stir bar sorptive extraction (SBSE) with isotope dilution direct analysis in real time (DART) Orbitrap™ mass spectrometry (OT-MS) is presented for the extraction of phosphoric acid alkyl esters (tri- (TnBP), di- (HDBP), and mono-butyl phosphate (H2MBP)) from aqueous samples. First, SBSE of phosphate esters was performed using a Twister™ coated with 24 μL of polydimethylsiloxane (PDMS) as the extracting phase. SBSE was optimized for extraction pH, phase ratio (PDMS volume/aqueous phase volume), stirring speed, extraction time and temperature. Then, coupling of SBSE to DART/Orbitrap-MS was achieved by placing the Twister™ in the middle of an open-ended glass tube between the DART and the Orbitrap™. The DART mass spectrometric response of phosphate esters was probed using commercially available and synthesized alkyl phosphate ester standards. The positive ion full scan spectra of alkyl phosphate triesters (TnBP) was characterized by the product of self-protonation [M + H]{sup +} and, during collision-induced dissociation (CID), the major fragmentation ions corresponded to consecutive loss of alkyl chains. Negative ionization gave abundant [M − H]{sup −} ions for both HDnBP and H2MnBP. Twisters™ coated with PDMS successfully extracted phosphate acid esters (tri-, di- and mono-esters) granted that the analytes are present in the aqueous solution in the neutral form. SBSE/DART/Orbitrap-MS results show a good linearity between

  14. Integration of Quantitative Positron Emission Tomography Absolute Myocardial Blood Flow Measurements in the Clinical Management of Coronary Artery Disease.

    Science.gov (United States)

    Gewirtz, Henry; Dilsizian, Vasken

    2016-05-31

    In the >40 years since planar myocardial imaging with(43)K-potassium was introduced into clinical research and management of patients with coronary artery disease (CAD), diagnosis and treatment have undergone profound scientific and technological changes. One such innovation is the current state-of-the-art hardware and software for positron emission tomography myocardial perfusion imaging, which has advanced it from a strictly research-oriented modality to a clinically valuable tool. This review traces the evolving role of quantitative positron emission tomography measurements of myocardial blood flow in the evaluation and management of patients with CAD. It presents methodology, currently or soon to be available, that offers a paradigm shift in CAD management. Heretofore, radionuclide myocardial perfusion imaging has been primarily qualitative or at best semiquantitative in nature, assessing regional perfusion in relative terms. Thus, unlike so many facets of modern cardiovascular practice and CAD management, which depend, for example, on absolute values of key parameters such as arterial and left ventricular pressures, serum lipoprotein, and other biomarker levels, the absolute levels of rest and maximal myocardial blood flow have yet to be incorporated into routine clinical practice even in most positron emission tomography centers where the potential to do so exists. Accordingly, this review focuses on potential value added for improving clinical CAD practice by measuring the absolute level of rest and maximal myocardial blood flow. Physiological principles and imaging fundamentals necessary to understand how positron emission tomography makes robust, quantitative measurements of myocardial blood flow possible are highlighted. PMID:27245647

  15. Staphylococcus aureus DNA in human venous blood detected by real-time quantitative PCR assay%实时定量PCR检测人全血中金黄色葡萄球菌DNA方法的建立

    Institute of Scientific and Technical Information of China (English)

    康军仁; 马恩陵; 房嘉宾; 崔希增

    2014-01-01

    目的 建立实时定量PCR (RQ-PCR)快速检测人全血中金黄色葡萄球菌DNA的方法,以便早期定量评估肠屏障损伤所致肠道内细菌移位引起或加重的全身感染.方法 对15份模拟全血标本及26份外科发热患者全血标本进行RQ-PCR检测.选择金黄色葡萄球菌高度保守的看家基因fem A基因作为靶基因设计引物和Taqman探针,建立20μl的RQ-PCR反应体系,采用含靶基因扩增片段的重组质粒建立标准曲线,提取血标本中的细菌基因组DNA.结果 引物和探针特异性好,检测限为100拷贝/μ1 (103 CFU/ml),灵敏度为99.7%,特异度为94.6%.标准曲线线性关系好,R2在0.9918 ~0.9997.不同浓度的金黄色葡萄球菌样本检测的平均准确性为(96.25±2.26)%,批内及批间重复性的平均变异系数分别为(8.06±0.07)%和(10.01±4.40)%.全血标本中金黄色葡萄球菌DNA平均回收率为(111.72±20.72)%.临床血标本RQ-PCR检测阳性率为15.4% (4/26),血培养结果皆为金黄色葡萄球菌阴性.结论 RQ-PCR可用于定量检测全血标本中的金黄色葡萄球菌DNA含量,具有快速、灵敏、特异性强、重复性好的优点.%Objective To establish a rapid real-time quantitative polymerase chain reaction (RQ-PCR)assay in quantifying and detecting Staphylococcus aureus DNA from human venous blood samples,so as to quantificationally evaluate the systemic infection caused or deteriorated by intestinal bacteria translocation.Methods Totally 26 clinical blood samples and 15 simulation blood samples were detected.The primers and TaqMan probe were designed targeting the highly conserved house-keeping femA gene of Staphylococcus aureus,and a 20 μl RQ-PCR amplification reaction system was established.The standard curve was built based on the recombinant plasmid DNA containing the amplicon of the target gene,and genomic DNA was extracted using QIAamp DNA Blood Mini Kit.Results The specificity of primers and probe was excellent

  16. Modular specification of real-time systems

    DEFF Research Database (Denmark)

    Inal, Recep

    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...... is presented through a top-level specification of requirements for a simple air traffic monitoring system, and reasoning is illustrated by a refinement towards a design...

  17. Modular specification of real-time systems

    OpenAIRE

    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 is presented through a top-level specification of requirements for a simple air traffic monitoring system, and reasoning is illustrated by a refinement towards a design

  18. 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...

  19. Real-time Transmission Over Internet

    OpenAIRE

    Gao, Qi

    2004-01-01

    With the Internet expansion, real-time transmission over Internet is becoming a new promising application. Successful real-time communication over IP networks requires reasonably reliable, low delay, low loss date transport. Since Internet is a non-synchronous packet switching network, high load and lack of guarantees on data delivery make real-time communication such as Voice and Video over IP a challenging application to become realistic on the Internet. This thesis work is composed of two...

  20. Real-time graphics rendering engine

    CERN Document Server

    Bao, Hujun

    2011-01-01

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

  1. 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...... Bertrand case, welfare is the same with all or no consumers on smart meters....... 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. In the...

  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. De toekomst van Real Time Intelligence

    OpenAIRE

    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 van TNO is de kracht van real-time intelligence, dat het bijdraagt aan het creëren van een beter beeld van de situatie waarin ingegrepen moet worden. Real-time intelligence kan op die manier een gr...

  4. Evaluation of a real-time two-step RT-PCR assay for quantitation of Chronic bee paralysis virus (CBPV) genome in experimentally-infected bee tissues and in life stages of a symptomatic colony.

