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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

    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

    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

    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

    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

    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.

    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.

    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

    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.

    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

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

    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.

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

    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.

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

    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

    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

    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

    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

    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

    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

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

    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.

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

    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

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

    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

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

    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

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

    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...

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

    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...

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

    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

    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

    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.

    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

    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

    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

    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.

    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

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

    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...

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

    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...

  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

    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

    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

    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

    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.

    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.

    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.

    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.

    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

    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

    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

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

    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

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

    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.

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

    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”

    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

    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

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

    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...

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

    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

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

    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)

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

    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.

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

    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

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

    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.

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

    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

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

    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

    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.

    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

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

    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.

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

    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.

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

    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.

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

    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.

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

    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)

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

    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.

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

    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

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

    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...

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

    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...

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

    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...

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

    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...

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

    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

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

    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.

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

    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...

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

    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.

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

    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

    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

    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

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

    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.

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

    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...

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

    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

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

    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...

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

    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...

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

    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 ...

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

    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...

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

    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...

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

    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...

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

    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...

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

    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,...

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

    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...

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

    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...

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

    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

    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

    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.

    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

    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.

    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.

    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.

    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

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

    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...

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

    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...

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

    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...

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

    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.

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

    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...

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

    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

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

    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...

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

    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...

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

    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.

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

    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

    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.

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

    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.

    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

    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.

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

    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

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

    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

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

    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

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

    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.

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

    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

    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

    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.

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

    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

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

    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

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

    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

    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

    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.

    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

    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

    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.

    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

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

    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)

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

    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

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

    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.

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

    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

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

    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.

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

    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

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

    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.

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

    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.

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

    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.

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

    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.

    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

    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

    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

    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

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

    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

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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

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

    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

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

    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.

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

    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.

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

    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

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

    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

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

    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

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

    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

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

    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

    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

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

    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

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

    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...

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

    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...

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

    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.

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

    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

    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

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

    Qiusheng Kong

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

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

    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

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

    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

    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.

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

    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.

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

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

    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)

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

    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.

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

    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

    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.

    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

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

    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...

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

    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...

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

    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

    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.

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

    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...

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

    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...

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

    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

    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

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

    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...

  2. [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].

    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

  3. 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

    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...

  4. Real-Time PCR

    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

    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.

    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.

    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.

    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

    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.

    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

    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

    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.

    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.

    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

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

    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 ...

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

    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...

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

    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, ...

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

    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...

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

    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...

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

    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...

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

    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....

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

    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

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

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

    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...

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

    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 ...

  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

    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

    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

    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.)

    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.

    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.

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

    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

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

    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.

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

    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

    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

    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

    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.

    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.

    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. 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

    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

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

    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

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

    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. 

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

    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

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

    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

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

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

    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

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

    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.

  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.

    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

    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....