    OpenAIRE

    Blanchard, Philippe; Ribière, Magali; Celle, Olivier; Lallemand, Perrine; Schurr, Frank; Olivier, Violaine; Iscache, Anne Laure; Faucon, Jean Paul

    2007-01-01

    A two-step real-time RT-PCR assay, based on TaqMan technology using a fluorescent probe (FAM-TAMRA) was developed to quantify Chronic bee paralysis virus (CBPV) genome in bee samples. Standard curves obtained from a CBPV control RNA and from a plasmid containing a partial sequence of CBPV showed that this assay provided linear detection over a 7-log range (R(2)>0.99) with a limit of detection of 100 copies, and reliable inter-assay and intra-assay reproducibility. Standardisation including RN...

  5. Real-time immunohistochemistry analysis of embedded tissue

    International Nuclear Information System (INIS)

    We present a novel analysis of membrane-protein expression in tissue sections based on semi-automatic real-time measurement using LigandTracer technology. A commercial antiHER2 antibody developed for immunohistochemistry used in this setup was revealed to have sub-optimal interaction with tissue when analyzed as recommended for immunohistochemistry. We therefore think that real-time measurement of tissue, offering direct and quantitative membrane-protein interaction analysis, can lead to improved reproducibility and eliminate the subjective operator dependences that classical immunohistochemsitry suffers from.

  6. Detection of pathogenic elephant endotheliotropic herpesvirus in routine trunk washes from healthy adult Asian elephants (Elephas maximus) by use of a real-time quantitative polymerase chain reaction assay

    Science.gov (United States)

    Stanton, Jeffrey J.; Zong, Jian-Chao; Latimer, Erin; Tan, Jie; Herron, Alan; Hayward, Gary S.; Ling, Paul D.

    2013-01-01

    Objective To investigate the pathogenesis and transmission of elephant endotheliotropic herpesvirus (EEHV1) by analyzing various elephant fluid samples with a novel EEHV1-specific real-time PCR assay. Animals 5 apparently healthy captive Asian elephants (Elephas maximus) from the same herd. Procedures A real-time PCR assay was developed that specifically detects EEHV1. The assay was used to evaluate paired whole blood and trunk-wash samples obtained from the 5 elephants during a 15-week period. Deoxyribonucleic acid sequencing and viral gene subtyping analysis were performed on trunk-wash DNA preparations that had positive results for EEHV1. Viral gene subtypes were compared with those associated with past fatal cases of herpesvirus-associated disease within the herd. Results The PCR assay detected viral DNA to a level of 1,200 copies/mL of whole blood. It was used to detect EEHV1 in trunk secretions of 3 of the 5 elephants surveyed during the 15-week period. Viral gene subtyping analysis identified 2 distinct elephant herpesviruses, 1 of which was identical to the virus associated with a previous fatal case of herpesvirus-associated disease within the herd. Conclusions and Clinical Relevance EEHV1 was shed in the trunk secretions of healthy Asian elephants. Trunk secretions may provide a mode of transmission for this virus. Results of this study may be useful for the diagnosis, treatment, and management of EEHV1-associated disease and the overall management of captive elephant populations. PMID:20673092

  7. Comparative study of bovine and porcine derived materials in hydrolysate samples by real-time fluorescence quantitative PCR and general PCR%实时荧光定量PCR和常规PCR检测肝水解肽样品中牛、猪源性成分的对比研究

    Institute of Scientific and Technical Information of China (English)

    余燕; 何素婷; 王自强; 邓锋

    2015-01-01

    Objective To compare real-time fluorescence quantitative PCR with general PCR in detecting bovine and porcine derived materials in hydrolysate samples.Methods DNA were extracted from hydrolysate samples which prepared by different steps by real-time fluorescence quantitative PCR and general PCR.Results DNA of bovine and porcine could be detected by real-time fluorescence quantitative PCR and general PCR in samples prepared in the processes before enzymolysis solution, but not detected in samples from supermatant to the fourth ultrafiltrate.Conclusion Both real-time fluorescence quantitative PCR and general PCR can be applied to detect the fragments in hydrolysate samples.And real-time fluorescence quantitative PCR has the advantage such as rapid,convenient, non-environment-polluted, good repeatability, which improves the quality and efficiency.%目的:对比实时荧光定量PCR与常规PCR检测肝水解肽样品中牛、猪源性成分的有效性。方法对牛、猪源性肝水解肽样品肝脏至上清液工艺段样品进行DNA提取,采用实时荧光定量PCR和常规PCR同时检测样品中的DNA。结果实时荧光定量PCR和常规PCR在猪源、牛源肝水解肽从肝脏到酶解液的各工艺步骤段样品中,均检测到动物源性DNA,在上清液至超滤液四中,均未检测出动物源性DNA。结论两种方法均可用在检测肝水解肽样品中牛、猪源性成分。实时荧光定量PCR同时还具有快速、简便、不污染环境、重复性好的特点,可明显提高工作质量及效率。

  8. Real-time Pricing in Power Markets

    DEFF Research Database (Denmark)

    Boom, Anette; Schwenen, Sebastian

    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. In the...

  9. Validation of RNAi by real time PCR

    DEFF Research Database (Denmark)

    Josefsen, Knud; Lee, Ying Chiu

    2011-01-01

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

  10. A Real-time Network at Home

    NARCIS (Netherlands)

    Hanssen, Ferdy; Hartel, Pieter; Jansen, Pierre; Scholten, Hans; Vervoort, Wiek

    2001-01-01

    This paper proposes a home network which integrates both real-time and non-real-time capabilities for one coherent, distributed architecture. Such a network is not yet available. Our network will support inexpensive, small appliances as well as more expensive, large appliances. The network is based

  11. Storm real-time processing cookbook

    CERN Document Server

    Anderson, Quinton

    2013-01-01

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

  12. 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...

  13. 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...

  14. 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.

  15. Hybrid Multiprocessor Real-Time Scheduling Approach

    Directory of Open Access Journals (Sweden)

    Ali A Safaei

    2011-03-01

    Full Text Available Real-time scheduling is one of the most important aspects of a real-time system design. To achieve a real-time system's requirement, especially to be fast, multiprocessor systems are used. Generally, multiprocessor real-time scheduling algorithms fall into one the two well-known approaches: Partitioning or Global. The partitioning approach has acceptable overhead for underlying system but can NOT guarantee to provide an optimal schedule. The global approach can provide this guarantee by holding some preconditions and considerable overheads. In this paper, an intermediate hybrid multiprocessor real-time scheduling approach is proposed in which optimality will be reached via the minimum overheads for underlying system. Presenting and analyzing different feasible paradigms for combination of the two existing approaches, the proposed hybrid approach satisfies the two major goals of this combination: optimality and lightweightness. Experimental results show that the hybrid approach outperforms the two existing ones.

  16. Real-time change detection in data streams with FPGAs

    International Nuclear Information System (INIS)

    Highlights: • Automatic recognition of changes in data streams of multidimensional signals. • Detection algorithm based on testing exchangeability on-line. • Real-time and off-line applicability. • Real-time implementation in FPGAs. - Abstract: The automatic recognition of changes in data streams is useful in both real-time and off-line data analyses. This article shows several effective change-detecting algorithms (based on martingales) and describes their real-time applicability in the data acquisition systems through the use of Field Programmable Gate Arrays (FPGA). The automatic event recognition system is absolutely general and it does not depend on either the particular event to detect or the specific data representation (waveforms, images or multidimensional signals). The developed approach provides good results for change detection in both the temporal evolution of profiles and the two-dimensional spatial distribution of volume emission intensity. The average computation time in the FPGA is 210 μs per profile

  17. Development and application of absolute quantitative detection by duplex chamber-based digital PCR of genetically modified maize events without pretreatment steps.

    Science.gov (United States)

    Zhu, Pengyu; Fu, Wei; Wang, Chenguang; Du, Zhixin; Huang, Kunlun; Zhu, Shuifang; Xu, Wentao

    2016-04-15

    The possibility of the absolute quantitation of GMO events by digital PCR was recently reported. However, most absolute quantitation methods based on the digital PCR required pretreatment steps. Meanwhile, singleplex detection could not meet the demand of the absolute quantitation of GMO events that is based on the ratio of foreign fragments and reference genes. Thus, to promote the absolute quantitative detection of different GMO events by digital PCR, we developed a quantitative detection method based on duplex digital PCR without pretreatment. Moreover, we tested 7 GMO events in our study to evaluate the fitness of our method. The optimized combination of foreign and reference primers, limit of quantitation (LOQ), limit of detection (LOD) and specificity were validated. The results showed that the LOQ of our method for different GMO events was 0.5%, while the LOD is 0.1%. Additionally, we found that duplex digital PCR could achieve the detection results with lower RSD compared with singleplex digital PCR. In summary, the duplex digital PCR detection system is a simple and stable way to achieve the absolute quantitation of different GMO events. Moreover, the LOQ and LOD indicated that this method is suitable for the daily detection and quantitation of GMO events. PMID:27016439

  18. Modelling the world in real time: how robots engineer information.

    Science.gov (United States)

    Davison, Andrew J

    2003-12-15

    Programming robots and other autonomous systems to interact with the world in real time is bringing into sharp focus general questions about representation, inference and understanding. These artificial agents use digital computation to interpret the data gleaned from sensors and produce decisions and actions to guide their future behaviour. In a physical system, however, finite computational resources unavoidably impose the need to approximate and make selective use of the information available to reach prompt deductions. Recent research has led to widespread adoption of the methodology of Bayesian inference, which provides the absolute framework to understand this process fully via modelling as informed, fully acknowledged approximation. The performance of modern systems has improved greatly on the heuristic methods of the early days of artificial intelligence. We discuss the general problem of real-time inference and computation, and draw on examples from recent research in computer vision and robotics: specifically visual tracking and simultaneous localization and mapping. PMID:14667303

  19. Real-time Crane Control via PC

    OpenAIRE

    Arm, J

    2015-01-01

    In this paper, creation of SCADA system, which controls and monitors model of gantry crane in real time, is described. This crane will be controlled via process station communicating with PC via Modbus TCP protocol. Moreover, HTTP server will be created to monitor crane state over intranet. On this work, the difference between real-time and fast execution will be described and it will be shown on the built system that the fast execution system can behave like a real-time system under specific...

  20. Ada in Real-time Embedded System

    OpenAIRE

    Elnaz Ghodsvali; Rabee Sharifi Rad; Sima Sinaei

    2013-01-01

    Ada has an important role in the real-time/embedded/safety-critical areas. It is the only ISO-standard, object-oriented, concurrent, real-time programming language. Ada is used as a usual language for application areas such as defense embedded systems that reliability and efficiency are very essential. One of the main Ada’s characteristics in compare with other programming languages is that, Ada was developed from the ground up with capabilities that provide real-time requirements. In this st...

  1. Interactive Real-time Magnetic Resonance Imaging

    DEFF Research Database (Denmark)

    Brix, Lau

    Real-time acquisition, reconstruction and interactively changing the slice position using magnetic resonance imaging (MRI) have been possible for years. However, the current clinical use of interactive real-time MRI is limited due to an inherent low spatial and temporal resolution. This PhD project...... regard to optimal sampling strategy for detecting motion in four different anatomies on two different MRI scanner brands. A fully implemented interactive real-time MRI system was exploited in a group of healthy fetuses and proved its eligibility as an alternative diagnostic tool for fetal imaging...

  2. 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.

  3. Visualization in Real-Time Experiment Project

    Data.gov (United States)

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

  4. Lymphoid follicles of the ileal Peyer's patch of lambs express low levels of PrP, as demonstrated by quantitative real-time RT-PCR on microdissected tissue compartments, in situ hybridization and immunohistochemistry.

    Science.gov (United States)

    Austbø, Lars; Espenes, Arild; Olsaker, Ingrid; Press, Charles McL; Skretting, Grethe

    2006-11-01

    The expression level of normal cellular prion protein (PrP(C)) is thought to influence the transmission of transmissible spongiform encephalopathies (TSEs) from the peripheral entry site to the site of pathological changes in the central nervous system. In many TSEs, the clinical disease is preceded by a period in which the agent accumulates in lymphoid organs, particularly in association with follicular dendritic cells of lymphoid follicles. As the probable route of entry of the TSE agent is via the gut, the expression profile of PrP was examined in well-developed gut-associated lymphoid tissue of lambs, the ileal Peyer's patch, by laser microdissection and real-time RT-PCR. Lymphoid follicles were found to have very low levels of expression, whilst highest levels were detected in the outer submucosa and the muscular layer. These findings were supported by in situ hybridization and immunohistochemistry, which showed specific labelling in nerve cells in ganglia of the submucosal (Meissner's) and myenteric (Auerbach's) plexi of the enteric nervous system. Based on the assumption that potential sites for conversion to the scrapie-related prion protein (PrP(Sc)) should display high levels of expression of PrP(C), this study suggests that the accumulation of PrP(Sc) in the lymphoid follicles of the Peyer's patch is not preceded by PrP conversion in the same tissue compartment. PMID:17030883

  5. Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

    KAUST Repository

    Abu Samra, Dina Bashir Kamil

    2015-06-29

    Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow onand off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Identification of the Homozygosis of Genetically Modified Crop by Real-time Quantitative PCR%实时定量 PCR 鉴定转基因作物纯合体

    Institute of Scientific and Technical Information of China (English)

    张丽; 刘丽丽; 梁晓声; 王海英

    2015-01-01

    Rice endogenous reference gene phospholipase D gene ( PLD) and genetically modified ( GM) rice TT51-1 event-specific flanking sequence were used as PCR detection targets.And the homozygosis of GM rice TT51-1 plants originated from single plant seeds were analyzed through real-time PCR assays, which analyzed the Ct value of the endogenous reference gene and the flanking sequences.The reliability of this method was verified by calculation of GM copy number ratio based on the standard curves.It was concluded that the method was simple and accurate to identify the homozygosis of GM crops.%以水稻内标准基因磷脂酶D基因( phospholipase D, PLD)和转基因水稻TT51-1特异性旁侧序列为检测靶标,对单株转基因水稻的种子播种后长出的单株进行了荧光定量PCR,以其内标准基因和旁侧序列Ct值的差值判断了植株的纯合体,并用标准曲线计算了转基因含量,证实了此法的可靠性,说明此法用于鉴定植株的纯合体简便准确。

  7. Risk management using big real time data

    OpenAIRE

    Cheng, Jie

    2014-01-01

    This thesis focuses on risk management of flight delay area using big real time data. It proposes two different prediction models, one is called General Long Term Departure Prediction Model and the other is named as Improved Real Time Arrival Prediction Model. By studying the main factors lead to flight delay, this thesis takes weather, carrier, National Aviation System, security and previous late aircraft as analysis factors. By utilizing our models can do not only long time b...

  8. Integrated Control and Real-Time Scheduling

    OpenAIRE

    Cervin, Anton

    2003-01-01

    The topic of the thesis is codesign of flexible real-time control systems. Integrating control theory and real-time scheduling theory, it is possible to achieve higher resource utilization and better control performance. The integration requires new tools for analysis, design, and implementation. The problem of scheduling the individual parts of a control algorithm is studied. It is shown how subtask scheduling can reduce the input-output latency in a set of control tasks. Deadline ass...

  9. Interferometer real time control development for SIM

    Science.gov (United States)

    Bell, Charles E.

    2003-02-01

    Real Time Control (RTC) for the Space Interferometry Mission will build on the real time core interferometer control technology under development at JPL since the mid 1990s, with heritage from the ground based MKII and Palomar Testbed Interferometer projects developed in the late '80s and early '90s. The core software and electronics technology for SIM interferometer real time control is successfully operating on several SIM technology demonstration testbeds, including the Real-time Interferometer Control System Testbed, System Testbed-3, and the Microarcsecond Metrology testbed. This paper provides an overview of the architecture, design, integration, and test of the SIM flight interferometer real time control to meet challenging flight system requirements for the high processor throughput, low-latency interconnect, and precise synchronization to support microarcsecond-level astrometric measurements for greater than five years at 1 AU in Earth-trailing orbit. The electronics and software architecture of the interferometer real time control core and its adaptation to a flight design concept are described. Control loops for pointing and pathlength control within each of four flight interferometers and for coordination of control and data across interferometers are illustrated. The nature of onboard data processing to fit average downlink rates while retaining post-processed astrometric measurement precision and accuracy is also addressed. Interferometer flight software will be developed using a software simulation environment incorporating models of the metrology and starlight sensors and actuators to close the real time control loops. RTC flight software and instrument flight electronics will in turn be integrated utilizing the same simulation architecture for metrology and starlight component models to close real time control loops and verify RTC functionality and performance prior to delivery to flight interferometer system integration at Lockheed Martin

  10. The LAA real-time benchmarks

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-04-01

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

  11. Real Time Information Fusion in Military Systems

    Directory of Open Access Journals (Sweden)

    E. Bhagiratharao

    1990-01-01

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

  12. REAL TIME WIRELESS AIR POLLUTION MONITORING SYSTEM

    OpenAIRE

    Raja Vara Prasad Y; Mirza Sami Baig; Mishra, Rahul K; Rajalakshmi, P.; U. B. Desai; S. N. Merchant

    2011-01-01

    Air pollution has significant influence on the concentration of constituents in the atmosphere leading to effects like global warming and acid rains. To avoid such adverse imbalances in the nature, an air pollution monitoring system is utmost important. This paper attempts to develop an effective solution for pollution monitoring using wireless sensor networks (WSN) on a real time basis namely real time wireless air pollution monitoring system. Commercially available discrete gas sensors for ...

  13. Real time monitoring of electron processors

    International Nuclear Information System (INIS)

    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)

  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 Scheduling for Software Prototyping

    OpenAIRE

    Luqi; Shing, M

    1996-01-01

    This paper presents several real-time scheduling algorithms developed to support rapid prototyping of embedded systems using the Computer Aided Prototyping System (CAPS). The CAPS tools are based on the Prototyping System Description Language (PSDL), which is a high-level language designed specifically to support the conceptual modeling of real-time embedded systems. This paper describes the scheduling algorithms used in CAPS along with the associated timing constraint and hardwar...

  16. Towards validated real-time software

    OpenAIRE

    Bertin, V.; Poize, M.; Pulou, J.; Sifakis, J

    2000-01-01

    We present a tool for the design and validation of embedded real time applications. The tool integrates two approaches: the use of the synchronous programming language, ESTEREL for design, and the application of model checking techniques for validation of real time properties. Validation is carried out on a global formal model (timed automata) taking into account the effective implementation of the application on the target hardware architecture as well as its external environment behavior

  17. Benefits of real-time gas management

    International Nuclear Information System (INIS)

    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

  18. Development of an isoform-specific tandem mass spectrometry assay for absolute quantitation of maize lipid transfer proteins.

    Science.gov (United States)

    Stevenson, Severin E; McClain, Scott; Thelen, Jay J

    2015-01-28

    Precise and accurate quantitation of maize grain allergens is important for seed and food industries. The major allergen in maize grain is Zea m 14, a lipid transfer protein (LTP). The B73 maize genome encodes for at least six LTPs sharing 15%-87% sequence identity to Zea m 14. Phylogenetic analysis of the maize LTP family revealed one gene that corresponds to Zea m 14 (denoted as LTPa) and two other genes sharing 43% (LTPc) and 74% (LTPb) identity with Zea m 14 that are putative homologues. Using stable isotope peptide mimics as internal standards for LTPs, we present a multiple reaction monitoring mass spectrometry approach for multiplexed, absolute quantitation of all three LTP proteins and alternative transcript models therein. To validate quantitative accuracy, a redundant peptide, simultaneously representing the two most abundant LTPs, was included. Analysis of 21 maize varieties revealed LTPa was most prominently expressed in maize grain, ranging from 9 to 32 μg LTP/mg protein. Proteins belonging to the LTPb and LTPc gene models were also expressed but at approximately 10- and 100-fold lower levels than LTPa, respectively. The quantitative results provided by the redundant peptide show around 95% agreement with the sum of the two unique peptides, thus providing support for the LTP gene models and validating the accuracy of this method. Though not all Zea m 14-related LTPs are abundant in grain, their high sequence homology and detectable expression in maize grain signify that LTPb and LTPc are putative allergens and should be accounted for in any quantitation strategy for maize LTP allergens. PMID:25540820

  19. A Novel Pretreatment-Free Duplex Chamber Digital PCR Detection System for the Absolute Quantitation of GMO Samples

    Directory of Open Access Journals (Sweden)

    Pengyu Zhu

    2016-03-01

    Full Text Available Digital polymerase chain reaction (PCR has developed rapidly since it was first reported in the 1990s. However, pretreatments are often required during preparation for digital PCR, which can increase operation error. The single-plex amplification of both the target and reference genes may cause uncertainties due to the different reaction volumes and the matrix effect. In the current study, a quantitative detection system based on the pretreatment-free duplex chamber digital PCR was developed. The dynamic range, limit of quantitation (LOQ, sensitivity and specificity were evaluated taking the GA21 event as the experimental object. Moreover, to determine the factors that may influence the stability of the duplex system, we evaluated whether the pretreatments, the primary and secondary structures of the probes and the SNP effect influence the detection. The results showed that the LOQ was 0.5% and the sensitivity was 0.1%. We also found that genome digestion and single nucleotide polymorphism (SNP sites affect the detection results, whereas the unspecific hybridization within different probes had little side effect. This indicated that the detection system was suited for both chamber-based and droplet-based digital PCR. In conclusion, we have provided a simple and flexible way of achieving absolute quantitation for genetically modified organism (GMO genome samples using commercial digital PCR detection systems.

  20. A Novel Pretreatment-Free Duplex Chamber Digital PCR Detection System for the Absolute Quantitation of GMO Samples.

    Science.gov (United States)

    Zhu, Pengyu; Wang, Chenguang; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

    2016-01-01

    Digital polymerase chain reaction (PCR) has developed rapidly since it was first reported in the 1990s. However, pretreatments are often required during preparation for digital PCR, which can increase operation error. The single-plex amplification of both the target and reference genes may cause uncertainties due to the different reaction volumes and the matrix effect. In the current study, a quantitative detection system based on the pretreatment-free duplex chamber digital PCR was developed. The dynamic range, limit of quantitation (LOQ), sensitivity and specificity were evaluated taking the GA21 event as the experimental object. Moreover, to determine the factors that may influence the stability of the duplex system, we evaluated whether the pretreatments, the primary and secondary structures of the probes and the SNP effect influence the detection. The results showed that the LOQ was 0.5% and the sensitivity was 0.1%. We also found that genome digestion and single nucleotide polymorphism (SNP) sites affect the detection results, whereas the unspecific hybridization within different probes had little side effect. This indicated that the detection system was suited for both chamber-based and droplet-based digital PCR. In conclusion, we have provided a simple and flexible way of achieving absolute quantitation for genetically modified organism (GMO) genome samples using commercial digital PCR detection systems. PMID:26999129

  1. Development and Application of Real-time Quantitative Polymerase Chain Reaction for the Detection of Human Hepatocellular Carcinoma Related Novel Gene HSF1 Expression%肝细胞癌患者外周血HSF1基因表达的检测及应用价值

    Institute of Scientific and Technical Information of China (English)

    卢鹏; 冯薇; 李霄; 杨平; 寇明文; 胡延忠; 宋振顺

    2012-01-01

    目的:建立检测HSF1 mRNA的real-time PCR的方法,了解肝细胞癌患者外周血中HSF1的表达水平及其与各临床病理特征之间的关系.方法:利用real-time PCR的方法检测20例肝细胞癌患者及20例正常人群外周血中HSF1 mRNA的表达量.结果:肝细胞癌患者外周血中的HSFl mRNA表达量显著高于正常人群(P<0.05);肝细胞癌患者外周血中HSFI mRNA的表达水平在不同性别、肿瘤大小、门静脉侵犯情况、HbsAg水平及AFP水平的患者中的差异无统计学意义(P>0.05);在不同病理分化程度、TNM分期的患者中的差异有统计学意义(P<0.05).结论:real-time PCR技术可以成功检测外周血中HSFl mRNA的表达量,HSF1可能与肝细胞癌的发生发展密切相关.%Objective: To establish a quantitative real-time PCR method for detection of HSF1 gene expression level in human hepatocellular carcinoma and to investigate the relationship between HSFl mRNA expression level and the clinicopathological characters. Methods: Real-time PCR was employed to determine the expression level of HSF1 mRNA in peripheral blood of 20 specimens of human hepatocellular carcinoma patients and normal human. Results: Comparing with normal human, HSF1 present higher expressions in human hepatocellular carcinoma patients (P0.05). There is statistical differences of HSF1 mRNA expression level between the group with different differentiations and TNM stages (P>0.05). Conclusion: The established quantitative real-time PCR method can successfully detect the expression level of HSF1 mRNA, which may be closely related to the occurrence and development of human hepatocellular carcinoma.

  2. Interference Between D and M Types of Plum pox virus in Japanese Plum Assessed by Specific Monoclonal Antibodies and Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction.

    Science.gov (United States)

    Capote, Nieves; Gorris, M Teresa; Martínez, M Carmen; Asensio, Margarita; Olmos, Antonio; Cambra, Mariano

    2006-03-01

    ABSTRACT The dynamics of virus interference between two isolates of Plum pox virus (PPV) belonging to the main PPV types, D and M, were analyzed in Japanese plum (Prunus salicina) by challenge inoculations. To assess the consequences of a PPV-M infection on plum already infected with PPV-D, and vice versa (predominance of one of the strains, recombination, synergism, symptoms aggravation, and so on), 30 Japanese plum trees were graft inoculated with PPV-D or PPV-M isolates in quarantine conditions. One year postinoculation, in the event that the inoculated isolates were detected in the whole plant, a second challenge inoculation (PPV-M or PPV-D, respectively) was performed by grafting. The presence of PPV-D, PPV-M, or both was monitored for 7 years by double-antibody sandwich indirect enzyme-linked immunosorbent assay using specific monoclonal antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) with D- and M-specific primers confirmed the serological typing. Real-time RT-PCR assays were performed using D- and M-specific fluorescent 3' minor groove binder-DNA probes, which were able to detect and quantify PPV populations in the inoculated plants with greater precision. The presence of PPV-D in Japanese plum did not cross-protect the trees against PPV-M infection. In PPV-D-infected plants, the PPV-M strain used as challenge inoculum behaved differently depending on the plum cultivar assayed. In cv. Black Diamond, PPV-M invaded the plant progressively, displacing the previous PPV-D population; whereas, in cv. Sun Gold, both PPV isolates coexisted in the plant. In contrast, the PPV-D isolate used was unable to infect plants of both cultivars in which a PPV-M population already was established. After 7 years, no synergism was observed and no recombination event between PPV-D and PPV-M genomes was detected. PMID:18944448

  3. SYBR GreenI实时荧光定量PCR检测小麦纹枯病菌体系的建立和应用%Establishment of SYBR Green I Real-Time PCR for Quantitatively Detecting Rhizoctonia cerealis in Winter Wheat

    Institute of Scientific and Technical Information of China (English)

    孙炳剑; 陈清清; 袁虹霞; 施艳; 李洪连

    2015-01-01

    [Objective]Wheat sharp eyespot (WSE) caused by Rhizoctonia cerealis is one of the most important soil-born diseases on wheat in China. Early accurate quantitative detection is a foundation of forecast and control. Traditional method of organization isolation and identification of pathogen is time consuming, complicated and can’t be accurately quantified. In order to implement the early and quick quantitative determination of wheat sharp eyespot, a SYBR Green I real-time PCR method of R. cerealis was established based on the pathogen sequence information. [Method] Based on the β-tubilin of R. cerealis, a pair of specific primers was designed. The SYBR Green I real-time PCR reaction system was established and optimized. The sensitivity, specificity and repeatability of the system were also evaluated, and R. cerealis, R. solani, AG-A, AG-F, Gaeumannomyces graminis var. tritici, Bipolaris sorokiniana, Fusarium graminearum, F. pseudograminearum were used for control fungi. The indoor potted plants of wheat which infected by R. cerealis were detected with optimized reaction system after inoculated for 5, 10 and 60 days, respectively.[Result]The primers were of great specificity, the specific PCR fragment was amplified from the DNA of R. cerealis isolates, but not from the DNA of other fungal isolates by conventional PCR. The real-time PCR assays also did not amplify DNA from control fungi. The sensitivity of conventional PCR was 6.5×103 copies/μL plasmid, while the sensitivity of real-time PCR was 6.5×102 copies/μL. The standard curve established by recombinant plasmid showed a fine linear relationship between threshold cycle and template concentration. The melt curve was specific with the correlation coefficient of 0.997 and with high amplification efficiency (0.91). For the indoor potted experiments, the detection results of real-time PCR of infected wheat samples, were showed a significant positive correlation with disease index and inoculum, respectively

  4. [Real time 3D echocardiography in congenital heart disease].

    Science.gov (United States)

    Acar, P; Dulac, Y; Taktak, A; Villacèque, M

    2004-05-01

    The introduction of the 3D mode in echocardiography has led to its use in everyday clinical practice. One hundred and fifty real time 3D echocardiographic examinations were performed in 20 foetus, 110 children and 20 adults with various congenital heart lesions (shunts, valvular lesions, aortic diseases). The 4x matricial probe enables the instantaneous acquisition of transthoracic volumes. Four modes of 3D imaging were used: real time, total volume, colour Doppler and biplane. Quantitative measurements were performed at an outlying station. The feasibility of the method in the foetus, the child and the adult was respectively 90%, 99% and 85%. Real time 3D echocardiography did not affect the diagnoses made by standard echocardiography. The 3D imaging gave a more accurate description of atrial septal defects and congenital valvular lesions. Biplane imaging was decisive in the quantitative approach to aortic dilatation of Marfan's syndrome and in segmental analysis of the foetal heart. 3D colour Doppler imaging has been disappointing but the possibilities of volumic quantification of blood flow are very promising. The present limitations of the method are the inadequate resolution in the small child and the absence of quantitative measurement on the echograph. The facility of utilisation of the matricial probe should lead to routine usage of 3D echocardiography as with 2D and the Doppler modes. Its value should be decisive in many congenital cardiac lesions requiring surgery or interventional catheterisation. PMID:15214550

  5. 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.

  6. Real-time control software on NSTX

    Energy Technology Data Exchange (ETDEWEB)

    Mastrovito, D. E-mail: dmastrovito@pppl.gov; Ferron, J.; Gates, D.; Gibney, T.; Johnson, R

    2004-06-01

    The National Spherical Tokamak Experiment (NSTX) depends on high speed, real-time control to establish initial magnetic field configurations, drive plasma current, and control plasma position and shape. The control required on NSTX is achieved through the combined efforts of several applications developed collaboratively including the power supply real-time controller (PSRTC) and the plasma control system (PCS). The PSRTC communicates with the PCS receiving from the PCS real-time voltage controls and is responsible for supplying dc power to the NSTX poloidal field, toroidal field and Ohmic heating coils. The PCS, which was designed at general atomics, controls the data acquisition, gas injection, plasma current, and plasma shape, including a real-time EFIT and isoflux algorithm for equilibrium reconstruction. Modular control algorithms, specific to NSTX, were written and incorporated into the PCS. The NSTX control system is equipped with 160 channels of fast real-time data acquisition and eight 333 MHz G4 processors, which perform the supervision, position, shape, and performance control. After a discharge, the acquired data and setup parameters are transferred to an MDSPlus central archive for later inspection and can be used to repeat previous machine configurations.

  7. 并行工程中下游阶段启动时间的实时确定%QUANTITATIVE APPROACH TO REAL TIME STARTUP TIME DETERMINATION OF DOWNSTREAM PHASES IN CONCURRENT ENGINEERING

    Institute of Scientific and Technical Information of China (English)

    吕常魁; 姜澄宇; 王宁生

    2004-01-01

    Implementation of simultaneous execution phases in the concurrent engineering (CE) needs careful planning when the downstream phase could be activated as the upstream phase developed to a certain point. The determination of startup time of overlapping jobs in CE has long been a disturbance in manufacturing industry implementing CE programs. A novel model based on both fuzzy logic and neural network is proposed to mathematically formulate the inter-connective information between the two coupled phases in CE projects, and to determine the startup time of downstream phases in real time. The information transferring between the two coupled phases is quantified by using the negative Shannon entropy. Based on this algorithm, a PDM-based framework is proposed to narrow the gap between pro-duct design and manufacture, in which five modules are built to monitor, reshuffle and implement the simultaneous executing processes. Finally, an example is given to illustrate applications of the algorithm in the real world.%在并行工程的实施过程中,上下游阶段交叠形式的不合理安排将会给产品的开发带来不可预见的风险.如何决定各并行阶段的启动时间一直是困扰实施并行工程企业的问题.对于这方面的研究工作目前大多只限于概念模型分析的层面,而没有具体的数学模型.文中应用 Shannon信息熵量化了上下游阶段的交互信息和系统的有序度,建立了基于多层次模糊综合评判和BP神经网络的动态模型来实时确定下游阶段的启动时间.最后给出了基于PDM的实施框架和该模型的应用实例.

  8. Shape based kinetic outlier detection in real-time PCR

    Directory of Open Access Journals (Sweden)

    D'Atri Mario

    2010-04-01

    Full Text Available Abstract Background Real-time PCR has recently become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR assumes that the compared samples have similar PCR efficiency. However, many factors present in biological samples affect PCR kinetic, confounding quantification analysis. In this work we propose a new strategy to detect outlier samples, called SOD. Results Richards function was fitted on fluorescence readings to parameterize the amplification curves. There was not a significant correlation between calculated amplification parameters (plateau, slope and y-coordinate of the inflection point and the Log of input DNA demonstrating that this approach can be used to achieve a "fingerprint" for each amplification curve. To identify the outlier runs, the calculated parameters of each unknown sample were compared to those of the standard samples. When a significant underestimation of starting DNA molecules was found, due to the presence of biological inhibitors such as tannic acid, IgG or quercitin, SOD efficiently marked these amplification profiles as outliers. SOD was subsequently compared with KOD, the current approach based on PCR efficiency estimation. The data obtained showed that SOD was more sensitive than KOD, whereas SOD and KOD were equally specific. Conclusion Our results demonstrated, for the first time, that outlier detection can be based on amplification shape instead of PCR efficiency. SOD represents an improvement in real-time PCR analysis because it decreases the variance of data thus increasing the reliability of quantification.

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

    NARCIS (Netherlands)

    Dijksman J.F.; Pierik, A.

    2012-01-01

    Real Time Array PCR is a recently developed biochemical technique that measures amplification curves (like quantitative real time Polymerase Chain Reaction (qPCR)) of a multitude of different templates ina sample. It combines two different techniques to profit from theadvantages of both techniques,

  10. Scene independent real-time indirect illumination

    DEFF Research Database (Denmark)

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

    2005-01-01

    A novel method for real-time simulation of indirect illumination is presented in this paper. The method, which we call Direct Radiance Mapping (DRM), is based on basal radiance calculations and does not impose any restrictions on scene geometry or dynamics. This makes the method tractable for rea......, which can draw further advantages from upcoming GPU functionalities. The method has been tested for moderately sized scenes with close to real-time frame rates and it scales with interactive frame rates for more complex scenes.......-time rendering of arbitrary dynamic environments and for interactive preview of feature animations. Through DRM we simulate two diffuse reflections of light, but can also, in combination with traditional real-time methods for specular reflections, simulate more complex light paths. DRM is a GPU-based method...

  11. Testing of real-time-software

    International Nuclear Information System (INIS)

    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.)

  12. Development of Real Time Multitask Kernel

    Directory of Open Access Journals (Sweden)

    B. Sahli

    2008-01-01

    Full Text Available This study describes real-time kernel essential mechanisms and deals with the implementation of a real-time multitasks executive. To make more advantage of microprocessors in applications involving many functions simultaneously, our real-time kernel provides a multiprogramming environment in which many independent multitasking application programmes may execute and provides facilities to manage efficiently the processes (tasks and communicate between them. These facilities are provided by system calls that handle data structures namely tasks, semaphores, messages, events flag, resources, mail-boxes, queues and interruptions. Present kernel is preemptive and priorities assigned to tasks are dynamic, the kernel manages up to 63 task levels (63 is the lowest priority level assigned to the idle task. Round robin scheduling is not used here.

  13. Deterministic Real-time Thread Scheduling

    CERN Document Server

    Yun, Heechul; Sha, Lui

    2011-01-01

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

  14. Absolute calibration of the Jenoptik CHM15k-x ceilometer and its applicability for quantitative aerosol monitoring

    Science.gov (United States)

    Geiß, Alexander; Wiegner, Matthias

    2014-05-01

    The knowledge of the spatiotemporal distribution of atmospheric aerosols and its optical characterization is essential for the understanding of the radiation budget, air quality, and climate. For this purpose, lidar is an excellent system as it is an active remote sensing technique. As multi-wavelength research lidars with depolarization channels are quite complex and cost-expensive, increasing attention is paid to so-called ceilometers. They are simple one-wavelength backscatter lidars with low pulse energy for eye-safe operation. As maintenance costs are low and continuous and unattended measurements can be performed, they are suitable for long-term aerosol monitoring in a network. However, the signal-to-noise ratio is low, and the signals are not calibrated. The only optical property that can be derived from a ceilometer is the particle backscatter coefficient, but even this quantity requires a calibration of the signals. With four years of measurements from a Jenoptik ceilometer CHM15k-x, we developed two methods for an absolute calibration on this system. This advantage of our approach is that only a few days with favorable meteorological conditions are required where Rayleigh-calibration and comparison with our research lidar is possible to estimate the lidar constant. This method enables us to derive the particle backscatter coefficient at 1064 nm, and we retrieved for the first time profiles in near real-time within an accuracy of 10 %. If an appropriate lidar ratio is assumed the aerosol optical depth of e.g. the mixing layer can be determined with an accuracy depending on the accuracy of the lidar ratio estimate. Even for 'simple' applications, e.g. assessment of the mixing layer height, cloud detection, detection of elevated aerosol layers, the particle backscatter coefficient has significant advantages over the measured (uncalibrated) attenuated backscatter. The possibility of continuous operation under nearly any meteorological condition with temporal

  15. 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

  16. Real Time Implementation Of Face Recognition System

    Directory of Open Access Journals (Sweden)

    Megha Manchanda

    2014-10-01

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

  17. Machine vision for real time orbital operations

    Science.gov (United States)

    Vinz, Frank L.

    1988-01-01

    Machine vision for automation and robotic operation of Space Station era systems has the potential for increasing the efficiency of orbital servicing, repair, assembly and docking tasks. A machine vision research project is described in which a TV camera is used for inputing visual data to a computer so that image processing may be achieved for real time control of these orbital operations. A technique has resulted from this research which reduces computer memory requirements and greatly increases typical computational speed such that it has the potential for development into a real time orbital machine vision system. This technique is called AI BOSS (Analysis of Images by Box Scan and Syntax).

  18. 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.

  19. Quantitative vapor-phase IR intensities and DFT computations to predict absolute IR spectra based on molecular structure: I. Alkanes

    Science.gov (United States)

    Williams, Stephen D.; Johnson, Timothy J.; Sharpe, Steven W.; Yavelak, Veronica; Oates, R. P.; Brauer, Carolyn S.

    2013-11-01

    Recently recorded quantitative IR spectra of a variety of gas-phase alkanes are shown to have integrated intensities in both the C3H stretching and C3H bending regions that depend linearly on the molecular size, i.e. the number of C3H bonds. This result is well predicted from CH4 to C15H32 by density functional theory (DFT) computations of IR spectra using Becke's three parameter functional (B3LYP/6-31+G(d,p)). Using the experimental data, a simple model predicting the absolute IR band intensities of alkanes based only on structural formula is proposed: For the C3H stretching band envelope centered near 2930 cm-1 this is given by (km/mol) CH_str=(34±1)×CH-(41±23) where CH is number of C3H bonds in the alkane. The linearity is explained in terms of coordinated motion of methylene groups rather than the summed intensities of autonomous -CH2-units. The effect of alkyl chain length on the intensity of a C3H bending mode is explored and interpreted in terms of conformer distribution. The relative intensity contribution of a methyl mode compared to the total C3H stretch intensity is shown to be linear in the number of methyl groups in the alkane, and can be used to predict quantitative spectra a priori based on structure alone.

  20. Statistical analysis of real-time PCR data

    Directory of Open Access Journals (Sweden)

    Reed Ann

    2006-02-01

    Full Text Available Abstract Background Even though real-time PCR has been broadly applied in biomedical sciences, data processing procedures for the analysis of quantitative real-time PCR are still lacking; specifically in the realm of appropriate statistical treatment. Confidence interval and statistical significance considerations are not explicit in many of the current data analysis approaches. Based on the standard curve method and other useful data analysis methods, we present and compare four statistical approaches and models for the analysis of real-time PCR data. Results In the first approach, a multiple regression analysis model was developed to derive ΔΔCt from estimation of interaction of gene and treatment effects. In the second approach, an ANCOVA (analysis of covariance model was proposed, and the ΔΔCt can be derived from analysis of effects of variables. The other two models involve calculation ΔCt followed by a two group t-test and non-parametric analogous Wilcoxon test. SAS programs were developed for all four models and data output for analysis of a sample set are presented. In addition, a data quality control model was developed and implemented using SAS. Conclusion Practical statistical solutions with SAS programs were developed for real-time PCR data and a sample dataset was analyzed with the SAS programs. The analysis using the various models and programs yielded similar results. Data quality control and analysis procedures presented here provide statistical elements for the estimation of the relative expression of genes using real-time PCR.

  1. Kinetic Measurement and Real Time Visualization of Somatic Reprogramming.

    Science.gov (United States)

    Quintanilla, Rene H; Asprer, Joanna; Sylakowski, Kyle; Lakshmipathy, Uma

    2016-01-01

    Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming. PMID:27500543

  2. Phase-shifting real-time holography with photorefractive crystals

    Science.gov (United States)

    Gesualdi, M. R. R.; Soga, D.; Muramatsu, M.

    2006-01-01

    The phase-shifting interferometry techniques is a well-known technique which has been used with great success in optical profilers, micro-displacements, micro-deformations and others applications in Non-Destructive Test in basic research, engineering and biotechnology areas. This work presents our Advances in Phase-Shifting Real-Time Holography using Photorefractive Sillenite. And we have obtained quantitative results in many applications in measurements of micro-rotation, micro-displacements, deformation, surface contouring and whole lens wave-optics. The real-time holography process is doing using the photorefractive Bi 12SiO 20 crystal recording medium, where the phase-shifting 4-frames method for obtained the phase map, this was filtered by sin/cos filter and was applied the unwrapping process. The experimental results agree with the expected one in these applications and with promises potentialities of this method for studies with in situ visualization, monitoring and analysis.

  3. Real Time Engineering Analysis Based on a Generative Component Implementation

    DEFF Research Database (Denmark)

    Kirkegaard, Poul Henning; Klitgaard, Jens

    2007-01-01

    The present paper outlines the idea of a conceptual design tool with real time engineering analysis which can be used in the early conceptual design phase. The tool is based on a parametric approach using Generative Components with embedded structural analysis. Each of these components uses the...... geometry, material properties and fixed point characteristics to calculate the dimensions and subsequent feasibility of any architectural design. The proposed conceptual design tool provides the possibility for the architect to work with both the aesthetic as well as the structural aspects of architecture...... without jumping from aesthetics to structural digital design tools and back, but to work with both simultaneously and real time. The engineering level of knowledge is incorporated at a conceptual thinking level, i.e. qualitative information is used in stead of using quantitative information. An example...

  4. Refactoring Real-Time Java Profiles

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  5. [Real-time ultrasonography in neonatal diagnosis].

    Science.gov (United States)

    Nogués, A; Morales, A; Munguía, C; Pagola, C; Arena, J

    1982-11-01

    Real time ultrasonography is a diagnostic technique very widely used in pediatrics and with specific applications in neonatology. Bedside its use in Neonatal I.C.U. it has many interesting aspects for intraabdominal and intracranial pathology. In some particular conditions this procedure can be the first diagnostic tool. Conventional X-rays can be performed after sonographic data have been analyzed. PMID:7168508

  6. Refactoring Real-Time Java Profiles

    DEFF Research Database (Denmark)

    Søndergaard, Hans; Thomsen, Bent; Ravn, Anders P.;

    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...

  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. Testing Real-Time Systems Using UPPAAL

    DEFF Research Database (Denmark)

    Hessel, Anders; Larsen, Kim Guldstrand; Mikucionis, Marius; Nielsen, Brian; Pettersson, Paul; Skou, Arne

    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...... objectives, derive test sequences, apply these to the system under test, and assign a verdict....

  9. The Real-Time Multitask Threading Control

    OpenAIRE

    Han, Shuang

    2007-01-01

    In this master thesis, we design and implemented a super mode for multiple streaming signal processing applications, and got the timing budget based on Senior DSP processor. This work presented great opportunity to study the real-time system and firmware design knowledge on embedded system.

  10. Ray Tracing for Real-time Games

    NARCIS (Netherlands)

    Bikker, J.

    2012-01-01

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

  11. Real-Time Blackboards For Sensor Fusions

    Science.gov (United States)

    Johnson, Donald H.; Shaw, Scott W.; Reynolds, Steven; Himayat, Nageen

    1989-09-01

    Multi-sensor fusion, at the most basic level, can be cast into a concise, elegant model. Reality demands, however, that this model be modified and augmented. These modifications often result in software systems that are confusing in function and difficult to debug. This problem can be ameliorated by adopting an object-oriented, data-flow programming style. For real-time applications, this approach simplifies data communications and storage management. The concept of object-oriented, data-flow programming is conveniently embodied in the black-board style of software architecture. Blackboard systems allow diverse programs access to a central data base. When the blackboard is described as an object, it can be distributed over multiple processors for real-time applications. Choosing the appropriate parallel architecture is the subject of ongoing research. A prototype blackboard has been constructed to fuse optical image regions and Doppler radar events. The system maintains tracks of simulated targets in real time. The results of this simulation have been used to direct further research on real-time blackboard systems.

  12. Solar neutrinos: Real-time experiments

    Science.gov (United States)

    Totsuka, Yoji

    1993-04-01

    This report outlines the principle of real-time solar neutrino detection experiments by detecting electrons with suitable target material, via Charged-Current (CC) reaction using conventional counting techniques developed in high-energy physics. Only B-8 neutrinos can be detected by minimum detectable energy of several MeV. The MSW (Mikheyev, Smirnov, Wolfenstein) effect not only distorts the energy spectrum but also induces new type of neutrinos, i.e. mu-neutrinos or tau-neutrinos. These neutrinos do not participate in the CC reaction. Therefore real-time experiment is to be sensitive to Neutral Current (NC) reactions. It is a challenge to eliminate environment background as much as possible and to lower the minimum detectable energy to several 100 keV, which will enable observation of Be-7 neutrinos. Target particles of real-time experiments currently running and under construction or planning are electron, deuteron, or argon. The relevant reactions corresponding to CC reaction and some relevant comments on the following targets are described: (1) electron target; (2) deuteron target; and (3) argon target. On-going experiment and future experiments for real-time neutron detection are also outlined.

  13. 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.

  14. Compiling models into real-time systems

    International Nuclear Information System (INIS)

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

  15. Compiling models into real-time systems

    International Nuclear Information System (INIS)

    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

  16. Real time estimates of GDP growth

    NARCIS (Netherlands)

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

    2005-01-01

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

  17. Real time estimates of GDP growth

    NARCIS (Netherlands)

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

    2005-01-01

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

  18. 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…

  19. RTMOD: Real-Time MODel evaluation

    DEFF Research Database (Denmark)

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

    2000-01-01

    The 1998 - 1999 RTMOD project is a system based on an automated statistical evaluation for the inter-comparison of real-time forecasts produced by long-range atmospheric dispersion models for national nuclear emergency predictions of cross-boundaryconsequences. The background of RTMOD was the 1994...... probably contaminated areas....

  20. The Power of Real-Time PCR

    Science.gov (United States)

    Valasek, Mark A.; Repa, Joyce J.

    2005-01-01

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